Vila-Cejudo Marta, Ibañez Elena, Santalo Josep
Unitat de Biologia Cellular, Facultat de Biociències, Universitat Autònoma de Barcelona.
Unitat de Biologia Cellular, Facultat de Biociències, Universitat Autònoma de Barcelona;
J Vis Exp. 2017 Aug 20(126):56171. doi: 10.3791/56171.
Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.
小鼠胚胎干细胞（mESC）的分离是从植入前胚胎建立多能细胞系的过程。这些细胞系在特定条件下保留自我更新或分化的能力。由于这些特性，mESC在再生医学、疾病建模和组织工程研究中是一种有用的工具。本文描述了一种简单的方案，通过在添加白血病抑制因子的限定培养基中，将来自允许性小鼠品系的囊胚培养在饲养细胞上，以获得高分离效率（60 - 80%）的mESC系。通过在分离培养基（2i培养基）中简单添加两种小分子抑制剂的混合物，该方案也可有效地从非允许性小鼠品系中分离出mESC系。文中提供了饲养细胞的制备和培养、小鼠胚胎的收集和培养以及mESC系的分离和培养的详细步骤。该方案不需要专门的设备，任何具备基本哺乳动物细胞培养专业知识的实验室都可以进行。
