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Apr 2018
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Human Endothelial Cell Spheroid-based Sprouting Angiogenesis Assay in Collagen
在胶原蛋白中分析人内皮细胞基于球体的出芽式血管生成   

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Abstract

Angiogenesis, the formation of new blood vessels from pre-existing ones plays an important role during organ development, regeneration and tumor progression. The spheroid-based sprouting assay is a well-established and robust method to study the influence of genetic alterations or pharmacological compounds on capillary-like tube formation of primary cultured endothelial cells. A major advantage of this assay is the possibility to study angiogenesis in a 3D environment. Endothelial cells are cultured as hanging drops to form spheroids. Those spheroids are embedded into a collagen matrix and tube formation is analyzed 24 h later. By analyzing sprout number and sprout length the effects of genetic manipulation or drug treatment on angiogenesis can be investigated.

Keywords: Endothelial cells (内皮细胞), Angiogenesis (血管生成), 3D cell culture (三维细胞培养), Capillary sprout formation (毛细血管出芽形式), Spheroids (球形体), Vascular (血管)

Background

Blood vessels supply organs with oxygen and nutrients. In cases the local demands are not met anymore, cells secrete vascular endothelial growth factor (VEGF) to induce the formation of new blood vessels. The new vessel sprout is composed of a leading tip cell which is trailed by stalk cells (Potente and Makinen, 2017). Angiogenesis occurs under physiological conditions (e.g., growth of muscle and adipose tissue) as well as pathological conditions (e.g., wound healing, macular degeneration, and tumor growth). As such, there is a great need to decipher the basic mechanisms coordinating angiogenesis and to test compounds that interfere with pathological angiogenesis.

The spheroid-based sprouting assay, which was developed by Dr. Thomas Korff and Dr. Hellmut Augustin in the late 90s (Korff and Augustin, 1999), enables researchers to investigate the effects of drugs or genetic manipulations on sprouting angiogenesis in a fast and robust manner (Heiss et al., 2015). One great advantage of the spheroid-based sprouting assay is the analysis of sprout formation in a 3D environment. This promotes cell-cell signaling between endothelial cells. Upon stimulation with VEGF endothelial cells degrade the surrounding matrix and invade it. This mimics the situation in vivo. Thereby, this assay better reflects in vivo angiogenesis than other well known in vitro angiogenesis assays such as 2D tube formation on Matrigel (Nowak-Sliwinska et al., 2018).

Sprout number and length are read-outs for the angiogenic potential. In addition, this assay can be used to analyze competition for the tip cell position. Therefore, genetically modified endothelial cells, which are labeled with different fluorophores, are mixed before spheroid formation (Figure 1). Thereby, it can be analyzed, which genetic manipulation leads to a preference for the tip or the stalk position (Tetzlaff et al., 2018). With live-cell imaging it is possible to investigate the migration of endothelial cells and the dynamic competition of endothelial cells for the tip position.


Figure 1. Endothelial cells expressing either mCherry or GFP mixed before spheroid formation. Spheroid was embedded in a collagen matrix and sprouting was analyzed 24 h later by using a fluorescence microscope. Scale bar = 100 µm.

Materials and Reagents

  1. Pipette tips (10 µl, 200 µl, 1,000 µl) (Starlab, catalog numbers: S1111-3700 , S1111-0706 , S1111-6701 )
  2. Reservoir (Thermo Fisher Scientific, catalog number: 95128093 )
  3. Serological pipettes (5 ml, 10 ml, 25 ml) (Corning, catalog numbers: 4487 , 4488 , 4489 )
  4. 10 cm square Petri dish (Greiner Bio One International, catalog number: 688102 )
  5. 6-well plate (Corning, catalog number: 3516 )
  6. 24-well plate for suspension culture (Greiner Bio One International, catalog number: 662102 )
  7. 15 ml conical tube (Corning, catalog number: 352096 )
  8. 50 ml conical tube (Corning, catalog number: 352070 )
  9. Human umbilical vein endothelial cells: freshly isolated from three different donors (also commercially available)
  10. Rat tails
  11. Acetic acid (Carl Roth, catalog number: 3738.2 )
  12. Endopan 3 Basal Medium (Pan-Biotech, catalog number: P04-0010B )
  13. Endopan 3 Medium plus Supplements (Pan-Biotech, catalog number: P04-0010K )
  14. Ethanol (VWR, catalog number: 20821.330 )
  15. FBS (Merck, Biochrom, catalog number: S 0615 )
  16. FGF2 (R&D Systems, catalog number: 234-FSE-025 )
  17. 10x Medium 199 (Sigma-Aldrich, catalog number: M0650 )
  18. Methyl cellulose, 4,000 centipoises (Sigma-Aldrich, catalog number: M0512 )
  19. Sodium hydroxide (Sigma-Aldrich, catalog number: 30620 )
  20. Paraformaldehyde (Sigma-Aldrich, catalog number: 158127 )
  21. PBS (Thermo Scientific Fisher, catalog number: 14190169 )
  22. 0.05% trypsin-EDTA (Thermo Scientific Fisher, catalog number: 25300054 )
  23. VEGF-A165 (R&D Systems, catalog number: 293-VE-010 )
  24. Methocel stock solution (see Recipes)
  25. Collagen stock solution (see Recipes)

