在体外分化的小鼠胚胎干细胞(MES)使用悬滴法

Biology

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Summary

本视频演示了如何在小鼠胚胎干细胞体外分化胚体采用悬滴法进行。

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Wang, X., Yang, P. In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method. J. Vis. Exp. (17), e825, doi:10.3791/825 (2008).

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Abstract

干细胞具有显着的潜力发展成许多不同的细胞类型。当一个干细胞分裂时,每个新细胞的潜力仍然是一个干细胞,或成为另一个类型的细胞与一个更专业的功能,这是有前途的科学顶尖科学家研究细胞疗法来治疗疾病的可能性。当没有暂停antidif​​ferentiation因素文化时,胚胎干细胞自发分化和形成立体的多细胞聚集。这些细胞聚集被称为胚体(EB)。悬滴文化是一种广泛使用的EB形成诱导方法。悬滴的圆形底部,使胚胎干细胞,它可以提供一个良好的环境,形成EBS mES细胞的聚集。在悬滴aggregatied的ES细胞的数量是可以控制的不同细胞的数量在最初的细胞悬液,将挂在培养皿的盖子下降。使用这种方法,我们可以再现的形式从同质分化的ES细胞的预定人数。

Protocol

  1. 涂胶T75烧瓶中,在37℃,5%的CO 2组织培养箱培养过夜之一的前一天使用0.1%的明胶溶液和孵化板。
  2. 取出孵化mES细​​胞,吸出培养基,用PBS冲洗胚胎干细胞培养,加入0.05%胰蛋白酶溶液大衣的菜(2 ml/100mm板)底部。
  3. 孵育在37 ° C约1分钟,待细胞塌板。
  4. 轻轻磨碎(吸管向上和向下)的4至6倍,以分散与巴斯德吸管插入的ES细胞的胰酶消化细胞,。分散的ES细胞转移到15毫升锥形离心管预热(37℃)MES中等。
  5. 离心收集细胞。吸弃上清液,加入10毫升的MES培养基和吸管向上和向下,形成一个单细胞悬液。
  6. 转移细胞悬液,在37 °成T75容量瓶中,用0.1%的明胶和孵化预涂C的5%CO 2为一小时

    *一小时后,成纤维细胞附着到板材,但干细胞的培养基中保持。移液器中期收集干细胞。

  7. 在5分钟1000转的旋转细胞和吸MES的介质。然后添加另一个MES分化培养基10毫升和重复吹打重悬细胞,直到出现被罚款的细胞悬浮液。
  8. 计数与血球使用分化培养基中的细胞,干细胞悬液,以稀释浓度20ul(20ul/drop)的400至500个细胞,在无菌盆地。
  9. 提起盖子,小心地倒置和地方菜含有10毫升的PBS上。使用多道移液器,20ul滴行的组织培养皿的盖子向上翻内表面上。
  10. 2天,小心地在孵化器的菜。两天后,仔细地翻板盖,吸180 UL新鲜分化培养基放入96孔超低附件板以及数滴。然后用吸管和转让滴,一个接一个,96孔板皮卡下降。放入3天的孵化器不受干扰的板块。
  11. 大衣,每孔48孔组织培养板,0.1%的明胶300ul。加入明胶后,板一夜之间在37 ° C提前一天之前转移的EBS。
  12. 第二天,从48孔板吸明胶。然后分化培养基加入300 UL每口井。从96孔板的EBS转移到48以及明胶涂层板一。更改介质的第二天,然后改变中期隔日维持细胞。
  13. 自发cardiaomyocyte收缩应在7天明显。

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Discussion

悬滴法的缺点如下:不超过50ul,由于维持表面张力的盖子上悬滴一滴的液体的体积是有限的,它不可能改变中期挂滴。形成胚滴直接用显微镜观察也很难在种植。更进一步,悬滴法是由两个步骤组成,因此,悬滴法的一系列步骤可能会很麻烦。

