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Rat Aortic Ring Model to Assay Angiogenesis ex vivo
利用大鼠主动脉环的体外活体模型测定血管生成   

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Abstract

Angiogenesis is a multifactorial event which requires the migration, proliferation, differentiation and structure rearrangement of endothelial cells. This angiogenic process has been commonly studied using in vitro assays such as Boyden chamber assay, wound healing assay and tube formation assay. These assays mainly use monolayers of endothelial cells which are modified by repeated passages and are fully proliferative, a situation far away from physiology. In addition, not only endothelial cells are involved in this process but surrounding cells (such as pericytes, smooth muscle cells, fibroblasts) and the supporting matrix are also major players.
The three-dimensional ex vivo aortic ring model recapitulates the complexities of angiogenesis and combines the advantages of in vitro and in vivo models. The aortic ring is cultivated in a chemically defined culture environment. Microvessels which grow in this system are lumenized vessels with surrounding supporting cells and are essentially indistinguishable from microvessels formed during angiogenesis in vivo. The efficacy of pro-or anti-angiogenic factors can be determined in the absence of serum molecules which may otherwise interfere with the substances being tested (Nicosia and Ottinetti, 1990). However, this system requires access to fresh rat tissue but several samples can be prepared from one aorta.

Keywords: Aortic ring(主动脉环), Angiogenesis(血管生成), Endothelial cells(内皮细胞), Ex vivo model(体外模型)

Materials and Reagents

  1. Aluminium foils
  2. Cell culture plates 48 well (VWR International, catalog number: 734-2157 )
  3. Petri dishes (VWR International, catalog number: 734-2374 )
  4. LEW/Ss NHsd males rats 8-10 weeks old (Harlan Laboratories)
  5. Endothelial Cell Growth Medium (Lonza, catalog number: CC-3162 ) + 1% penicillin/streptomycin (P/S) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  6. Collagen R (2 mg/ml) (SERVA Electrophoresis GmbH, catalog number: 47254 )
  7. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S-5761 )
  8. 10x MEM (Thermo Fisher Scientific, GibcoTM, catalog number: 21430-020 )
  9. NaOH (Merck Millipore Corporation, catalog number: 1.06498 )

Equipment

  1. Dissecting microscope (Motic Stereomicroscope K-400L)
  2. Laminar flow hood
  3. Inverted light microscopes
  4. 50 ml Beaker
  5. 25 mm magnetic bar
  6. Magnetic stirrer
  7. Bleue extra gillette razor blades
  8. Scissors (Fine Science Tools, catalog number: 14090-11 )
  9. Vannas-Tübingen spring scissors (Fine Science Tools, catalog number: 15003-08 )
  10. 2 Microdissection forceps (VWR International, catalog number: 734-0448 )
  11. Dissecting boards and pins

Procedure

The day before
Wrapped dissecting board in 2 aluminium foils
Sterilize: Beaker, magnetic bar, blades and dissecting materials
Put in a fridge: 200 µl tips box, Cell culture plates 48 well

On the day

  1. Aortic ring preparation
    1. Sacrifice rat by CO2 inhalation and decapitation.
    2. Fix the rat on his back on a dissecting board.
    3. Disinfect skin with 80% ethanol.
    4. Make a Y-shape incision.
    5. Open the abdominal cavity.
    6. Cut the sternal plate and the diaphragm with scissors to expose the thoracic cavity.
    7. Displace the intestines, liver, stomach and spleen to the right side.
    8. Remove the lungs.
    9. The aorta is visible along the vertebral column.
    10. Excise the aorta starting at the point where abdominal aorta divides into two arteries up to the aortic arch using scissors placed parallel to the vertebral column and tweezers (do not stretch and mechanically damage the aorta).
    11. Transfer the aorta to a Petri dish filled with serum-free EBM-2 medium supplemented with 1% P/S on ice. All of the following steps will be performed under sterile conditions.
    12. Prior to pinning the aorta to the dissecting board remove the first sheet of aluminium foil and make sure that the aorta doesn’t dry by regularly adding serum-free EBM-2 medium + P/S.
    13. Under a dissecting microscope, remove the periaortic fibroadipose tissue, stumps of collateral vessels and blood clots with Vannas-Tübingen spring scissors and microdissection forceps.
    14. When the aorta is clean, transfer it to a clean Petri dish containing cold serum-free EBM-2 medium and keep it on ice.
    15. Prepare the collagen mix before cross-sectioning the thoracic aorta.
    16. Whilst in the Petri dish, Cross-section the thoracic aorta into rings that are 1 to 2 mm in length (20 to 25 rings/aorta) using a razor blade.
    17. Discard the aortic arch and the abdominal part of the aorta.
    18. Replace the medium 5 times with cold serum-free EBM-2 medium in the Petri-dish to rinse the rings.