Equipment

  1. 500 ml bottle (Fisher Scientific, catalog number: FB800500 )
  2. Magnetic stirrer (Heidolph Instruments, catalog number: 505-20000-00 )
  3. 12-channel pipette (Volume 10-100 μl) (Eppendorf, catalog number: 3125000044 )
  4. Hemacytometer; Neubauer counting chamber (BRAND, catalog number: 717805 )
  5. Pipette-aid (BRAND, catalog number: 26304 )
  6. Autoclave (VWR, Tuttnauer, catalog number: 481-0585 )
  7. Balance (KERN & SOHN, catalog number: PBJ 4200-2M )
  8. Centrifuge with swinging-bucket rotor and adaptors for 15 ml and 50 ml conical tubes (Eppendorf, model: 5810 , catalog number: 5810000320) 
  9. Humidified cell culture incubator set to 37 °C and 5% CO2 (Thermo Fisher Scientific, HeracellTM 150CU , catalog number: 50116047 )
  10. Light microscope (Leica Microsystems, model: Leica DM IRB )
  11. Safety cabinet (Thermo Fisher Scientific, model: Safe 2020 , catalog number: 51026638)
  12. Water bath (GFL, catalog number: 1012 )

Software

  1. FIJI (Curtis Rueden, University of Wisconsin-Madison, Laboratory for Optical and Computational Instrumentation)

Procedure

  1. Preparation of hanging drops
    Culture human umbilical vein endothelial cells (HUVEC) in Endopan 3 medium containing supplements and FBS until cells are confluent. This protocol is adjusted to running the experiment under three conditions: basal, VEGF stimulation, FGF2 stimulation.
    1. Wash HUVEC twice with PBS.
    2. Detach cells from cell culture plate using trypsin-EDTA.
    3. Stop reaction using PBS containing 10% FBS, spin down cells at 200 x g for 5 min, discard supernatant and re-suspend cells in cell culture medium.
    4. Count cells using a hemacytometer.
      Note: Twenty thousand cells are needed per condition. Due to loss of cells during the whole procedure, calculate to ensure you have an extra 20,000 cells. In total, 80,000 cells are needed for the preparation of the hanging drops.
    5. Transfer 80,000 cells to a fresh 15 ml conical tube. Add cell culture medium (Endopan 3 medium plus supplements and FBS) to a total volume of 4 ml.
    6. Add 1 ml of methocel stock solution, which can improve spheroid formation.
      Note: Some researchers do not use methocel at this step, however in our hands, this improves spheroid formation, which was also reported elsewhere (Leung et al., 2015).
    7. Mix solution carefully and transfer it to a sterile multichannel pipette reservoir.
    8. Using a 12-channel pipette, pipet 25 μl drops of the solution onto a 10 cm square Petri dish. Take care to avoid air bubbles (Figure 2A) (Video 1). 

      Video 1. Preparation of hanging drops. Endothelial cell-methocel-solution is pipetted on a square Petri dish for hanging drop formation.

    9. To form spheroids, incubate drops upside-down in a humidified cell culture incubator set at 37 °C and 5% CO2 for 24 h (Figure 2B).

Note: Hereon prepare the methocel solution containing 20% FBS which is needed on the next day. Mix carefully the methocel stock solution with 20% FBS. Avoid air bubbles. In case air bubbles are formed, check whether they disperse on the next day. If not, use another batch.


