人类胚胎干细胞传代的色调线与胰蛋白酶

Published 10/12/2006
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Summary

在这个视频中,我们展示了我们的实验室常规通道与胰蛋白酶色调人类胚胎干细胞系。

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Trish, E., Dimos, J., Eggan, K. Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.. J. Vis. Exp. (1), e49, doi:10.3791/49 (2006).

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Abstract

在这个视频中,我们展示了我们的实验室常规通道与胰蛋白酶色调人类胚胎干细胞系。人类胚胎干细胞是细胞培养的产物,并倾向于收购与高人口倍增的异常核型。正确的传代是保持一个健康的,未分化,核型正常的色调的人类胚胎干细胞培养的关键。首先,扩大文化是在PBS洗涤,以去除残留的媒体和的细胞碎片,然后将细胞覆盖一个温暖的0.05%胰蛋白酶EDTA的最小量。胰蛋白酶是留给长达五分钟的细胞,然后细胞被轻轻抛下一个2ML血清吸管。细胞悬液与色调媒体的大量收集和混合,然后收集细胞,轻轻离心。胰蛋白酶灭活媒体混合物被删除,细胞悬浮预热色调媒体。一个适当的分流比计算(一般为1:10至1:20),和细胞到1-2天老照射的小鼠胚胎成纤维细胞饲养层细胞单层板重新镀金。新种子的色调培养板,左为48小时不受干扰,然后媒体是每天更换其后。重要的是不要trpsinize单细胞悬液,因为这增加了引进核型异常的风险。

Protocol

一般

我们建议解冻在一个给定的的时间不超过一个样本,以确保容易处理。处理程序应注意不要长时间留在室温和低二氧化碳的文化。所有活细胞的离心是在室温下5分钟在500-600 XG。

MEFs(小鼠饲养层细胞)

  1. 预温MEF的媒体至37 ° C。删除MEF的小瓶从-80 ° C,并立即淹没在37℃水浴试管底部的一半。细胞解冻80%(左小冻结部分)之前,需要大约30-45秒。
  2. 迅速地把管层流罩,喷以​​70%酒精,细胞转移预热媒体。
  3. 吸板准备较早的明胶溶液。
  4. 分装2毫升MEF的一个明智的方式下降到每个六口井解决方案。请务必均匀分发以及MEFs。日期的MEF板,并在37℃培养箱中放置过夜,让MEFs板块的重视。
  5. 将被连接到6个小时后,MEFs板。最好是使用MEF板电镀后的24-48小时。不要超过4天的使用MEF板。
  6. 前暖色调媒体和0.05%胰蛋白酶/ EDTA至37 ° C下的引擎盖,prelabel之一无菌50毫升锥形管。
  7. 小心吸从文化的色调媒体被分裂。轻轻地用2ML 1X磷酸盐缓冲液(PBS),每口井的井。吸的PBS。添加0.75毫升0.05%胰蛋白酶/ EDTA井。
  8. 更换盖子,4倍的放大倍率下观察细胞。 MEFs周围色调殖民地开始收回。当MEFs有足够的圆润色调殖民地的边界粗糙,返回发动机罩板。这过程中应采取约5分钟,绝对不长于10分钟,或你的细胞,不会重新长出。
  9. 轻轻吸管,洗井的底部,直到MEF单层已完全脱离(单层粘和可能保持在一块)。
  10. 将细胞悬液50毫升锥形管中包含额外的色调媒体。旋转5分钟500xg细胞管,吸媒体,小心不要打扰细胞沉淀。
  11. 到一个新的MEFs板的板,添加细胞额外的色调媒体确定适当的量,并自动吸管,吸管的解决方案的5-7倍。
  12. 删除从一个新鲜的MEFs板的MEF媒体。
  13. 分装的色调明智的解决方案,下降到每口井,确保分发以及滴均匀。不摇板,仔细细胞返回到37 ° C培养箱过夜,让色调殖民地种子。
  14. 色调细胞分裂的行为同样解冻。

