A comparative study of protocols for mouse embryonic stem cell culturing.
A comparative study of protocols for mouse embryonic stem cell culturing.
复制标题
DOI：
10.1371/journal.pone.0081156
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Annerén C
中科院分区：
文献类型：
--
作者：
Tamm C;Pijuan Galitó S;Annerén C
大多数干细胞实验室仍然依赖于旧的培养方法来支持小鼠胚胎干细胞（ES）的扩增和维持。这些包括在小鼠胚胎成纤维细胞饲养细胞上或在补充有胎牛血清和白血病抑制因子（LIF）的培养基中的明胶上生长细胞。然而，这些技术具有几个缺点，包括需要饲养细胞和/或使用含有动物来源组分的不确定培养基。在不确定的条件下培养干细胞可诱导自发分化并降低实验的可重复性。近年来，一些新的ES细胞培养方案，使用更明确的条件，已经公布，我们已经比较了标准培养方案与两个新描述的方案：1）在含有两种小分子抑制剂的培养基中半贴壁生长细胞（CHIR 99021，PD 0325901）和; 2）在含有LIF和bFGF的确定成分培养基中的球状体悬浮培养物中生长细胞。在研究中使用了两种饲养层依赖性小鼠ES（mES）细胞系和两种适应饲养层非依赖性生长的细胞系。总的目标不仅是比较自我更新和差异化能力，而且还比较易用性和成本效益。我们发现，贴壁生长的胚胎干细胞增殖速度远远快于在悬浮液中生长的自由浮动的球体，独立的媒体使用。尽管所有测试的培养方案都可以在长时间培养后保持持续的多能性，但我们的数据证实了先前的报道，其显示与标准mES培养基相比，含有两种化学抑制剂的培养基产生更纯的干细胞培养物，自发分化的迹象可忽略不计。此外，我们表明，这种培养基有效地挽救和清理已经开始恶化的培养物，以及允许饲养层依赖性mES细胞系的有效适应，以维持在无饲养层条件下。
Most stem cell laboratories still rely on old culture methods to support the expansion and maintenance of mouse embryonic stem (ES) cells. These involve growing cells on mouse embryonic fibroblast feeder cells or on gelatin in media supplemented with fetal bovine serum and leukemia inhibitory factor (LIF). However, these techniques have several drawbacks including the need for feeder-cells and/or use of undefined media containing animal derived components. Culture of stem cells under undefined conditions can induce spontaneous differentiation and reduce reproducibility of experiments. In recent years several new ES cell culture protocols, using more well-defined conditions, have been published and we have compared the standard culture protocols with two of the newly described ones: 1) growing cells in semi-adherence in a medium containing two small molecule inhibitors (CHIR99021, PD0325901) and; 2) growing cells in a spheroid suspension culture in a defined medium containing LIF and bFGF. Two feeder-dependent mouse ES (mES) cell lines and two cell lines adapted to feeder-independent growth were used in the study. The overall aim has not only been to compare self-renewal and differentiation capacity, but also ease-of-use and cost efficiency. We show that mES cells when grown adherently proliferate much faster than when grown in suspension as free-floating spheres, independent of media used. Although all the tested culture protocols could maintain sustained pluripotency after prolonged culturing, our data confirm previous reports showing that the media containing two chemical inhibitors generate more pure stem cell cultures with negligible signs of spontaneous differentiation as compared to standard mES media. Furthermore, we show that this medium effectively rescues and cleans up cultures that have started to deteriorate, as well as allow for effective adaption of feeder-dependent mES cell lines to be maintained in feeder-free conditions.
登录
查看更多内容
影响因子：
1.5
作者：
Cheng, J;Dutra, A;Schwartzberg, PL
通讯作者：
Schwartzberg, PL
影响因子：
4
作者：
Tamm, Christoffer;Bower, Nathalie;Anneren, Cecilia
通讯作者：
Anneren, Cecilia
影响因子：
64.8
作者：
SMITH, AG;HEATH, JK;ROGERS, D
通讯作者：
ROGERS, D
影响因子：
0.7
作者：
Eiselleova, Livia;Peterkova, Iveta;Dvorak, Petr
通讯作者：
Dvorak, Petr
影响因子：
2.5
作者：
Meng, G. L.;Nieden, N. I. Zur;Rancourt, D. E.
通讯作者：
Rancourt, D. E.