Figure 2. Preparation of hanging drops for spheroid formation. A. Petri dish with hanging drops. B. Representative image of a spheroid in a hanging drop 24 h after upside-down incubation. Scale bar = 100 µm.


  1. Embedding of spheroids
    Note: Before continuing with the protocol, check whether spheroids formed overnight. In case multiple small cell clumps have been formed, discard them.
    1. Place collagen stock solution, 10x Medium 199, 0.2 N NaOH and methocel containing 20% FBS on ice.
    2. Using a 10 ml serological pipette gently wash off the hanging drops (containing the spheroids) with 10 ml PBS by pipetting up and down. Transfer solution into a 15 ml conical tube (Video 2).

      Video 2. Transfer of spheroids. Hanging drops containing spheroids are washed off and solution is transferred to a 15 ml conical tube.

    3. Spin down spheroids at 200 x g for 5 min. Then aspirate supernatant and add 2 ml of methocel containing 20% FBS (Video 3).

      Video 3. Mixing of spheroids and collagen. Spheroids are suspended in methocel containing 20% FBS and mixed with collagen. Solution is added to wells of a 24-well plate.

    4. Prepare collagen medium: Mix 4 ml collagen stock solution with 0.5 ml 10x Medium 199 on ice. Adjust pH value by adding dropwise sterile ice-cold 0.2 N NaOH. Subsequently invert the tube carefully to ensure thorough mixing. Add NaOH until the pH indicator of the collagen medium changes the color from yellow to orange (approximately 250-500 µl).
      Important: Prepare the collagen medium on ice to prevent collagen polymerization.
    5. Add 2 ml of the collagen from Step B5 to the spheroids which were re-suspended in methocel containing 20% FBS.
      Note: Mix the solution carefully. Avoid air bubbles.
    6. Add 1 ml of the spheroid-collagen solution per well of a 24-well plate.
    7. Incubate the plate in a humidified cell culture incubator set at 37 °C and 5% CO2 for 30 min to allow polymerization of the collagen matrix (Figure 3).


      Figure 3. Collagen matrices in which spheroids are embedded were added to wells of a 24-well suspension culture plate

    8. Stimulate spheroids with 100 µl of basal medium, 25 ng/ml VEGF-A (in basal medium) and 25 ng/ml FGF2 (in basal medium) by adding it dropwise on the collagen matrix.
      Note: The volume of the added medium can be changed if necessary.
    9. Incubate collagen matrix for 24 h in a humidified cell culture incubator.
    10. Stop the sprouting assay by adding 1 ml of 10% paraformaldehyde and store plates at 4 °C (for up to 4 weeks).

Data analysis

  1. To analyze the pro- or anti-angiogenic effect of a gene mutation or a chemical compound, the number of sprouts and the length of the sprouts have to be determined.
    1. Aspirate the paraformaldehyde from the collagen matrix.
    2. Place the 24-well plate under an inverted microscope.
    3. Acquire images of 10 randomly selected spheroids.
    4. Count the number of sprouts per spheroid and measure the sprout length of all sprouts per spheroid using the imaging software FIJI (Figure 4).


      Figure 4. Sprouting analysis of a HUVEC spheroid. A. Image of a HUVEC spheroid embedded in a collagen matrix after 24 h of sprouting. B. Image analysis: Measuring the sprout length of all sprouts of the spheroid by using FIJI software. A line was manually drawn, and the length of the line was determined. Scale bar = 100 µm.

  2. The following parameters are used as readout for the angiogenic activity:
    1. Number of sprouts per spheroid.
    2. Average sprout length of the sprouts per spheroid.
    3. Cumulative sprout length of all sprouts per spheroid.
  3. The parameters of ten technical replicates are averaged and at least 3 biological replicates should be analyzed. Spheroids stimulated with VEGF-A or FGF2 should show a clearly increased sprouting ability compared to basal spheroids (Figure 5).


    Figure 5. HUVEC spheroid embedded in a collagen matrix after 24 h of sprouting. The assay was performed under basal conditions (A), with 25 ng/ml VEGF-A stimulation (B) and with 25 ng/ml FGF2 stimulation (C). Scale bar = 100 µm.

