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Evaluation of Angiogenesis Inhibitors Using the HUVEC Fibrin Bead Sprouting Assay
采用HUVEC纤维蛋白珠催芽试验评估血管生成抑制因子   

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Abstract

Angiogenesis, the growth of new blood vessels from pre-existing vessels, is a critical process that occurs during normal development and tumor formation. Targeting tumor angiogenesis by blocking the activity of vascular endothelial growth factor (VEGF) has demonstrated some clinical benefit; nevertheless there is a great need to target additional angiogenic pathways. We have found that the human umbilical vein endothelial cell (HUVEC) fibrin bead sprouting assay (FBA) is a robust and predictive in vitro assay to evaluate the activity of angiogenesis inhibitors. Here, we describe an optimized FBA protocol for the assessment of biological inhibitors of angiogenesis and the automated quantification of key endpoints.

Background

Angiogenesis, the growth of new blood vessels from pre-existing vessels, is a physiological process that occurs during wound healing and normal development. Angiogenesis is a complex and highly regulated process involving the tight coordination of endothelial cell proliferation, differentiation, migration, matrix adhesion, and cell-to-cell signaling. Angiogenesis is also critically involved in tumor development and metastasis. Indeed, targeting tumor angiogenesis by blocking the activity of vascular endothelial growth factor (VEGF) has demonstrated clinical benefit. Since tumors do eventually develop resistance to VEGF-targeted therapy, there is a great need to target additional angiogenic pathways. We have found that the human umbilical vein endothelial cell (HUVEC) fibrin bead sprouting assay (FBA) (Nakatsu et al., 2007; Nakatsu and Hughes, 2008; Nehls and Drenckhahn, 1995) is a robust and predictive in vitro assay to evaluate the activity of angiogenesis inhibitors. This assay recapitulates key aspects of angiogenesis such as lumen formation, endothelial cell polarization and dependency on stromal cells, and is correlative with the activities of angiogenesis inhibitors as observed in in vivo tumor studies (Figures 1 and 2) (Eichten et al., 2013; Holash et al., 2012; Kuhnert et al., 2015; Noguera-Troise et al., 2006). Here we describe an optimized FBA protocol for the assessment of biological inhibitors of angiogenesis and the automated quantification of key endpoints, such as the number of endothelial cells or branch points, as well as sprout length and area (Figure 3). To illustrate the spectrum of treatment outcomes in the FBA, the effects of three different angiogenesis inhibitors [aflibercept, Dll4 blocking monoclonal antibody (Dll4 MAB) and anti-Integrin a6 antibody GOH3] on endothelial sprouting have been included in the protocol.

Materials and Reagents

  1. 50 ml conical tubes (Corning, Falcon®, catalog number: 352070 )
  2. Cytodex-3 beads (GE Healthcare, Amersham Pharmacia Biotech, catalog number: 17-0485-01 )
  3. Aspirator
  4. FACS tubes (5 ml) (Corning, Falcon®, catalog number: 352063 )
  5. 15 ml conical tubes (Conring, Falcon®, catalog number: 352099 )
  6. 1.5 ml centrifuge tube
  7. 24 well plate (Corning, Falcon®, catalog number: 351147 )
  8. 0.22 μm filter (VWR, catalog number: 28145-501 )
  9. Human umbilical vein endothelial cells (HUVEC) and HUVEC complete media (Lonza, catalog numbers: C2517A and CC3162 )
    Note: Optimal at passage 2-4.
  10. Normal human lung fibroblasts (NHLF) (Lonza, catalog number: CC2512 )
  11. Sigmacote siliconizing reagent (Sigma-Aldrich, catalog number: SL2-25ML )
  12. Distilled water (Thermo Fisher Scientific, GibcoTM, catalog number: 15230-162 )
  13. DPBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14040-141 )
  14. Fibrinogen from bovine plasma (Sigma-Aldrich, catalog number: F8630-1G )
  15. Thrombin from bovine plasma (Sigma-Aldrich, catalog number: T3399-1KU )
    Note: This product has been discontinued.
  16. Aprotinin (Sigma-Aldrich, catalog number: A1153-10MG )
  17. Clonetics EGM-2 bullet kit (Lonza, catalog number: CC3162 )
  18. Trypsin/EDTA (0.025% trypsin/0.75 mM EDTA) (EMD Millipore, catalog number: SM-2004-C )
  19. Aflibercept (VEGF-Trap) (Regeneron Pharmaceuticals)
  20. Dll4 blocking monoclonal antibody (Dll4 MAB) (Regeneron Pharmaceuticals)
  21. Rat anti-human integrin α-6 antibody, GoH3 (BD, BD PharmingenTM, catalog number: 555734 )
  22. Paraformaldehyde (PFA) (16%) (Electron Microscopy Sciences, catalog number: 15710 )
  23. Triton X-100 (Sigma-Aldrich, catalog number: T8787-100ML )
  24. Phalloidin-Tetramethylrhodamine B isothiocyanate (Phalloidin-TRITC) (Sigma-Aldrich, catalog number: P1951-.1MG )
  25. FGM-2 bullet kit (Lonza, catalog number: CC3132 )
  26. Hoechst 33258, pentahydrate, bis-benzimide (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: H3569 )
  27. Fibrinogen solution (see Recipes)
  28. Thrombin stock solution (see Recipes)
  29. Aprotinin stock solution (see Recipes)