  2. Embedding of aortic rings in collagen gels
    1. Homogenously spread 200 µl of collagen solution onto the bottom of each well in a pre-chilled 48-well plate using pre-chilled tips.
    2. Put the plate into a CO2 incubator (37 °C) for 30 min to allow polymerization.
    3. Take a ring with a forceps.
    4. Dry the ring by carefully dabbing it on the side of an empty Petri dish.
    5. Place the ring in the middle of the well at the top of the polymerized collagen.
    6. Repeat this process for all the rings, but be as quick as possible to make sure the rings do not dry out.
    7. Add a second layer of 200 µl collagen mix at the top of the aorta ring using pre-chilled tips.
    8. Place the plate back in the incubator.
    9. After 30 min, add 500 µl of serum-free EBM-2 medium supplemented or not with the drug of interest.

  3. Quantification
    Quantification of microvessel outgrowth is done manually from pictures obtained after 7-8 days of culture with a 2.5x magnification on an inverted microscope. The quantification involves counting the number of vessels and branch points (Nicosia and Ottinetti, 1990). A visualization of the counting process is presented in Aplin et al. (2008).

Representative data


Figure 1. Representative pictures of one aortic ring at different times in culture (top left to bottom left) and pictures of aortic rings treated with a growing concentration of an angiogenic agent (from left to right) at 8 days post-isolation

Notes

  1. Some isolated cells and vessels start to be present around 3-4 days. A well-developed network is visible around 7 to 9 days before regression.
  2. In this study, commercial collagen matrice has been used but it can be replaced by fibrin or Matrigel. One should note, however, that non-endothelial cells including fibroblasts have been shown to organize into networks in Matrigel (Donovan et al., 2001).

Recipes

  1. Collagen solution
    The collagen solution is prepared in a 50 ml beaker with pre-chilled reagents under slow agitation to avoid air bubbles.
    1. Mix 1 volume 10x MEM with 1.5 volumes 186 mM NaHCO3 until the color of the solution changes to orange.
    2. Add 7.5 volumes 2 mg/ml collagen R.
    3. Add few drops of 1 M NaOH to reach pH 7.4 (checked by the color indicator in the solution).

Acknowledgments

This protocol was adapted from Nicosia and Ottinetti (1990) and Blacher et al. (2001). This study was supported by the Society for Research on Cardiovascular Diseases and The Ministry of Culture, Higher Education and Research of Luxembourg.

References

  1. Aplin, A. C., Fogel, E., Zorzi, P. and Nicosia, R. F. (2008). The aortic ring model of angiogenesis. Methods Enzymol 443: 119-136.
  2. Blacher, S., Devy, L., Burbridge, M. F., Roland, G., Tucker, G., Noel, A. and Foidart, J. M. (2001). Improved quantification of angiogenesis in the rat aortic ring assay. Angiogenesis 4(2): 133-142.
  3. Donovan, D., Brown, N. J., Bishop, E. T. and Lewis, C. E. (2001). Comparison of three in vitro human 'angiogenesis' assays with capillaries formed in vivo. Angiogenesis 4(2): 113-121.
  4. Nicosia, R. F. and Ottinetti, A. (1990). Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro. Lab Invest 63(1): 115-122.

