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In vitro Human Umbilical Vein Endothelial Cells (HUVEC) Tube-formation Assay

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Feb 2012


Angiogenesis is involved not only in pathological conditions including cancer biology and non-neoplastic diseases, but also many biological processes including reproduction, development and repair. During angiogenesis, endothelial cells (ECs) undergo activation after binding of angiogenic factors to their receptors, release of proteases to dissolve the basement membrane, migration towards an angiogenic signal, proliferation, and an increase in cell number for new blood vessel formation. Finally, reorganization of ECs forms the three-dimensional vasculature. HUVEC tube-formation assay is one of the simple, but well-established in vitro angiogenesis assays based on the ability of ECs to form three-dimensional capillary-like tubular structures, when cultured on a gel of growth factor-reduced basement membrane extracts. During the assay, ECs differentiate, directionally migrate to align, branch, and form the tubular polygonal networks of blood vessels.

Keywords: HUVEC (人脐静脉内皮细胞), Angiogenesis assay (血管生成实验), Tube formation assay (管形成实验)

Materials and Reagents

  1. Target cell lines can be those with and without drug treatment or expressing the gene of interest.
    Note: Our example includes four cell lines that are engineered to have inducible expression of tumor suppressor gene and the corresponding vector alone controls.
  2. Growth factor-reduced BD Matrigel (BD Biosciences, catalog number: 354230 )
  3. Dulbecco’s Modified Eagle Medium (DMEM) (High Glucose L-Glutamine, 500 ml) (Life Technologies, Invitrogen™, catalog number: 11965-092 )
  4. Fetal bovine serum (Life Technologies, Invitrogen™, catalog number: 26140-079 )
  5. Primary Human Umbilical Vein Endothelial Cell (HUVEC) (Life Technologies, Invitrogen™, catalog number: C-003-5C )
  6. Medium 200PRF (Life Technologies, Invitrogen™, catalog number: M-200PRF-500 )
  7. Low serum growth supplement (LSGS) (Life Technologies, Invitrogen™, catalog number: S-003-10 )
  8. Conditioned medium (CM)


  1. Tissue culture setup
  2. Inverted microscope with digital camera (Nikon TMS)
  3. Scion Image software downloaded from the NIH website
  4. 96-well plates
  5. Centrifuge
  6. Cell counter
  7. T75 tissue culture flask


  1. Preparation of conditioned medium from target cell line(s)
    1. To prepare conditioned medium (CM), seed target cells and grow to 30-40% confluence (depending on the growth rate of the cell lines), replace growth medium with serum-free DMEM (e.g. 10 ml for a T75 tissue culture flask; it does not matter for the presence or absence of antibiotics) for 24 h, then harvest the CM, when cells reach 60-80% confluence in a T75 tissue culture flask.
    2. Make 0.5 ml aliquots of the CM and store at -80 °C if it is not immediately used after collection of CM.

  2. Preparation of Human Umbilical Vein Endothelial Cells (HUVEC) cells
    1. Prepare the HUVEC Medium 200PRF supplement with LSGS according to the product insert.
    2. Seed HUVEC cells in T25 or T75 flask, as needed, so that it reaches 70-80% confluence by the next day.
    3. Serum starve the HUVEC cells for 3-6 h just prior to performing the tube formation assay in Medium 200PRF without antibiotics (e.g. 5 ml for a T25 tissue culture flask).
      Note: At the end of serum starvation, go to procedure C step 1.
    4. After coating the 96-well plate with growth factor-reduced Matrigel, trypsinize to dislodge the cells from the surface of the flask, neutralize with DMEM with serum, pellet down the cells at room temperature by centrifugation at 1,200 rpm ( 276 x g) for 3 min, resuspend in 2-3 ml serum-free DMEM, determine the concentration of HUVEC by counting the cells, and resuspend HUVEC cells at 4 x 105 per ml serum-free DMEM by pipetting up and down several times to ensure a homogeneous single-cell suspension.
      Note: For the growth factor-reduced Matrigel, thaw/frozen cycle should be reduced as much as possible.
    5. Mix thoroughly while pipetting 500 μl of the HUVEC cell suspension into a 1.5 ml tube. Spin down the cells at 4,000 rpm (1,100 rcf) for 3 min using a bench-top centrifuge. Carefully aspirate the supernatant without disturbing the cell pellet. Remove as much of the supernatant as possible. Prepare the appropriate number of HUVEC cells in 1.5 ml tubes, according to the number of target cell lines to be used.
      Note: Per target cell line CM, one tube of HUVEC will be suspended, and per suspended HUVEC, 3 triplicate wells will be dispensed. So, in total, 3 wells will be used per target cell line.
    6. Thaw a 0.5 ml aliquot of the CM of target cell line(s) collected from procedure A step 2 and supplement it with fetal bovine serum to a final concentration of 1% for resuspending the HUVEC cell pellet(s) in procedure B step 5.
      Note: Each target cell line should be done in triplicate (each well requires 100 μl of the HUVEC cell suspension).