Equipment

  1. Siliconized glass bottles (Corning, PYREX®, catalog number: 1395-100 )
  2. Laminar flow hood
  3. Water bath (PolyScience, catalog number: WB05A11B )
  4. T25 flasks (Corning, catalog number: 430639 )
  5. Centrifuge
  6. Incubator
  7. P1000 pipette
  8. 24 well glass bottom sensoPlate (Greiner Bio One, catalog number: 662892 )
  9. Cell counter (Nexcelom BioScience, model: Cellometer Auto 1000 )
  10. Microscope (Nikon, model: Eclipse Ti-S )
  11. ImageXpress® MICRO XL (Molecular Devices)

Software

  1. MetaXpress (Molecular Devices)
    Note: MetaXpress software (MX) from Molecular Devices is optimized to perform with the ImageXpressMICRO imaging systems. MX is used both to control image acquisition and to perform image analysis. Necessary features of MX utilized in FBA image acquisition and analysis are: 1) Image acquisition: Laser-based autofocus, Z-stack acquisition; 2) Image analysis: 'Tube Formation' image analysis application module, an interactive Custom Module.

Procedure

  1. Preparation of reagents
    1. Siliconization and sterilization
      1. Add 5 ml of Sigmacote to clean glass bottles in order to prevent the beads from sticking to the glassware.
      2. Rotate the vessel or glassware to ensure that the Sigmacote covers the entire surface of the glass bottle.
      3. Aspirate excess Sigmacote from the glassware and allow to air dry in a laminar flow hood.
      4. Thoroughly wash the glassware in deionized tissue culture grade water. A minimum of two washings is suggested.
      5. Sterilize glassware by autoclaving.
      Note: Once glassware has been siliconized, it is not necessary to treat prior to each use.
    2. Cytodex bead preparation
      1. In a 50 ml Falcon tube, hydrate 0.5 g dry beads in 50 ml PBS (pH = 7.4). Place on a rocker for at least 3 h at RT.
      2. Let beads settle (~15 min). Aspirate the supernatant using a pipette and wash 3 x 5 min in 50 ml fresh PBS using a rocker at RT. Do not vortex.
      3. Aspirate PBS using a pipette and replace with fresh PBS (50 ml) such that the bead concentration is 10 mg/ml or 30,000 beads/ml.
      4. Transfer the bead suspension in a siliconized glass bottle.
      5. Sterilize the beads by autoclaving for 15 min at 115 °C.
      6. Store at 4 °C.
    3. Fibrinogen solution preparation (see Recipes)
    4. Thrombin stock solution preparation (see Recipes)
    5. Aprotinin stock solution preparation (see Recipes)

  2. Preparation of cells
    Change the growth media for HUVECs and fibroblasts to EGM-2 media 1 day before use. For HUVECs, switch medium to EGM-2 the day before beading. For fibroblasts, switch the FGM-2 medium to EGM-2 the day before embedding in fibrin. Beading requires ~400 HUVECs per bead. Use 20,000 fibroblasts per well of a 24-well plate.

  3. Coating the beads with HUVEC (Day-1)
    1. Warm EGM-2 media in a water bath set to 37 °C.
    2. Trypsinize HUVECs which should be 80% confluent. Wash cells with PBS and then add 3 ml of trypsin. Allow trypsin to remain on cells for approximately 2 min or until cells begin to detach from the flask. Add EGM-2 media to neutralize the trypsin and bring the total volume to 10 ml. Spin cells in a centrifuge, remove supernatant with an aspirator, and adjust cell concentration to 2 x 106 cells/ml.
    3. Under sterile conditions, place 170 μl bead solution (stock concentration: 30,000 beads/ml) in a FACS tube. This should be ~5,000 beads. Allow beads to settle (do not centrifuge), aspirate the supernatant, and wash the beads in 2 ml of warm EGM-2 medium. Remove media using a pipette and add fresh warm media for a total volume of 2 ml.
    4. Add 2 x 106 HUVEC (1 ml of 2 x 106 cells/ml) to 5,000 beads (in 2 ml) in the FACS tube (total volume 3 ml). Place tube vertically in the incubator. This will be enough for ~20 wells.
    5. Incubate for 4 h at 37 °C, inverting and mixing the tube every 20 min. Beads should look like mini golf balls after beading (Figure 1).
    6. After 4 h, transfer the coated beads to a T25 tissue culture flask and leave overnight in a total volume of 5 ml of EGM-2 at 37 °C and 5% CO2.