Notes

  1. Methocel stock solution: Proper centrifugation of methyl cellulose solution is important to remove debris. Otherwise, cells can stick to the plate resulting in multiple small spheroids instead of a single one. Stock solution can be stored at 4 °C for up to 6 months.
  2. Collagen stock solution: Prepare the collagen stock solution four weeks in advance, since freshly isolated collagen can cause higher inter-experimental variation. Stock solution can be stored at 4 °C for at least 6 months.
  3. Embedding of spheroids: After neutralization, collagen medium should be clear.

Recipes

  1. Methocel stock solution
    1. Weigh 6 g of methyl cellulose, transfer it to a 500 ml bottle and add a clean magnetic stirrer. Autoclave it at 121 °C for 20 min
    2. Heat up 250 ml Endopan 3 basal medium to 60 °C and add it to the autoclaved methyl cellulose
    3. Stir the solution for 20 min at room temperature
    4. Add 250 ml Endopan 3 basal medium (room temperature) and stir the solution overnight at 4 °C
    5. Aliquot the stock solution into 50 ml conical tubes
    6. Centrifuge methocel solution for 2 h at room temperature and 5,000 x g
    7. Use supernatant of the methocel solution for spheroid culture
  2. Collagen stock solution
    Note: A detailed video protocol has been published by (Bruneau et al., 2010).
    1. Place two rat tails in 500 ml of 70% ethanol and incubate for 20 min at room temperature
    2. Cut the skin by using a scalpel and peel off the skin from the tail root to the tail tip
    3. Wash tails in 70% ethanol
    4. Break every second vertebral and isolate the tendons 
      Be careful: Do not isolate the attached connective tissue.
    5. Collect tendons and incubate them in 500 ml of 70% ethanol for 20 min
    6. Dry sterilized tendons in a safety cabinet for 30-60 min
    7. Transfer tendons into 250 ml of 0.1 % sterile acetic acid (v/v in H2O) and incubate them for 48 h at 4 °C
    8. Aliquot the collagen solution and centrifuge the aliquots at 4 °C and 17,000 x g for 90 min
    9. Collect the clear supernatant, aliquot it into 50 ml conical tubes and store it at 4 °C
    10. For equilibration, add 0.5 ml of ten-fold Medium 199 to 4 ml of collagen solution, mix and incubate it for at least 15 min on ice. In case the mixed solution solidifies, dilute the collagen stock solution with 0.1% sterile acetic acid until the Medium 199-collagen-solution stays liquid.

Acknowledgments

The assay was originally developed by Dr. T. Korff and Dr. H.G. Augustin (Korff and Augustin, 1999; Pfisterer and Korff, 2016).
This work was supported by the Deutsche Forschungsgemeinschaft (SFB-TR23, project A7) and the Helmholtz society to A.F.

Competing interests

The authors declare that they have no conflict of interest.

References

  1. Bruneau, A., Champagne, N., Cousineau-Pelletier, P., Parent, G. and Langelier, E. (2010). Preparation of rat tail tendons for biomechanical and mechanobiological studies. J Vis Exp(41).
  2. Heiss, M., Hellstrom, M., Kalen, M., May, T., Weber, H., Hecker, M., Augustin, H.G. and Korff, T. (2015). Endothelial cell spheroids as a versatile tool to study angiogenesis in vitro. FASEB J 29(7): 3076-3084.
  3. Korff, T. and Augustin, H. G. (1999). Tensional forces in fibrillar extracellular matrices control directional capillary sprouting. J Cell Sci 112 (Pt 19): 3249-3258.
  4. Leung, B. M., Lesher-Perez, S. C., Matsuoka, T., Moraes, C. and Takayama, S. (2015). Media additives to promote spheroid circularity and compactness in hanging drop platform. Biomater Sci 3(2): 336-344.
  5. Nowak-Sliwinska, P., Alitalo, K., Allen, E., Anisimov, A., Aplin, A.C., Auerbach, R., Augustin, H.G., Bates, D.O., van Beijnum, J. R., Bender, R. H. F., Bergers, G., Bikfalvi, A., Bischoff, J., Böck, B. C. and Brooks, P. C. (2018). Consensus guidelines for the use and interpretation of angiogenesis assays. Angiogenesis 21: 425-532.
  6. Pfisterer, L. and Korff, T. (2016). Spheroid-Based in vitro angiogenesis model. Methods Mol Biol 1430: 167-177.
  7. Potente, M. and Makinen, T. (2017). Vascular heterogeneity and specialization in development and disease. Nat Rev Mol Cell Biol 18(8): 477-494.
  8. Tetzlaff, F., Adam, M. G., Feldner, A., Moll, I., Menuchin, A., Rodriguez-Vita, J., Sprinzak, D. and Fischer, A. (2018). MPDZ promotes DLL4-induced Notch signaling during angiogenesis. Elife 7: e32860.