  3. Coating of 96-well plate with growth factor-reduced Matrigel
    1. Thaw an appropriate volume of growth factor-reduced Matrigel one day before use at 4 °C.
    2. Pre-chill the 96-well plate and the pipet tips at -20 °C for 2-3 h.
    3. Near the end of serum starvation of HUVEC cells and before counting HUVEC cells, aliquot 50 μl of growth factor-reduced Matrigel per well of the pre-chilled 96-well plate on ice. Swirl the plate until the gel is evenly distributed over the whole well. It is very important to avoid bubble formation. Allow it to polymerize at room temperature for 1 h on a level surface.

  4. Dispensing HUVEC cells in onto coated 96-well plate
    1. Dispense 100 μl of the HUVEC cell suspension obtained in procedure B step 6 with thorough mixing into the labeled wells of a 96-well plate. Incubate the plate at 37 °C, 5% CO2 for 4-6 h.
    2. Visualize the cells using a light microscope. Take pictures of the images of the capillary network and count the tube lengths using the Scion Image software.

      Figure 1. Representative image of tubes forms on the growth factor-reduced Matrigel by HUVEC after incubation with CM for 6 h (100x, size bar = 100 μm)


The protocol was established based on modifications from previous publications: Chan et al. (2011) and Kong et al. (2007).


  1. Chan, K. C., Ko, J. M., Lung, H. L., Sedlacek, R., Zhang, Z. F., Luo, D. Z., Feng, Z. B., Chen, S., Chen, H., Chan, K. W., Tsao, S. W., Chua, D. T., Zabarovsky, E. R., Stanbridge, E. J. and Lung, M. L. (2011). Catalytic activity of Matrix metalloproteinase-19 is essential for tumor suppressor and anti-angiogenic activities in nasopharyngeal carcinoma. Int J Cancer 129(8): 1826-1837.
  2. Kong, D., Li, Y., Wang, Z., Banerjee, S. and Sarkar, F. H. (2007). Inhibition of angiogenesis and invasion by 3,3'-diindolylmethane is mediated by the nuclear factor-kappaB downstream target genes MMP-9 and uPA that regulated bioavailability of vascular endothelial growth factor in prostate cancer. Cancer Res 67(7): 3310-3319.


血管生成不仅涉及包括癌症生物学和非肿瘤疾病的病理状况,而且涉及许多生物过程,包括繁殖,发育和修复。 在血管生成期间,内皮细胞(EC)在血管生成因子与其受体结合之后经历活化,释放蛋白酶以溶解基底膜,向血管生成信号迁移,增殖和用于新血管形成的细胞数目的增加。 最后,EC的重组形成三维脉管系统。 当在生长凝胶上培养时,HUVEC管形成测定是基于EC形成三维毛细管样管状结构的能力的简单的,但是良好建立的体外血管生成测定法之一 因子减少基底膜提取物。 在测定期间,ECs分化,定向迁移以排列,分支并形成管状多边形网络的血管。

关键字:人脐静脉内皮细胞, 血管生成实验, 管形成实验


  1. 靶细胞系可以是具有和不具有药物处理或表达目标基因的细胞系。
  2. 生长因子减少的BD Matrigel(BD Biosciences,目录号:354230)
  3. Dulbecco改良的Eagle培养基(DMEM)(高葡萄糖L-谷氨酰胺,500ml)(Life Technologies,Invitrogen TM,目录号:11965-092)
  4. 胎牛血清(Life Technologies,Invitrogen TM,目录号:26140-079)
  5. 原发性人脐静脉内皮细胞(HUVEC)(Life Technologies,Invitrogen TM,目录号:C-003-5C)
  6. 培养基200PRF(Life Technologies,Invitrogen TM,目录号:M-200PRF-500)
  7. 低血清生长补充剂(LSGS)(Life Technologies,Invitrogen TM,目录号:S-003-10)
  8. 条件培养基(CM)


  1. 组织培养设置
  2. 带数码相机(尼康TMS)的倒置显微镜
  3. 从NIH网站下载的Scion Image软件
  4. 96孔板
  5. 离心机
  6. 单元格计数器
  7. T75组织培养瓶