  4. Embedding coated beads in fibrin gel (Day 0)
    1. Add 0.15 Units/ml of aprotinin to 15 ml of fibrinogen solution.
    2. Check HUVEC coated beads. Transfer 5 ml of coated beads to a 15 ml conical tube and let the beads settle. Use additional 5 ml of media to wash off beads that are stuck to flask.
    3. Let beads settle to the bottom of the tube. Aspirate off media. Re-suspend beads in 1 ml of EGM-2 and transfer to a 1.5 ml centrifuge tube.
    4. Wash the beads 2 x with 1 ml of EGM-2, mixing by pipetting up and down very slowly/carefully (beads are fragile) with a P1000 pipette.
    5. Add 6.25 μl of thrombin stock solution (50 U/ml) to the center of each well of a 24-well plate. This will result in 0.625 U/ml once 0.5 ml of fibrinogen/bead suspension is added in the next step.
    6. Get fibrinogen/bead mixture into solution by pipetting up and down gently. Add 0.5 ml of fibrinogen/bead suspension to each well.
    7. Mix the thrombin and the fibrinogen/beads by pipetting up and down gently ~five times. Change the pipette tip for each well in order to prevent clotting in the pipette tip between wells. Avoid creating bubbles in the fibrin gel (may hinder visibility in gel). Do not move the plate in order to avoid tearing of the fibrin gel.
      Note: Usually, when the fibrin gel is formed, tiny bubbles will be present in the gel. They will disappear in 3 to 4 days.
    8. Allow the fibrinogen/bead solution to solidify for 5 min at RT in hood. It is important that the plate is not disturbed during the first 5 min of clotting because sheared fibrin reduces sprouting. After 5 min, check under microscope to see if beads are evenly distributed (not clumped together). Ideally there should be 200-250 beads per well.
      Note: Increasing the number of beads per well results in earlier anastomosis.
    9. Then incubate at 37 °C and 5% CO2 for 10-15 min.
    10. While waiting, trypsinize fibroblasts and re-suspend in EGM adjusted to 1 x 106 cell/ml (or 1,000 cells/μl)
      Note: Fibroblasts will proliferate, migrate and form a monolayer on the fibrin gel.
    11. Prepare the appropriate amount of EGM-2 media containing the angiogenesis inhibitor of choice (aflibercept at 50 μg/ml, Dll4 MAB at 50 μg/ml, Integrin α-6 antibody GoH3 at 10 μg/ml).
    12. After incubation, add 1 ml of EGM-2 prepared to each well slowly (drop by drop). If media is added too fast, it could tear the gel.
    13. Seed 20 μl of NHLF cell solution (20,000 cells) on top of the clot into each well. Add directly into the center of well and into the media.
    14. Replace with fresh EGM-2 medium every other day (day 2, 5, 7, etc.) until desired growth (see notes below) is achieved and capture images accordingly.
      Notes:
      1. Budding/sprouting should be apparent between day 2 and 4.
      2. Lumen formation begins around day 4 to 5 and sprouts continue to elongate.
      3. Newly formed tubes begin to branch around day 4 to 6.
      4. By day 6 to 7, the microvessel-like structures begin to anastomose (connection of two structures) with adjoining tubes.

  5. Gel processing and sprout staining for DNA and actin
    1. To stop the assay, 500 μl of 2% paraformaldehyde (PFA) is added to the gels for 1 h at 37 °C, followed by overnight incubation at room temperature (RT). Wash twice with PBS.
    2. To stain the endothelial sprouts, gels are permeabilized with 500 μl of 0.5% Triton X-100 for 20 min, washed twice with PBS.
    3. Incubate with the mixture of 500 μl of Phalloidin-TRITC (1:1,000) and Hoechst 33258 (1:2,000) in PBS for 1 h at RT.
    4. Wash twice with PBS.
    Note: For qualitative assessment of endothelial sprouting, EGM-2 medium-covered 24-well plates can be imaged with an inverted microscope under bright field settings at desired time points (Figure 2).

  6. Image acquisition on ImageXpressMICRO XL
    1. Fibrin beads assays are performed in 24 well optical plates (Sensoplate).
    2. Images of whole wells are acquired with 10x objective using automated microscope ImageXpressMICRO XL and MetaXpress (MX) software, as follows: for each site, proprietary Z-stack journal is used to collect sixteen focus planes separated by 10 μm. Proprietary Z-stack journal is a configured journal (macro) written in MX software. The configured journal consists of a 'command' guiding the stage move in Z-plane, followed by a command guiding collection of 16 images separated by the distance of 10 μm, followed by a command to create max. intensity projection of these 16 images.
    3. Planes are collapsed into one final image using MX Maximum Intensity Projection algorithm.


