Name: shrinky-dink hanging drops: a simple way to form and culture embryoid bodies
Uploaded: 2008-03-05T22:18:00
Description: Watch this Scientific Journal Video about Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies at JoVE.com

我们要感谢支持这项工作摆围。安德拉什纳吉博士在西奈山医院，多伦多，安大略省的细胞系的慷慨捐赠。

1。制作Shrinky，丁克模具<ol><li> shrinky，丁克表使用一个很好的定义打印机打印所需的图案。 </li><li>烘烤shrinky丁克表在163℃，约10分钟，或直至完全萎缩，并取得了一个常规的形状。 </li><li> shrinky，丁克模具冷却下来后，把它浸入异丙醇洗澡，直到完整的表面几乎覆盖。 </li><li>小心，喷在模具丙酮和摇晃容器几次。添加更多的异丙醇洗出丙酮过剩，重复此步骤几次，直到shrinky模看上去干净。 </li...

<ol>
	<li>Keller, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+embryonic+stem+cells.">In vitro differentiation of embryonic stem cells.</a> Curr Opin Cell Biol. 7, 862-869 (1995).</li><li>Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W., Kemler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vitro+development+of+blastocyst-derived+embryonic+stem+cell+lines:+Formation+of+visceral+yolk+sac,+blood+islands+and+myocardium.">The in vitro development of blastocyst-derived embryonic stem cell lines: Formation of visceral yolk sac, blood islands and myocardium.</a> Journal of Embryology and Experimental Morphology. 87, 27-45 (1985).</li><li>Park, J., Cho, C. H., Parashurama, N., Li, Y., Berthiaume, F., Toner, M., Tilles, A. W., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabrication-based+modulation+of+embryonic+stem+cell+differentiation.">Microfabrication-based modulation of embryonic stem cell differentiation.</a> Lab Chip. 7, 1018-1028 (2007).</li><li>Koike, M., Sakaki, S., Amano, Y., Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+embryoid+bodies+of+mouse+embryonic+stem+cells+formed+under+various+culture+conditions+and+estimation+of+differentiation+status+of+such+bodies.">Characterization of embryoid bodies of mouse embryonic stem cells formed under various culture conditions and estimation of differentiation status of such bodies.</a> J Biosci Bioeng. 104, 294-299 (2007).</li><li>Hwang, N. S., Varghese, S., Elisseeff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+differentiation+of+stem+cells.">Controlled differentiation of stem cells.</a> Advanced Drug Delivery Reviews. 60, 199-204 (2008).</li><li>Adelman, C. A., Chattopadhyay, S., Bieker, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BMP/BMPR/Smad+pathway+directs+expression+of+the+erythroid-specific+EKLF+and+GATA1+transcription+factors+during+embryoid+body+differentiation+in+serum-free+media.">The BMP/BMPR/Smad pathway directs expression of the erythroid-specific EKLF and GATA1 transcription factors during embryoid body differentiation in serum-free media.</a> Development. 129, 539-549 (2002).</li><li>Tanaka, N., Takeuchi, T., Neri, O. V., Sills, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-assisted+blastocyst+dissection+and+subsequent+cultivation+of+embryonic+stem+cells+in+a+serum/cell+free+culture+system:+applications+and+preliminary+results+in+a+murine+model.">Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model.</a> J Transl Med. 4, (2006).</li></ol>

我们已经开发出一种简单，快速，可伸缩的培养方法加载到微井（Shrinky - Dinks成型）预先定义的细胞数目和他们保持胚体的发​​展。最后，这些细胞是方便作进一步的分析和实验。这种方法适合任何实验室，不需要专用的设备，因为我们排除光刻需要。我们可以改变的微孔的大小以及细胞/井的浓度改变胚体的数量和大小。

对于MESC媒体（McCloskey的实验室协议）：（媒体准备总额：50ML;百毫升）KO的DMEM：40.8毫升;81.6毫升15％KSR：7.5毫升;15毫升1X宾夕法尼亚链球菌：0.5毫升;1毫升2mm的L -谷氨酰胺： .5毫升;1毫升NEAA：0.5毫升;1毫升LIF：100ul 200ul BMP - 4（在10ng/ml）：50ul; 100ul摊薄BME：50ul; 100ul（PBS和注射器过滤器5毫升消毒无菌过滤的BME 35ul丢弃2周后的解决方案中的最终浓度是0.1毫米。）

shrinky-dink hanging drops: a simple way to form and culture embryoid bodies

胚体（EB）的胚胎干细胞的聚集。创建这些聚集的最常用的方法是悬滴法，孔板移液到任意数量的细胞费力的方法。被迫彼此接近的干细胞之间的相互作用，促进了EBS的一代。因为在每个井的媒体每天要手动交换，这种做法是手动密集。 此外，因为环境参数，包括细胞，细胞可溶性因子的相互作用，pH值，和氧气供应的EB大小的功能，从传统的挂滴获得的细胞群可以相差很大，即使在相同条件下培养。的确，最近的研究表明，初步形成总额的细胞数量有重大影响干细胞分化。我们已经开发出一种简单，快速，可伸缩的文化的方法加载到预先定义的数字，微型井细胞胚体的发展和维护。最后，这些细胞是方便作进一步的分析和实验。这种方法适合任何实验室，无需专用设备。我们通过这种方法使用红色荧光的小鼠细胞株（129S6B6 F1），胚体。

Watch this Scientific Journal Video about Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies at JoVE.com

Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies

我们展示一个简单而快速的方法加载到预先定义的数字，微型井细胞胚体的发展和维护。

Shrinky丁克挂滴：一个简单的方法，形式和文化胚体

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