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Discussion

在这个视频中,我们证明我们定期通过色调如何在我们的实验室中的人类胚胎干细胞系。高效和定期的分裂和传代是保持一个健康的人类胚胎干细胞系的关键。胰蛋白酶介导的通过是一个色调细胞系的显着优势,但是,重要的是不要过分trypsinize文化或有一个单细胞的过程中再platting大的比重。

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Materials

Name Company Catalog Number Comments
HuES media 651.5 ml
KO-DMEM 500 ml
PenStrep 6.5 ml
GlutaMAXTM 6.5 ml
NEAA 6.5 ml
2-mercapt–thanol 0.5ul
KO Serum Replacement 65 ml
Plasmanate 65 ml
bFGF2 (10 ng/mL final) 0.65 ml

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References

  1. Edmond, M. B. Nosocomial bloodstream infections in United States hospitals: a three-year analysis. Clin. Infect. Dis. 29, 239-244 (1999).
  2. Ramage, G., Bachmann, S., Patterson, T. F., Wickes, B. L., Lopez-Ribot, J. L. Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms. J. Antimicrob. Chemother. 49, 973-980 (2002).
  3. Srinivasan, A., Uppuluri, P., Lopez-Ribot, J., Ramasubramanian, A. K. Development of a High-Throughput Candida albicans Biofilm Chip. PLoS ONE. 6, 19036-19036 (2011).
  4. Ramage, G., Vandewalle, K., Wickes, B. L., Lopez-Ribot, J. L. Characteristics of biofilm formation by Candida albicans. Rev. Iberoam. Micol. 18, 163-170 (2001).
  5. Pierce, C. G. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat. Protoc. 3, 1494-1500 (2008).
  6. Lee, M. Y. Three-dimensional cellular microarray for high-throughput toxicology assays. Proc. Natl. Acad. Sci. U.S.A. 105, 59-63 (2008).
  7. Meyerhofer, D. Characteristics of resist films produced by spinning. Journal of Applied Physics. 49, (1978).
  8. Ramage, G., Vande Walle, K., Wickes, B. L., Lopez-Ribot, J. L. Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob. Agents Chemother. 45, 2475-2479 (2001).
  9. Chandra, J. Biofilm formation by the fungal pathogen Candida albicans: Development, architecture, and drug resistance. Journal of Bacteriology. 183, 5385-5394 (2001).
  10. Jabra-Rizk, M. A., Falkler, W. A., Meiller, T. F. Fungal biofilms and drug resistance. Emerg. Infect. Dis. 10, 14-19 (2004).
  11. Tobudic, S., Lassnigg, A., Kratzer, C., Graninger, W., Presterl, E. Antifungal activity of amphotericin B, caspofungin and posaconazole on Candida albicans biofilms in intermediate and mature development phases. Mycoses. 53, 208-214 (2010).

Comments

4 Comments

  1. That's very nice clip and  I have some question. Normally, HuES cells are very hard to form colony from single cell state. By treating with trypsin, I think, HuES colony will be dissociated into single cell state or very small colony state. How could you solve this problem? Which HuES cell line your lab are using? Looking forward to your response! Tran Ngoc Tung  

    Reply
    Posted by: Anonymous
    January 6, 2009 - 7:15 AM
  2. For Tran Njoc Tung, You are totally correct that HuES, in single cell state, form colonies with great difficulty. The trick to avoid making a single cell suspension is to minimize the time for which you trypsinze. As you put trypsin, observe the cells under the microscope and just when there are small aggregates of HuESC's remaninig add the media. This would not allow the formation f single cell suspension. It totally works for me. Hope it dŒs for you.   Sumit Rai

    Reply
    Posted by: Sumit R.
    April 7, 2009 - 4:06 AM
  3. Dear Sir/Madame Unfortunately I can"t see the movie. How can I overcome this problem? Thank you and regards Vitali Shilo vitali.shilo@gmail.com

    Reply
    Posted by: vitali s.
    January 22, 2009 - 8:05 AM
  4. Hello Vitali, Please try the steps on this page: http://www.jove.com/index/page.stp?name=FlashProblems and let me know if you have any trouble. Cheers, Nikita

    Reply
    Posted by: Anonymous
    January 22, 2009 - 9:33 AM

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