      Figure 1. Coating Cytodex beads with human umbilical vein endothelial cells. Bright field images of Cytodex beads before (A) and after coating with endothelial cells (B). Hoechst 33258 staining identifies endothelial nuclei on coated beads (C). Scale bar represents 100 μm.


      Figure 2. Effects of the angiogenesis inhibitors aflibercept and Dll4 MAB on HUVEC sprouting. Aflibercept treatment quantitatively suppressed HUVEC sprouting, while blockade of Dll4 with Dll4 MAB increased HUVEC sprout length and branch point number. Scale bar represents 100 μm. Asterisks mark branch points.


      Figure 3. Integrin α-6 antibody GOH3 significantly inhibits HUVEC sprouting. A. Representative images of stained sprouts in transmitted light channel (left panel) or TRITC channel (middle and right panels) are shown. Fluorescent images of fixed gels were acquired on ImageXpressMICRO automated microscope. Scale bar represents 100 μm. B. Sprout Area image (right panel) was derived by subtracting a circular shape corresponding to Bead Area from Total Segmented Area Image (Figure 3A, middle panel) using 'Subtract' function of MetXpress software.

Data analysis

  1. Image analysis using MetaXpressTM software (MX): To calculate the number of endothelial cells per bead, maximum intensity projection images acquired in transmitted light and DAPI channels were analyzed using configured MX Custom Module in combination with configured MX Multi-Wavelength Cell Scoring Module (Kuhnert et al., 2015; Sweet et al., 2012).
  2. To calculate the total length of sprouts, or the number of branch points, Maximum Intensity Projection images acquired in transmitted light and DAPI channels were analyzed using configured MX Custom Module in combination with configured MX Tube Formation Module. Statistical significance for quantitative data is determined by Student’s t-test in pairwise comparisons and one-way ANOVA for multiple comparisons (Kuhnert et al., 2015; Sweet et al., 2012).

Recipes

  1. Fibrinogen solution
    Dissolve 2 mg/ml fibrinogen in DPBS in a 37 °C water bath.
    Mix by inverting the tube.
    Do not vortex.
    Pass through a 0.22 μm filter to sterilize.
  2. Thrombin stock solution
    Reconstitute in sterile water at 50 U/ml and sterile filter through a 0.22 μm filter.
    Make aliquots of 0.5 ml each and store at -20 °C.
  3. Aprotinin stock solution
    Reconstitute lyophilized aprotinin at 4 U/ml in DI water and sterile filter through a 0.22 μm filter.
    Make aliquots of 0.5 ml each.
    Store at -20 °C.

Acknowledgments

This work was funded by Regeneron Pharmaceuticals, Inc.

References

  1. Eichten, A., Adler, A. P., Cooper, B., Griffith, J., Wei, Y., Yancopoulos, G. D., Lin, H. C. and Thurston, G. (2013). Rapid decrease in tumor perfusion following VEGF blockade predicts long-term tumor growth inhibition in preclinical tumor models. Angiogenesis 16(2): 429-441.
  2. Holash, J., Davis, S., Papadopoulos, N., Croll, S. D., Ho, L., Russell, M., Boland, P., Leidich, R., Hylton, D., Burova, E., Ioffe, E., Huang, T., Radziejewski, C., Bailey, K., Fandl, J. P., Daly, T., Wiegand, S. J., Yancopoulos, G. D. and Rudge, J. S. (2002). VEGF-Trap: a VEGF blocker with potent antitumor effects. Proc Natl Acad Sci U S A 99(17): 11393-11398.
  3. Kuhnert, F., Chen, G., Coetzee, S., Thambi, N., Hickey, C., Shan, J., Kovalenko, P., Noguera-Troise, I., Smith, E., Fairhurst, J., Andreev, J., Kirshner, J. R., Papadopoulos, N. and Thurston, G. (2015). Dll4 blockade in stromal cells mediates antitumor effects in preclinical models of ovarian cancer. Cancer Res 75(19): 4086-4096.
  4. Nakatsu, M. N., Davis, J. and Hughes, C. C. (2007). Optimized fibrin gel bead assay for the study of angiogenesis. J Vis Exp(3): 186.
  5. Nakatsu, M. N. and Hughes, C. C. (2008). An optimized three-dimensional in vitro model for the analysis of angiogenesis. Methods Enzymol 443: 65-82.
  6. Nehls, V. and Drenckhahn, D. (1995). A novel, microcarrier-based in vitro assay for rapid and reliable quantification of three-dimensional cell migration and angiogenesis. Microvasc Res 50(3): 311-322.
  7. Noguera-Troise, I., Daly, C., Papadopoulos, N. J., Coetzee, S., Boland, P., Gale, N. W., Lin, H. C., Yancopoulos, G. D. and Thurston, G. (2006). Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis. Nature 444(7122):1032-1037.
  8. Sweet, D. T., Chen, Z., Wiley, D. M., Bautch, V. L. and Tzima, E. (2012). The adaptor protein Shc integrates growth factor and ECM signaling during postnatal angiogenesis. Blood 119(8): 1946-1955.