简介

血管生成,从先前存在的血管形成新血管在器官发育,再生和肿瘤进展中起重要作用。 基于球体的发芽测定法是一种成熟且稳健的方法,用于研究遗传改变或药理学化合物对原代培养的内皮细胞的毛细血管样管形成的影响。 该测定的主要优点是可以在3D环境中研究血管生成。 将内皮细胞培养为悬滴以形成球状体。 将这些球状体嵌入胶原基质中,24小时后分析管形成。 通过分析发芽数和发芽长度,可以研究遗传操作或药物治疗对血管生成的影响。

【背景】血管为器官提供氧气和营养。在不再满足局部需求的情况下,细胞分泌血管内皮生长因子(VEGF)以诱导新血管的形成。新的容器芽由一个由茎细胞牵引的前端细胞组成(Potente和Makinen,2017)。血管生成在生理条件下(例如,肌肉和脂肪组织的生长)以及病理条件(例如,伤口愈合,黄斑变性和肿瘤生长)发生。因此,非常需要破译协调血管生成的基本机制并测试干扰病理性血管生成的化合物。

基于球体的发芽试验由Thomas Korff博士和Hellmut Augustin博士在90年代后期开发(Korff和Augustin,1999),使研究人员能够快速研究药物或基因操作对发芽血管生成的影响。稳健的方式(Heiss et al。,2015)。基于球体的发芽测定的一个重要优点是分析3D环境中的芽形成。这促进内皮细胞之间的细胞 - 细胞信号传导。在用VEGF内皮细胞刺激后,降解周围的基质并侵入其中。这模仿了体内的情况。因此,该测定比其他众所周知的体外血管生成测定更好地反映了体内血管生成,例如Matrigel上的2D管形成(Nowak-Sliwinska 等。,2018)。

芽的数量和长度是血管生成潜力的读数。此外,该测定可用于分析针尖细胞位置的竞争。因此,在球状体形成之前混合用不同荧光团标记的遗传修饰的内皮细胞(图1)。因此,可以分析哪种遗传操作导致对尖端或茎位置的偏好(Tetzlaff 等人,2018)。通过活细胞成像,可以研究内皮细胞的迁移和内皮细胞对尖端位置的动态竞争。


图1.在球状体形成前混合表达mCherry或GFP的内皮细胞。将球状体嵌入胶原基质中,24小时后用荧光显微镜分析发芽。比例尺=100μm。

关键字:内皮细胞, 血管生成, 三维细胞培养, 毛细血管出芽形式, 球形体, 血管

材料和试剂

  1. 移液器吸头(10μl,200μl,1000μl)(Starlab,目录号:S1111-3700,S1111-0706,S1111-6701)
  2. 水库(Thermo Fisher Scientific,目录号:95128093)
  3. 血清移液管(5 ml,10 ml,25 ml)(Corning,目录号:4487,4488,4489)
  4. 10平方厘米培养皿(Greiner Bio One International,目录号:688102)
  5. 6孔板(康宁,目录号:3516)
  6. 用于悬浮培养的24孔板(Greiner Bio One International,目录号:662102)
  7. 15毫升锥形管(康宁,目录号:352096)
  8. 50毫升锥形管(康宁,目录号:352070)
  9. 人脐静脉内皮细胞:从三个不同的供体新鲜分离(也可商购)
  10. 鼠尾巴
  11. 乙酸(Carl Roth,目录号:3738.2)
  12. Endopan 3基础培养基(Pan-Biotech,目录号:P04-0010B)
  13. Endopan 3 Medium plus Supplements(Pan-Biotech,目录号:P04-0010K)
  14. 乙醇(VWR,目录号:20821.330)
  15. FBS(Merck,Biochrom,目录号:S 0615)
  16. FGF2(R& D Systems,目录号:234-FSE-025)
  17. 10x Medium 199(Sigma-Aldrich,目录号:M0650)
  18. 甲基纤维素,4,000厘泊(Sigma-Aldrich,目录号:M0512)
  19. 氢氧化钠(Sigma-Aldrich,目录号:30620)
  20. 多聚甲醛(Sigma-Aldrich,目录号:158127)
  21. PBS(Thermo Scientific Fisher,目录号:14190169)
  22. 0.05%胰蛋白酶-EDTA(Thermo Scientific Fisher,目录号:25300054)
  23. VEGF-A 165 (R& D Systems,目录号:293-VE-010)
  24. Methocel原液(见食谱)
  25. 胶原蛋白原液(见食谱)