简介

血管生成是一种多因素事件,需要内皮细胞的迁移,增殖,分化和结构重排。这种血管生成过程已经使用体外试验例如Boyden腔室试验,伤口愈合试验和管形成试验进行了普遍研究。这些测定主要使用通过重复传代修饰的内皮细胞的单层,并且是完全增殖的,远离生理学的情况。此外,不仅内皮细胞参与这个过程,而且周围细胞(例如周细胞,平滑肌细胞,成纤维细胞)和支持基质也是主要的玩家。
三维主动脉环模型概括了血管生成的复杂性并结合了体外和体内模型的优点。主动脉环在化学上确定的培养环境中培养。在该系统中生长的微血管是具有周围支持细胞的管腔血管,并且与在体内血管生成期间形成的微血管基本上不可区分。可以在不存在血清分子的情况下测定促血管生成因子或抗血管生成因子的功效,否则血清分子可能干扰待测试的物质(Nicosia和Ottinetti,1990)。然而,该系统需要获得新鲜的大鼠组织,但是可以从一个主动脉制备几个样品。

关键字:主动脉环, 血管生成, 内皮细胞, 体外模型

材料和试剂

  1. 铝箔
  2. 细胞培养板48孔(VWR International,目录号:734-2157)
  3. 培养皿(VWR International,目录号:734-2374)
  4. LEW/Ss NHsd雄性大鼠8-10周龄(Harlan Laboratories)
  5. 内皮细胞生长培养基(Lonza,目录号:CC-3162)+ 1%青霉素/链霉素(P/S)(Thermo Fisher Scientific,目录号:15140-122) >
  6. 胶原R(2mg/ml)(SERVA Electrophoresis GmbH,目录号:47254)
  7. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S-5761)
  8. 10x MEM(Thermo Fisher Scientific,Gibco TM ,目录号:21430-020)
  9. NaOH(Merck Millipore Corporation,目录号:1.06498)

设备

  1. 解剖显微镜(Motic Stereomicroscope K-400L)
  2. 层流罩
  3. 倒置显微镜
  4. 50 ml烧杯
  5. 25 mm磁性杆
  6. 磁力搅拌器
  7. 漂白额外的gillette剃刀刀片
  8. 剪刀(Fine Science Tools,目录号:14090-11)
  9. Vannas-Tübingen弹簧剪刀(Fine Science Tools,目录号:15003-08)
  10. 2显微切割钳(VWR International,目录号:734-0448)
  11. 解剖板和针

程序

前一天
包裹解剖板在2个铝箔
消毒:烧杯,磁棒,刀片和解剖材料
放入冰箱:200μl提示盒,细胞培养板48孔

当日

  1. 主动脉环准备
    1. 牺牲鼠CO 2吸入和斩首
    2. 把老鼠的背部固定在解剖板上。
    3. 用80%乙醇消毒皮肤。
    4. 做一个Y形切口。
    5. 打开腹腔。
    6. 用剪刀剪切胸骨板和膈肌以暴露胸腔。
    7. 将肠,肝,胃和脾位于右侧
    8. 取出肺。
    9. 主动脉沿着脊柱可见。
    10. 消费税 ?主动脉开始于腹主动脉分成两部分的点 动脉到主动脉弓使用剪刀平行放置 脊柱和镊子(不伸展和机械损伤 主动脉)。
    11. 转移主动脉到充满培养皿 在冰上补加1%P/S的无血清EBM-2培养基。全部 以下步骤将在无菌条件下进行
    12. 先前 将主动脉固定到解剖板上去除第一张 铝箔,并确保主动脉不会定期干燥 加入无血清EBM-2培养基+ P/S
    13. 下解剖 显微镜,去除心脏主动脉纤维组织,残端 侧支血管和血栓与Vannas-Tübingen春天剪刀 和显微切割镊子
    14. 当主动脉清洁时,将其转移到含有冷无血清EBM-2培养基的干净培养皿中,并保存在冰上。
    15. 在切开胸主动脉之前准备胶原混合物。
    16. 同时 ?在培养皿中,将胸主动脉横切成环 ?1至2毫米长度(20至25环/主动脉)使用剃刀刀片
    17. 舍弃主动脉弓和主动脉的腹部。
    18. 在培养皿中用冷的无血清EBM-2培养基更换培养基5次,以冲洗环。