简介

血管发生,来自预先存在的血管的新血管的生长是在正常发育和肿瘤形成期间发生的关键过程。通过阻断血管内皮生长因子(VEGF)的活性靶向肿瘤血管生成已经证明了一些临床益处;然而,非常需要靶向额外的血管生成途径。我们已经发现,人脐静脉内皮细胞(HUVEC)纤维蛋白珠发芽测定(FBA)是评估血管生成抑制剂的活性的稳健的和预测性的体外测定。在这里,我们描述了用于评估血管生成的生物抑制剂和关键终点的自动定量的优化的FBA方案。

[背景] 血管发生,新血管的生长从先前存在的血管,是在伤口愈合和正常发育期间发生的生理过程。血管生成是一个复杂和高度调节的过程,涉及内皮细胞增殖,分化,迁移,基质粘附和细胞间的信号的紧密协调。血管发生也严重参与肿瘤发展和转移。事实上,通过阻断血管内皮生长因子(VEGF)的活性靶向肿瘤血管生成已经证明了临床益处。由于肿瘤最终对VEGF靶向治疗产生抗性,因此非常需要靶向额外的血管生成途径。我们已经发现人脐静脉内皮细胞(HUVEC)纤维蛋白珠发芽测定(FBA)(Nakatsu等人,2007; Nakatsu和Hughes,2008; Nehls和Drenckhahn,1995)和预测性体外测定来评价血管生成抑制剂的活性。该测定概括了血管生成的关键方面,例如管腔形成,内皮细胞极化和对基质细胞的依赖性,并且与在体内肿瘤研究中观察到的血管生成抑制剂的活性相关(图1和图2) (Eichten等人,2013; Holash等人,2012; Kuhnert等人,2015; Noguera-Troise等人al。,2006)。在这里我们描述了一个优化的FBA协议,用于评估血管生成的生物抑制剂和关键端点的自动定量,如内皮细胞或分支点的数量,以及发芽长度和面积(图3)。为了说明FBA中的治疗结果的谱,三种不同的血管生成抑制剂[阿柏西普,Dll4阻断单克隆抗体(Dll4 MAB)和抗整联蛋白α6抗体GOH3]对内皮发芽的影响已包括在方案中。

材料和试剂

  1. 50ml锥形管(Coring,Falcon ,目录号:352070)
  2. Cytodex-3珠(GE Healthcare,Amersham Pharmacia Biotech,目录号:17-0485-01)
  3. 吸气器
  4. FACS管(5ml)(Coring,Falcon ,目录号:352063)
  5. 15ml锥形管(Coring,Falcon ,目录号:352099)
  6. 1.5ml离心管
  7. 24孔板(Coring,Falcon ,目录号:351147)
  8. 0.22μm过滤器(VWR,目录号:28145-501)
  9. 人脐静脉内皮细胞(HUVEC)和HUVEC完全培养基(Lonza,目录号:C2517A和CC3162)
    注意:最好在2-4节。
  10. 正常人肺成纤维细胞(NHLF)(Lonza,目录号:CC2512)
  11. Sigmacote渗硅试剂(Sigma-Aldrich,目录号:SL2-25ML)
  12. 蒸馏水(Thermo Fisher Scientific,Gibco< sup> TM,目录号:15230-162)
  13. DPBS(Thermo Fisher Scientific,Gibco TM ,目录号:14040-141)
  14. 来自牛血浆的纤维蛋白原(Sigma-Aldrich,目录号:F8630-1G)
  15. 来自牛血浆的凝血酶(Sigma-Aldrich,目录号:T3399-1KU)
    注意:此产品已停产。
  16. 抑肽酶(Sigma-Aldrich,目录号:A1153-10MG)
  17. Clonetics EGM-2子弹试剂盒(Lonza,目录号:CC3162)
  18. 胰蛋白酶/EDTA(0.025%胰蛋白酶/0.75mM EDTA)(EMD Millipore,目录号:SM-2004-C)
  19. Aflibercept(VEGF-Trap)(Regeneron Pharmaceuticals)
  20. Dll4阻断单克隆抗体(Dll4 MAB)(Regeneron Pharmaceuticals)
  21. 大鼠抗人整联蛋白α-6抗体GoH3(BD,BD Pharmingen ,目录号:555734)
  22. 多聚甲醛(PFA)(16%)(Electron Microscopy Sciences,目录号:15710)
  23. Triton X-100(Sigma-Aldrich,目录号:T8787-100ML)
  24. 鬼笔环肽 - 四甲基罗丹明B异硫氰酸酯(Phalloidin-TRITC)(Sigma-Aldrich,目录号:P1951-.1MG)
  25. FGM-2子弹试剂盒(Lonza,目录号:CC3132)
  26. Hoechst 33258,五水合物,双 - 苯甲酰亚胺(Thermo Fisher Scientific,Molecular Probes TM ,目录号:H3569)
  27. 纤维蛋白原溶液(参见配方)
  28. 凝血酶储备溶液(见配方)
  29. 抑肽酶原液(参见配方)

