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分离人脐静脉内皮细胞(HUVEC)

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Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI)

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Cite this Article: 分离人脐静脉内皮细胞(HUVEC)

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).

Abstract: 分离人脐静脉内皮细胞(HUVEC)

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: 分离人脐静脉内皮细胞(HUVEC)

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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Disclosures: 分离人脐静脉内皮细胞(HUVEC)

Materials: 分离人脐静脉内皮细胞(HUVEC)

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

Ask the Author: 分离人脐静脉内皮细胞(HUVEC)

7 Comments

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

1

Reply

Posted by: loryMay 16, 2008, 5:47 AM

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

 

3

Reply

Posted by: RathnakumarJune 1, 2008, 11:25 AM

hallo,can you tell me what's the  PHEC+ ?thank you !

4

Reply

Posted by: wuqiongDecember 15, 2008, 6:27 AM

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

5

Reply

Posted by: KarinAugust 26, 2009, 8:06 AM

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.

5.1

Reply

Posted by: Linda Kalin HimSeptember 8, 2009, 5:45 AM

hi
how to i prepare collagenase for HUVEC isolation

6

Reply

Posted by: swati s.October 22, 2009, 1:45 PM

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

7

Reply

Posted by: Ge Z.January 11, 2010, 6:56 PM

To answer Ge's question, our lab lysate the HUVEC by adding lysate buffer 1x either in DPBS or water.

7.1

Reply

Posted by: AnonymousJanuary 20, 2010, 2:54 PM

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?

7.1.1

Reply

Posted by: Ge ZouJanuary 20, 2010, 3:09 PM

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:-)

8

Reply

Posted by: AnonymousAugust 3, 2012, 11:57 AM

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