  1. 从靶细胞系制备条件培养基
    1. 为了制备条件培养基(CM),种子靶细胞并生长至30-40%汇合(取决于细胞系的生长速率),用无血清DMEM代替生长培养基(例如 10ml 对于T75组织培养瓶;当存在或不存在抗生素时无关紧要)24小时,然后当细胞在T75组织培养瓶中达到60-80%汇合时收获CM。
    2. 使0.5ml等分的CM,并存储在-80°C,如果它不是立即使用后收集CM。

  2. 人脐静脉内皮细胞(HUVEC)细胞的制备
    1. 根据产品说明书,用LSGS制备HUVEC Medium 200PRF补充剂。
    2. 种子HUVEC细胞在T25或T75烧瓶中,根据需要,使其达到70-80%汇合第二天。
    3. 在不含抗生素的培养基200PRF(对于T25组织培养瓶,例如5ml)中,在进行管形成测定之前,血清使HUVEC细胞饥饿3-6小时。
    4. 在用生长因子减少的Matrigel包被96孔板后,用胰蛋白酶处理从烧瓶表面除去细胞,用血清用DMEM中和,在室温下通过在1,200rpm离心(276×g )3分钟,重悬于2-3ml无血清DMEM中,通过计数细胞测定HUVEC的浓度,并通过上下吹吸数次以4×10 5/ml无血清DMEM重悬HUVEC细胞次以确保均匀的单细胞悬浮 注意:对于生长因子减少的Matrigel,应尽可能减少解冻/冻结循环。
    5. 充分混匀,同时移取500微升的HUVEC细胞悬浮液到1.5毫升管。使用台式离心机以4,000rpm(1,100rcf)旋转细胞3分钟。小心吸出上清液,不要打扰细胞沉淀。尽可能多地去除上清液。根据要使用的目标细胞系的数量,在1.5毫升管中准备适当数量的HUVEC细胞。
    6. 解冻从程序A步骤2收集的靶细胞系的CM的0.5ml等分试样,并用胎牛血清补充至终浓度为1%,以在程序B步骤5中重悬HUVEC细胞沉淀。

  3. 用生长因子减少的Matrigel涂覆96孔板
    1. 在使用前一天在4℃下融化适当体积的生长因子减少的Matrigel
    2. 预冷96孔板和移液器吸头在-20°C 2-3小时
    3. >
    4. 接近HUVEC细胞的血清饥饿结束并且在计数HUVEC细胞之前,在冰上将预冷的96孔板的每孔等分50μl生长因子减少的Matrigel。旋转板直到凝胶均匀分布在整个孔上。避免气泡形成是非常重要的。使其在室温下在水平表面上聚合1小时

  4. 将HUVEC细胞分配到涂覆的96孔板中
    1. 将100μl在程序B步骤6中获得的HUVEC细胞悬浮液充分混合到96孔板的标记孔中。将板在37℃,5%CO 2孵育4-6小时
    2. 使用光学显微镜可视化细胞。拍摄毛细管网络的图像,并使用Scion Image软件计算管长度。





  1. Chan,KC,Ko,JM,Lung,HL,Sedlacek,R.,Zhang,ZF,Luo,DZ,Feng,ZB,Chen,S.,Chen,H.,Chan,KW,Tsao,SW,Chua,DT ,Zabarovsky,ER,Stanbridge,EJ和Lung,ML(2011)。 基质金属蛋白酶-19的催化活性对于鼻咽癌中的肿瘤抑制基因和抗血管生成活性是必需的。 Int J Cancer 129(8):1826-1837。
  2. Kong,D.,Li,Y.,Wang,Z.,Banerjee,S。和Sarkar,F.H。(2007)。 抑制血管生成和3,3'-二吲哚基甲烷的侵袭是由核因子kappaB下游介导的 目标基因MMP-9和uPA,其调节前列腺癌中血管内皮生长因子的生物利用度。 Cancer Res 67(7):3310-3319。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ko, J. M. and Lung, M. L. (2012). In vitro Human Umbilical Vein Endothelial Cells (HUVEC) Tube-formation Assay. Bio-protocol 2(18): e260. DOI: 10.21769/BioProtoc.260.



thank you for your informative protocol

i need to culture and assay angiogenic response of HUVEC for my ms theses.
i have read that for best results in vitro angiogenesis assays must be done at passage 2-6.my question is at 2-6 passages means from a cryopreserved cell line or from a primary isolated HUVEC cells?
9/30/2012 3:57:40 AM Reply