设备

  1. 硅化玻璃瓶(Corning,PYREX ,目录号:1395-100)
  2. 层流罩
  3. 水浴(PolyScience,目录号:WB05A11B)
  4. T25烧瓶(Corning,目录号:430639)
  5. 离心机
  6. 孵化器
  7. P1000移液器
  8. 24孔玻璃底部感光板(Greiner Bio One,目录号:662892)
  9. 细胞计数器(Nexcelom BioScience,型号:Cellometer Auto 1000)
  10. 显微镜(Nikon,型号:Eclipse Ti-S)
  11. ImageXpress ?MICRO XL (Molecular Devices)

软件

  1. MetaXpress(Molecular Devices)
    注意:Molecular Devices的MetaXpress软件(MX)经过优化,可与ImageXpress MICRO 成像系统配合使用。 MX用于控制图像采集和执行图像分析。在FBA图像采集和分析中使用的MX的必要特征是:1)图像采集:基于激光的自动对焦,Z堆叠采集; 2)图像分析:"管形成"图像分析应用模块,交互式自定义模块。

程序

  1. 试剂的制备
    1. 硅化和灭菌
      1. 加入5毫升的Sigmacote清洗玻璃瓶,以防止珠子粘在玻璃器皿。
      2. 旋转容器或玻璃器皿,以确保Sigmacote覆盖玻璃瓶的整个表面。
      3. 从玻璃器皿吸出过量的Sigmacote,并允许在层流罩中风干
      4. 在去离子组织培养级水中彻底清洗玻璃器皿。建议至少进行两次洗涤。
      5. 通过高压灭菌器灭菌玻璃器皿。
      注意:一旦玻璃器皿已经硅化,在每次使用之前不必进行处理。
    2. Cytodex珠准备
      1. 在50ml Falcon管中,在50ml PBS(pH = 7.4)中水合0.5g干珠。在室温下放置摇臂至少3小时。
      2. 让珠沉淀(?15分钟)。使用移液器吸取上清液,并使用摇床在室温下在50ml新鲜PBS中洗涤3×5分钟。不要涡旋。
      3. 用移液管吸取PBS,更换新鲜PBS(50ml),使珠浓度为10mg/ml或30,000珠/ml。
      4. 将珠悬浮液转移到硅化玻璃瓶中。
      5. 通过在115℃下高压灭菌15分钟来灭菌珠子
      6. 储存于4°C。
    3. 纤维蛋白原溶液制备(参见配方)
    4. 凝血酶储备液制备(参见配方)
    5. 抑肽酶原液制备(见配方)

