分离人脐静脉内皮细胞(HUVEC)

Biology

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Summary

这个视频协议说明了人脐静脉内皮细胞(HUVEC)的人脐血的分离和培养。一旦分离出这些细胞可以在体外血管生成像优化的纤维蛋白凝胶珠还展示了由休斯实验室的检测分析。

Cite this Article

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Davis, J., Crampton, S. P., Hughes, C. C. Isolation of Human Umbilical Vein Endothelial Cells (HUVEC). J. Vis. Exp. (3), e183, doi:10.3791/183 (2007).

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Abstract

血管生成是一个复杂的多步骤的过程,在血管生成刺激,从现有的血管创建新船。这些步骤包括:降解基底膜,增殖和迁移(发芽)内皮细胞到细胞外基质,对齐欧共体(EC)的成线,形成管腔吻合,并形成一个新的基底膜。在体外实验中有许多已开发研究这个过程,但大多只模仿血管生成的某些阶段,形态的船只往往并不像在体内的船只。在这里,我们证明,利用人脐静脉EC和成纤维细胞在体外血管生成试验进行了优化。这种模式概括所有血管生成的关键早期阶段,而重要的血管显示专利包围极化欧共体间流明。船只,可以很方便地观察相衬和时间推移显微镜,并在下游应用纯粹的形式收回。

Protocol

程序

  1. 放置于清洁垫和民建联多余的血线。线的两端的新鲜削减。
  2. 21 1 / 2 G针 ,静脉插入的塑料针鞘上。 (静脉是世界上最大的开放; 2较小的动脉)
  3. 用止血钳将针钳 ,重视汉克斯20CC的注射器针头。
  4. 通过适度的压力静脉推汉克斯 。与漂白水的烧杯中收集的废物。 (用单手的针和线的同时,也会防止针头静脉出现。)如果有很多在静脉取血,洗第二次。
  5. 莱垫线和钳的另一端的静脉。填写几毫升的汉克斯,检查有无泄漏,沿着电线。抽出5 ml和断开的底部钳。
  6. 断开20CC的注射器。删除从10CC的注射器的柱塞。附上10CC的注射器针头,倒在10ml胶原酶和更换柱塞。静脉胶原酶推入,直到你看到的第一个金额退出开放结束。钳的开口端,并填写胶原酶,直到有中度腹胀静脉。平滑肌污染太多腹胀结果。
  7. 轻轻按摩电源线
  8. 孵育线在DPBS(止血,针头和注射器附加)在37 ° C为15分钟。
  9. 在孵化过程中, 继续与第二线的步骤1-8。
  10. 孵育后,采取线的烧杯中,同时举行了50毫升管线以上的底部钳剪开结束。 务必收集管中的一切。其余胶原酶推通过线,然后将其附加的20CC的注射器,通过适度的压力,推动汉克斯。
  11. 在这个时候,如果没有平滑肌细胞需要,弃线。否则,将成为第二个50毫升管〜5毫升胶原酶孵育同一线在37 ° C为30-60分钟。
  12. 保持管,直到完成所有的电线。
  13. 自旋为5分钟〜1200 RPM
  14. 吸上清液(〜1-2毫升的除外)。重悬在一个T25 5mls PHEC颗粒+和板块。
  15. 在37℃,5%的CO 2过夜孵育
  16. 第二天,去除上清液,并更换新媒体。如果有很多的红细胞,洗一次与M199,然后添加PHEC +。继续培育像往常一样,直到该板块融合(1-4天)。拆分成一个糊化T75。
  17. 一旦T75汇合,分成三个T75s。冻结两小瓶每烧瓶。

注:(未激活)的内皮细胞有鹅卵石的外观。有时激活内皮细胞(长,尖尖的)后隔离,后一对夫妇通过他们通常会返回到一个unactive状态。

净化

  1. 非常仔细,处理锐器容器针。
  2. 处置中的生物危害大容器的注射器。
  3. 放入小生物危害袋的所有组织。关闭袋,并冻结在-20 °直到焚烧。
  4. 至少10分钟,浸泡在virucide的所有仪器。
  5. 新增孵化媒体浪费/漂白剂烧杯。让我们坐了至少10分钟。
  6. 生物危险废物弃置于工作台垫。
  7. 喷雾烧杯,台式水浴盖与vircide的内外。让我们坐10分钟,然后擦拭。
  8. 用温水冲洗烧杯和文书,并挂在机架干(印迹,使他们不生锈的文书)。
  9. 净化废物处置下来的水槽。
  10. 将未使用的媒体冰箱。
  11. 在生物危险废物手套处置。

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Materials

Name Company Catalog Number Comments
0.1% Collagenase 10ml per cord, warmed to 37°C
Tissue culture flasks T75, one per cord.
Hanks medium
scissors sterile
beaker sterile
50 ml tubes

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Comments

10 Comments

  1. may you tell me wath kind of collagenase do you use? thank you!

    Reply
    Posted by: Anonymous
    May 16, 2008 - 5:47 AM
  2. hi   i have been using dispase to isolate cells. iam succeeding in getting the cells but they are not spreading properly . even after two days iam not able to see  the typical cobble stone appearance. can you help.  Rathna  

    Reply
    Posted by: Anonymous
    June 1, 2008 - 11:25 AM
  3. hallo,can you tell me what's the  PHEC+ ?thank you !

    Reply
    Posted by: Anonymous
    December 15, 2008 - 6:27 AM
  4. Hi, do you have any idea how many cells I can get from a 90% confluent flask (²5 or 75 qcm)? Many thanks in advance, Karin

    Reply
    Posted by: Anonymous
    August 26, 2009 - 8:06 AM
  5. From my experience working with HUVEC, a confluent T²5 flask has about 400,000 to 600,000 million cells while a confluent T75 flask has approximately ²-4 millions cells. From this, you can probably roughly deduce how many cells are in a 90% confluent T²5/T75 flask. I hope this help.

    Reply
    Posted by: Anonymous
    September 8, 2009 - 5:45 AM
  6. hi
    how to i prepare collagenase for HUVEC isolation

    Reply
    Posted by: swati s.
    October 22, 2009 - 1:45 PM
  7. Hi,

    I was a synthetic organic chemist and am trying to conduct a bio-organic project. I will be very thankful if you would share some info with me about how to lysate the HUVECs.

    Ge

    Reply
    Posted by: Ge Z.
    January 11, 2010 - 6:56 PM
  8. To answer Ge's question, our lab lysate the HUVEC by adding lysate buffer 1x either in DPBS or water.

    Reply
    Posted by: Anonymous
    January 20, 2010 - 2:54 PM
  9. Thank you so much for the answer. May I have more info regarding the components of your lysate buffer and at what condition do you lysate HUVECs?

    Reply
    Posted by: Anonymous
    January 20, 2010 - 3:09 PM
  10. hi,

    Very Intresting way, but I wonder how many passages they can be usefull?
    Is there any difference between commercially available ones and freshly isolated ones(these ones)?
    Thanks in advance:-)

    Reply
    Posted by: Anonymous
    August 3, 2012 - 11:57 AM

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