设备

  1. 500毫升瓶(Fisher Scientific,目录号:FB800500)
  2. 磁力搅拌器(Heidolph Instruments,目录号:505-20000-00)
  3. 12通道移液器(容量10-100μl)(Eppendorf,目录号:3125000044)
  4. 血球; Neubauer计数室(BRAND,目录号:717805)
  5. 移液器(BRAND,目录号:26304)
  6. 高压灭菌器(VWR,Tuttnauer,目录号:481-0585)
  7. 平衡(KERN&SOHN,目录号:PBJ 4200-2M)
  8. 带有摆动转子和适配器的离心机,用于15 ml和50 ml锥形管(Eppendorf,型号:5810,目录号:5810000320) 
  9. 加湿的细胞培养箱设置为37°C和5%CO 2 (Thermo Fisher Scientific,Heracell TM 150CU,目录号:50116047)
  10. 光学显微镜(Leica Microsystems,型号:Leica DM IRB)
  11. 安全柜(Thermo Fisher Scientific,型号:Safe 2020,目录号:51026638)
  12. 水浴(GFL,目录号:1012)

软件

  1. FIJI(威斯康星大学麦迪逊分校柯蒂斯·鲁登,光学和计算仪器实验室)

程序

  1. 悬滴的制备
    在含有补充物和FBS的Endopan 3培养基中培养人脐静脉内皮细胞(HUVEC)直至细胞汇合。调整该方案以在三种条件下进行实验:基础,VEGF刺激,FGF2刺激。
    1. 用PBS洗涤HUVEC两次。
    2. 使用胰蛋白酶-EDTA从细胞培养板中分离细胞。
    3. 使用含有10%FBS的PBS停止反应,将细胞在200μL离心5分钟旋转5分钟,弃去上清液并将细胞重悬于细胞培养基中。
    4. 使用血细胞计数器计数细胞。
      注意:每种情况都需要2万个细胞。由于整个过程中细胞丢失,计算以确保您有额外的20,000个细胞。总共需要80,000个细胞来制备悬滴。
    5. 将80,000个细胞转移到新鲜的15ml锥形管中。加入细胞培养基(Endopan 3培养基加补充剂和FBS)至总体积为4ml。
    6. 加入1毫升甲基纤维素原液,可以改善球体的形成。
      注意:一些研究人员在此步骤中不使用methocel,但是在我们的手中,这会改善球体的形成,这在其他地方也有报道(Leung等,2015)。
    7. 仔细混合溶液并将其转移到无菌多通道移液器储液器中。
    8. 使用12通道移液器,将25μl溶液滴移到10cm见方的培养皿上。注意避免气泡(图2A)(视频1)。 

      视频1。
    9. 为了形成球状体,将液滴倒置在设定为37°C和5%CO 2 的潮湿细胞培养箱中24小时(图2B)。

注意:在此准备第二天需要的含有20%FBS的甲基纤维素溶液。将Methocel储备溶液与20%FBS小心混合。避免气泡。如果形成气泡,请检查它们是否在第二天消散。如果没有,请使用另一批。


图2.用于球状体形成的悬滴的制备。 A.带有悬滴的培养皿。 B.倒置孵育后24小时悬浮液中球状体的代表性图像。比例尺=100μm。


  1. 嵌入球状体
    注意:在继续使用该方案之前,请检查球状体是否在一夜之间形成。如果形成了多个小细胞团块,则丢弃它们。
    1. 将胶原储备液,10x培养基199,0.2N NaOH和含有20%FBS的甲基纤维素置于冰上。
    2. 使用10ml血清移液管,通过上下移液,用10ml PBS轻轻洗掉悬滴(含有球状体)。将溶液转移到15ml锥形管中(视频2)。