  2. 细胞的制备
    在使用前1天将HUVEC和成纤维细胞的生长培养基更换为EGM-2培养基。对于HUVEC,在珠粒前一天将培养基更换为EGM-2。对于成纤维细胞,在嵌入纤维蛋白前一天将FGM-2培养基切换到EGM-2。珠粒需要每个珠约400个HUVEC。在24孔板的每孔中使用20,000个成纤维细胞
  3. 用HUVEC(第1天)包被珠子
    1. 将温热的EGM-2培养基置于37℃的水浴中
    2. Trypsinize HUVECs应该是80%汇合。用PBS洗涤细胞,然后加入3毫升胰蛋白酶。使胰蛋白酶保留在细胞上约2分钟或直到细胞开始从烧瓶中分离。加入EGM-2培养基以中和胰蛋白酶并使总体积达到10ml。在离心机中旋转细胞,用吸气器除去上清液,并将细胞浓度调节至2×10 6个细胞/ml。
    3. 在无菌条件下,将170μl珠溶液(储备浓度:30,000个珠/ml)置于FACS管中。这应该?5,000珠。使珠沉降(不要离心),吸出上清液,并在2ml温热的EGM-2培养基中洗涤珠。使用移液管移除介质,添加新鲜温暖的媒体,总体积为2毫升
    4. 向FACS管(总体积3ml)中的5,000个珠(在2ml中)中加入2×10 6 HUVEC(1ml的2×10 6个细胞/ml) 。将管垂直放置在孵化器中。这对于?20口井就足够了。
    5. 在37°C孵育4小时,倒置和混合管每20分钟。珠子在珠粒后应该看起来像迷你高尔夫球(图1)。
    6. 4小时后,将包被的珠子转移到T25组织培养瓶中,并在37℃和5%CO 2的总体积为5ml的EGM-2中放置过夜。
  4. 将包被的珠子包埋在纤维蛋白凝胶(第0天)中
    1. 将0.15单位/ml抑肽酶加到15ml纤维蛋白原溶液中
    2. 检查HUVEC涂层珠。转移5毫升涂层的珠子到15毫升锥形管,让珠子沉降。使用额外的5ml培养基冲洗掉粘在烧瓶上的珠子。
    3. 让珠子沉淀到管的底部。吸出介质。将珠子重悬在1ml EGM-2中,并转移到1.5ml离心管中
    4. 用1ml EGM-2洗涤珠子2次,用P1000移液管非常缓慢/小心地上下吹打混合(珠子是脆弱的)。
    5. 向24孔板的每个孔的中心加入6.25μl凝血酶储备溶液(50U/ml)。这将导致0.625U/ml,一旦在下一步中加入0.5ml纤维蛋白原/珠悬浮液。
    6. 通过轻轻地向上和向下吸取纤维蛋白原/珠混合物溶液。向每个孔中加入0.5ml纤维蛋白原/珠悬浮液。
    7. 混合凝血酶和纤维蛋白原/珠通过轻轻吸取上下轻轻?五次。更换每个孔的移液器吸头,以防止吸头之间的孔凝结。避免在纤维蛋白凝胶中产生气泡(可能阻碍凝胶的可见性)。不要移动板,以避免撕裂纤维蛋白凝胶。
      注意:通常,当形成纤维蛋白凝胶时,凝胶中将存在微小的气泡。它们将在3到4天后消失。
    8. 允许纤维蛋白原/珠溶液在室温下在通风橱中固化5分钟。重要的是,在凝块的前5分钟期间板不被干扰,因为剪切的纤维蛋白减少发芽。 5分钟后,在显微镜下检查珠子是否均匀分布(不结块在一起)。理想地,每个孔应该有200-250个珠 注意:增加每个孔的珠数会导致更早的吻合。
    9. 然后在37℃和5%CO 2孵育10-15分钟
    10. 在等待时,胰蛋白酶消化成纤维细胞,并重新悬浮于调节至1×10 6细胞/ml(或1,000细胞/μl)的EGM中。
      注意:成纤维细胞会在纤维蛋白凝胶上增殖,迁移和形成单层。
    11. 制备适当量的含有选择的血管生成抑制剂(50μg/ml的阿柏西普,50μg/ml的Dll4 MAB,10μg/ml的整联蛋白α-6抗体GoH3)的EGM-2培养基。
    12. 孵育后,向每孔中缓慢加入1ml制备的EGM-2(逐滴)。如果添加的介质太快,可能会撕裂凝胶。
    13. 种子20微升的NHLF细胞溶液(20,000个细胞)的凝块顶部到每个孔。直接添加到孔的中心和介质中。
    14. 每隔一天(第2天,第5天,第7天,等)更换新鲜的EGM-2培养基,直到达到理想的生长(见下面的注释),并相应地捕获图像。 注意:
      1. 在第2天和第4天之间,显露/发芽应该是明显的。


      2. 。新建成的管子在第4天到第6天开始分支
      3. 在第6天到第7天,微血管样结构开始与邻接的管吻合(两个结构的连接)。

  5. 凝胶加工和DNA和肌动蛋白的新芽染色
    1. 为了停止测定,将500μl的2%多聚甲醛(PFA)在37℃下加入凝胶1小时,随后在室温(RT)下温育过夜。用PBS洗涤两次。
    2. 为了染色内皮新芽,用500μl0.5%Triton X-100将凝胶透化20分钟,用PBS洗涤两次。
    3. 在室温下与500μlPhalloidin-TRITC(1:1,000)和Hoechst 33258(1:2,000)在PBS中的混合物孵育1小时。
    4. 用PBS洗涤两次。
    注意:为了定性评估内皮萌芽,可以在明视野设置下在期望的时间点用倒置显微镜对EGM-2培养基覆盖的24孔板进行成像(图2)。