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Materials

Name Type Company Catalog Number Comments
DMEM Reagent GIBCO, by Life Technologies 11965-092
Fetal Bovine Serum(FBS) Reagent Hyclone SH30070.03
L-Glutamine Reagent GIBCO, by Life Technologies 25030
Non-Essential Amino Acids Reagent GIBCO, by Life Technologies 11140-050
Penicllin / Streptomycin Reagent GIBCO, by Life Technologies 15140-122
Sodium Pyruvate Reagent GIBCO, by Life Technologies 11360-70
β-mercapt–thanol Reagent Chemicon International ES-007-E
leukemia inhibitory factor(LIF) Reagent Chemicon International LIF2005
96-well ultralow attachment plate Tool Corning 3474

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References

  1. Wobus, A. M., Wallukat, G., Hescheler, J. Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers. Differentiation. 48, 173-182 (1991).
  2. Metzger, J. M., Lin, W. I., Samuelson, L. C. Transition in cardiac contractile sensitivity to calcium during the in vitro differentiation of mouse embryonic stem cells. J Cell Biol. 126, (1994).

Comments

7 Comments

  1. Dear Doctor Xiang Wang,  your technique appear useful and very interesting. I' m studying mES and I want to differentiate them into simple embryoid bodies, but not into predefinite cells. So I have a question for you. The differentiation medium could be a stem cell medium just without LIF? Or do I need to add some elements to my medium to produce embryoid bodies? Thank you very much and best regards,  Barbara Tondelli.

    Reply
    Posted by: Anonymous
    October 8, 2008 - 7:13 AM
  2. hi, i work together w/ xiang. he returned to china so i asked him to respond to you but in case he dŒsn't, let me answer: 1. yes. embryoid bodies can be made w/ the stem cell medium w/o lif. you need to create a voumetric clusters. this could be done using the hanging drop method or plate in cluster of cells. we have had best exerience making hanging drop. hope this helps.   phil

    Reply
    Posted by: Anonymous
    October 8, 2008 - 12:09 PM
  3. Hello I want to know the function and concentration here of sodium pyvurate. Can the heartbeat appear in a differentiation medium without sodium pyvurate? And if the plates are taken out sometimes during the suspension culture period, dŒs it matter so much? I've been trying but no cardiocytes seemed to appear, and I cannot find the key to this. Thank you a lot!  

    Reply
    Posted by: Anonymous
    November 7, 2008 - 3:02 AM
  4. Dear Huijun Zhu,   I use 1mM Sodium pyvurate for mESC culturing. Sodium pyvurate works mostly  as  an additional source  energy. It also have protective effects against hydrogen peroxide.  According to my experience,  Sodium pyruvate is not necessary for differentiation. It 's not good for moving  out of the incubate when the EBs are in hanging drop situation. It should be fine to take out the plate carefully and take a simple look once they were in ultralow attachment plates. Hope this helps, Good luck!   Xiang Wang  
     

    Reply
    Posted by: Anonymous
    November 10, 2008 - 8:00 AM
  5. Thank you Xiang Wang! The cardiaomyocyte contractions became obvious after 9days. But the contraction of a certain area became weaker and finally disappeared. Is it  normal or something gŒs wrong? And how can I purify the cardiaomyocytes? How can I increase the percent of the cardiomyocytes? Thank you a lot!

    Reply
    Posted by: Anonymous
    November 18, 2008 - 3:59 AM
  6. Dear Doctor Xiang Wang

    is necessary to add gelatin on the plate or not??

    Reply
    Posted by: Anonymous
    June 12, 2009 - 10:42 AM
  7. Dear Doctor Xiang Wang,

    is it possible to split the cardiomyocytes? Or how can i isolate them, to start my assays with them?
    Thank you very much.

    Kind regards

    Reply
    Posted by: Anonymous
    April 26, 2011 - 11:27 AM

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