  2. 将主动脉环嵌入胶原凝胶中
    1. 将200μl的胶原溶液均匀地铺在每个的底部 孔在预冷的48孔板中使用预冷的尖端
    2. 将板放入CO 2培养箱(37℃)中30分钟以允许聚合。
    3. 用镊子做一个戒指。
    4. 通过小心地将其擦在空培养皿的侧面来干燥环
    5. 将环放在聚合胶原的顶部的孔的中间
    6. 对所有环重复此过程,但要尽可能快,以确保环不会变干
    7. 使用预先冷却的提示,在主动脉环的顶部添加第二层200μl胶原混合物
    8. 将板放回培养箱中。
    9. 30分钟后,加入500μl无血清的EBM-2培养基补充或不补充感兴趣的药物。

  3. 量化
    通过在倒置显微镜上以2.5×放大率培养7-8天后获得的图片手动进行微血管增生的定量。定量包括计数血管和分支点的数量(Nicosia和Ottinetti,1990)。计数过程的可视化呈现在Aplin等人(2008)中。

代表数据


图1.分离后8天,培养中不同时间(左上至左下)的一个主动脉环的代表性图片和用生长浓度的血管生成剂治疗的主动脉环(从左到右)的图片

笔记

  1. 一些孤立的细胞和血管开始出现约3-4天。一个发达的网络在回归前约7至9天可见
  2. 在本研究中,商业胶原基质已被使用,但它可以被纤维蛋白或Matrigel替代。然而,应当注意,包括成纤维细胞的非内皮细胞已经显示在Matrigel中组织成网络(Donovan等人,2001)。

食谱

  1. 胶原蛋白溶液
    在缓慢搅拌下,在预先冷冻的试剂的50ml烧杯中制备胶原溶液,以避免气泡
    1. 将1体积10×MEM与1.5体积186mM NaHCO 3混合,直至溶液的颜色变为橙色。
    2. 加入7.5体积2mg/ml胶原蛋白R.
    3. 加入几滴1M NaOH以达到pH 7.4(通过溶液中的颜色指示剂检查)。

致谢

该方案改编自Nicosia和Ottinetti(1990)和Blacher等人(2001)。本研究得到了心血管疾病研究协会和卢森堡文化,高等教育和研究部的支持。

参考文献

  1. Aplin,A.C.,Fogel,E.,Zorzi,P。和Nicosia,R.F。(2008)。 血管生成的主动脉环模型 方法Enzymol 443 :119-136。
  2. Blacher,S.,Devy,L.,Burbridge,M.F.,Roland,G.,Tucker,G.,Noel,A.and Foidart,J.M。(2001)。 改进大鼠主动脉环测定中血管生成的定量。血管生成 em> 4(2):133-142。
  3. Donovan,D.,Brown,N.J.,Bishop,E.T.and Lewis,C.E。(2001)。 三种体外人类'血管生成'测定与毛细血管形成的比较< em 。 4(2):113-121。
  4. Nicosia,R.F.and Ottinetti,A。(1990)。 大鼠主动脉无血清基质培养中微血管的生长。体外血管生成的定量测定。实验室投资 63(1):115-122。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ernens, I., Lenoir, B., Devaux, Y. and Wagner, D. R. (2015). Rat Aortic Ring Model to Assay Angiogenesis ex vivo. Bio-protocol 5(20): e1622. DOI: 10.21769/BioProtoc.1622.
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