      视频2.转移球状体。将含有球状体的悬滴液洗掉,将溶液转移到15 ml锥形管中。

    3. 在200 x g 下旋转球状体5分钟。然后吸出上清液并加入2ml含20%FBS的甲基纤维素(视频3)。

      视频3.球状体和胶原蛋白的混合。将球状体悬浮在含有20%FBS的甲基纤维素中并与胶原蛋白混合。将溶液添加到24孔板的孔中。

    4. 准备胶原蛋白培养基:将4ml胶原蛋白原液与0.5ml 10x培养基199在冰上混合。通过滴加无菌冰冷的0.2N NaOH调节pH值。随后小心地倒转管子以确保充分混合。加入NaOH直至胶原培养基的pH指示剂颜色从黄色变为橙色(约250-500μl)。
      重要提示:在冰上准备胶原蛋白培养基以防止胶原蛋白聚合。
    5. 将2ml来自步骤B5的胶原蛋白加入到球状体中,将其重新悬浮在含有20%FBS的甲基纤维素中。
      注意:仔细混合溶液。避免气泡。
    6. 在24孔板的每个孔中加入1ml球状体 - 胶原蛋白溶液。
    7. 将培养板置于37℃和5%CO 2 的湿润细胞培养箱中孵育30分钟,使胶原基质聚合(图3)。


      图3.将嵌入球状体的胶原基质添加到24孔悬浮培养板的孔中

    8. 通过在胶原基质上滴加100μl基础培养基,25 ng / ml VEGF-A(基础培养基)和25 ng / ml FGF2(基础培养基)刺激球体。
      注意:如有必要,可以更改添加媒体的音量。
    9. 在潮湿的细胞培养箱中孵育胶原基质24小时。
    10. 通过加入1ml 10%多聚甲醛停止发芽测定,并将板在4℃下储存(最多4周)。

数据分析

  1. 为了分析基因突变或化合物的促血管生成或抗血管生成作用,必须确定芽的数量和芽的长度。
    1. 从胶原基质中吸出多聚甲醛。
    2. 将24孔板置于倒置显微镜下。
    3. 获取10个随机选择的球体的图像。
    4. 计算每个球体的芽数,并使用成像软件FIJI测量每个球体的所有芽的芽长(图4)。


      图4. HUVEC球体的发芽分析。 A.发芽24小时后嵌入胶原基质中的HUVEC球状体的图像。 B.图像分析:使用FIJI软件测量球体的所有芽的芽长。手动绘制线,并确定线的长度。比例尺=100μm。

  2. 以下参数用作血管生成活性的读数:
    1. 每个球状体的芽数。
    2. 每个球状体的芽的平均芽长。
    3. 每个球状体的所有芽的累积芽长度。
  3. 对10个技术重复的参数进行平均,并且应分析至少3个生物重复。与基底球体相比,用VEGF-A或FGF2刺激的球体应显示出明显增加的发芽能力(图5)。


    图5.发芽24小时后嵌入胶原基质中的HUVEC球状体。该测定在基础条件下进行(A),25 ng / ml VEGF-A刺激(B)和25 ng / ml FGF2刺激(C)。比例尺:100μm。

笔记

  1. Methocel原液:甲基纤维素溶液的正确离心对去除碎屑很重要。否则,细胞可能粘在板上,导致多个小球体而不是单个球体。储备溶液可以在4°C下储存长达6个月。
  2. 胶原蛋白原液:提前四周准备胶原蛋白原液,因为新鲜分离的胶原蛋白会导致更高的实验间差异。储备溶液可以在4°C下储存至少6个月。
  3. 球状体嵌入:中和后,胶原蛋白介质应清晰。