  6. 在ImageXpress MICRO XL
    上进行图像采集
    1. 纤维蛋白珠测定在24孔光学板(Sensoplate)中进行。
    2. 使用自动显微镜ImageXpress MICRO XL和MetaXpress(MX)软件,以如下方式用10x物镜获得整个孔的图像:对于每个位点,使用专有Z-堆叠日志收集十六个聚焦平面, 10μm。专有Z堆栈日志是用MX软件编写的配置日志(宏)。组态的日志包括一个"命令",引导舞台在Z平面中移动,然后是一个命令,引导收集16个图像,间隔10μm,然后命令创建最大。这些16幅图像的强度投影。
    3. 使用MX最大强度投影算法将平面折叠成一个最终图像。


      图1.用人脐静脉内皮细胞涂覆Cytodex珠。在涂覆内皮细胞(B)之前(A)和之后的Cytodex珠的明视野图像。 Hoechst 33258染色鉴定包被珠粒上的内皮细胞核(C)。比例尺表示100μm

      图2.血管生成抑制剂aflibercept和Dll4 MAB对HUVEC发芽的影响。Aflibercept治疗定量抑制HUVEC发芽,而用Dll4 MAB阻断Dll4增加HUVEC发芽长度和分支点数。比例尺表示100μm。星号标记分支点。


      图3.整联蛋白α-6抗体GOH3显着抑制HUVEC萌芽。显示了透射光通道(左图)或TRITC通道(中间和右图)中的染色芽的代表性图像。在ImageXpress MICRO自动显微镜上获得固定凝胶的荧光图像。比例尺表示100μm。 B.通过使用MetXpress软件的"Subtract"函数从总分割区域图像(图3A,中间图)减去对应于珠粒面积的圆形形状,得到芽芽区域图像(右图)。

数据分析

  1. 使用MetaXpress TM 软件(MX)进行图像分析:为了计算每个珠的内皮细胞的数量,使用配置的MX定制模块结合配置的MX分析在透射光和DAPI通道中获得的最大强度投影图像多波长细胞计分模块(Kuhnert等人,2015; Sweet 等人,2012)。
  2. 为了计算芽的总长度或分支点的数量,使用配置的MX定制模块与配置的MX管形成模块组合来分析在透射光和DAPI通道中获取的最大强度投影图像。通过成对比较中的Student's t检验和多重比较的单因素方差分析来确定定量数据的统计显着性(Kuhnert等人,2015; Sweet et al。 al 。,2012)。

食谱

  1. 纤维蛋白溶液
    在37℃水浴中将2mg/ml纤维蛋白原溶解在DPBS中 通过倒置管混合。
    不要涡旋。
    通过0.22μm过滤器进行灭菌
  2. 凝血酶储备液
    在无菌水中以50U/ml重构,并通过0.22μm过滤器无菌过滤 每份0.5ml,保存于-20℃
  3. 抑肽酶储备液
    在去离子水中以4U/ml重构冻干的抑肽酶,并通过0.22μm过滤器无菌过滤 分别制备0.5ml的等分试样。
    储存于-20°C。

致谢

这项工作由Regeneron Pharmaceuticals,Inc.

参考文献

  1. Eichten,A.,Adler,AP,Cooper,B.,Griffith,J.,Wei,Y.,Yancopoulos,GD,Lin,HC和Thurston,G。(2013)。  VEGF-Trap:具有有效抗肿瘤作用的VEGF阻断剂。 Proc Natl Acad Sci USA 99(17):11393-11398。
  2. Kuhnert,F.,Chen,G.,Coetzee,S.,Thambi,N.,Hickey,C.,Shan,J.,Kovalenko,P.,Noguera-Troise,I.,Smith,E.,Fairhurst,J 。Andreev,J.,Kirshner,JR,Papadopoulos,N。和Thurston,G。(2015)。  用于研究血管生成的优化的血纤维蛋白凝胶珠测定法。(3):186。
  3. Nakatsu,MN和Hughes,CC(2008)。  优化的三维体外模型用于血管生成的分析。


    方法 443:65-82。
  4. Nehls,V。和Drenckhahn,D。(1995)。  一种基于微载体的新颖体外试验,用于三维细胞迁移和血管生成的快速和可靠的定量。 Microvasc Res 50(3):311 -322。
  5. Noguera-Troise,I.,Daly,C.,Papadopoulos,NJ,Coetzee,S.,Boland,P.,Gale,NW,Lin,HC,Yancopoulos,GD和Thurston,G。 Dll4的阻断通过促进非生产性血管生成抑制肿瘤生长。 444(7122):1032-1037
  6. Sweet,DT,Chen,Z.,Wiley,DM,Bautch,VL和Tzima,E。(2012)。
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引用:Winters, L., Thambi, N., Andreev, J. and Kuhnert, F. (2016). Evaluation of Angiogenesis Inhibitors Using the HUVEC Fibrin Bead Sprouting Assay. Bio-protocol 6(19): e1947. DOI: 10.21769/BioProtoc.1947.
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