食谱

  1. Methocel原液解决方案
    1. 称取6 g甲基纤维素,将其转移至500 ml瓶中,加入干净的磁力搅拌器。在121℃下高压灭菌20分钟
    2. 将250ml Endopan 3基础培养基加热至60℃并加入高压灭菌的甲基纤维素中
    3. 在室温下搅拌溶液20分钟
    4. 加入250ml Endopan 3基础培养基(室温)并在4℃下搅拌溶液过夜
    5. 将储备溶液分装到50ml锥形管中
    6. 在室温下离心甲基纤维素溶液2小时,并且5,000 x g
    7. 使用甲基纤维素溶液的上清液进行球体培养
  2. 胶原蛋白原液解决方案
    注:详细的视频协议已由(Bruneau et al。,2010)发布。
    1. 将两只大鼠尾巴置于500ml 70%乙醇中,在室温下孵育20分钟
    2. 用手术刀切开皮肤,从尾根到尾尖剥去皮肤
    3. 用70%乙醇洗净尾巴
    4. 打破每一个椎骨并隔离肌腱。注意:不要隔离附着的结缔组织
    5. 收集肌腱并在500毫升70%乙醇中孵育20分钟
    6. 在安全柜中干燥灭菌的肌腱30-60分钟
    7. 将肌腱转移到250毫升0.1%无菌乙酸(v / v in H 2 O)中,并在4°C下孵育48小时
    8. 将胶原溶液分装并在4℃和17,000 x g 下离心等分试样90分钟
    9. 收集澄清的上清液,将其等分到50ml锥形管中并在4℃下储存
    10. 为了平衡,将0.5ml 10倍培养基199加入到4ml胶原溶液中,混合并在冰上孵育至少15分钟。在混合溶液固化的情况下,用0.1%无菌乙酸稀释胶原原液,直到培养基199-胶原溶液保持液体。

致谢

该试验最初由T. Korff博士和H.G. Augustin博士开发(Korff和Augustin,1999; Pfisterer和Korff,2016)。
这项工作得到了Deutsche Forschungsgemeinschaft(SFB-TR23,项目A7)和亥姆霍兹社团对A.F.的支持。

利益争夺

作者声明他们没有利益冲突。

参考

  1. Bruneau,A.,Champagne,N.,Cousineau-Pelletier,P.,Parent,G。和Langelier,E。(2010)。 制备用于生物力学和机械生物学研究的鼠尾肌腱。 J Vis Exp (41)。
  2. Heiss,M.,Hellstrom,M.,Kalen,M.,May,T.,Weber,H.,Hecker,M.,Augustin,H.G。和Korff,T。(2015)。 内皮细胞球体作为体外研究血管生成的多功能工具。 FASEB J 29(7):3076-3084。
  3. Korff,T。和Augustin,H.G。(1999)。 纤维状细胞外基质中的张力控制定向毛细血管发芽。 J Cell Sci 112(Pt 19):3249-3258。
  4. Leung,B.M.,Lesher-Perez,S.C.,Matsuoka,T.,Moraes,C。和Takayama,S。(2015)。 媒介添加剂,以促进悬滴平台的球体圆度和紧凑性。 Biomater Sci 3(2):336-344。
  5. Nowak-Sliwinska,P.,Alitalo,K.,Allen,E.,Anisimov,A.,Aplin,AC,Auerbach,R.,Augustin,HG,Bates,DO,van Beijnum,JR,Bender,RHF,Bergers, G.,Bikfalvi,A.,Bischoff,J.,Böck,BC和Brooks,PC(2018)。 血管生成检测的使用和解释的共识指南。 血管生成 21:425-532。
  6. Pfisterer,L。和Korff,T。(2016)。 基于球体的体外血管生成模型。 方法Mol Biol 1430:167-177。
  7. Potente,M。和Makinen,T。(2017)。 血管异质性和发育与疾病专业化。 Nat Rev Mol Cell Biol 18(8):477-494。
  8. Tetzlaff,F.,Adam,M.G.,Feldner,A.,Moll,I.,Menuchin,A.,Rodriguez-Vita,J.,Sprinzak,D。和Fischer,A。(2018)。 MPDZ在血管生成过程中促进DLL4诱导的Notch信号传导。 Elife 7:e32860。
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Copyright Tetzlaff and Fischer. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Tetzlaff, F. and Fischer, A. (2018). Human Endothelial Cell Spheroid-based Sprouting Angiogenesis Assay in Collagen. Bio-protocol 8(17): e2995. DOI: 10.21769/BioProtoc.2995.
  2. Tetzlaff, F., Adam, M. G., Feldner, A., Moll, I., Menuchin, A., Rodriguez-Vita, J., Sprinzak, D. and Fischer, A. (2018). MPDZ promotes DLL4-induced Notch signaling during angiogenesis. Elife 7: e32860.
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