Centre for Stem Cell Biology, Sheffield University, Sheffield S10 2TN, UK.
In Vitro Cell Dev Biol Anim. 2010 Apr;46(3-4):236-41. doi: 10.1007/s11626-010-9294-2. Epub 2010 Mar 12.
The conventional method for the derivation of human embryonic stem cells (hESCs) involves inner cell mass (ICM) co-culture with a feeder layer of inactivated mouse or human embryonic fibroblasts in an in vitro fertilisation culture dish. Growth factors potentially involved in primary derivation of hESCs may be lost or diluted in such a system. We established a microdrop method which maintained feeder cells and efficiently generated hESCs. Embryos were donated for stem cell research after fully informed patient consent. A feeder cell layer was made by incubating inactivated mouse embryonic fibroblasts (MEFs) feeder cells in a 50 microl drop of medium (DMEM/10% foetal calf serum) under mineral oil in a small tissue culture dish. MEFs formed a confluent layer and medium was replaced with human embryonic stem medium supplemented with 10% Plasmanate (Bayer) and incubated overnight. Cryopreserved embryos were thawed and cultured until the blastocyst stage and the zona pellucida removed with pronase (2 mg/ml; Calbiochem). A zona-free intact blastocyst was placed in the feeder microdrop and monitored for ES derivation with medium changed every 2-3 d. Proliferating hESCs were passaged into other feeder drops and standard feeder preparation by manual dissection until a stable cell line was established. Six hESC lines (Shef 3-8) were derived. From a total of 46 blastocysts (early to expanded), five hESC lines were generated (Shef 3-7). Shef 3-6 were generated on MEFs from 25 blastocysts. Shef7 was generated on human foetal gonadal embryonic fibroblasts from a further 21 blastocysts. From our experience, microdrop technique is more efficient than conventional method for derivation of hESCs and it is much easier to monitor early hESC derivation. The microdrop method lends itself to good manufacturing practice derivation of hESCs.
传统的人类胚胎干细胞（hESC）的分离方法涉及到将内细胞团（ICM）与失活的小鼠或人胚胎成纤维细胞的饲养层共培养，在体外受精培养皿中进行。在这种系统中，可能涉及到 hESC 初始分离的生长因子可能会丢失或稀释。我们建立了一种微滴方法，该方法维持了饲养细胞并有效地产生了 hESC。胚胎是在患者完全知情同意的情况下捐献用于干细胞研究的。将失活的小鼠胚胎成纤维细胞（MEF）饲养层在小组织培养皿中的 50 微升培养基（DMEM/10%胎牛血清）中的油滴中孵育，形成一层致密的饲养层。MEF 形成了一层致密的层，然后用含有 10%Plasmanate（拜耳）的人胚胎干细胞培养基替换培养基，并孵育过夜。冷冻保存的胚胎解冻并培养至囊胚阶段，用蛋白酶（2mg/ml；Calbiochem）去除透明带。将无透明带的完整囊胚放置在饲养层微滴中，并监测 ES 的衍生，每隔 2-3 天更换培养基。增殖的 hESC 通过手动分离传代到其他饲养层滴和标准饲养层制备中，直到建立稳定的细胞系。共衍生了 6 株 hESC 系（Shef3-8）。从总共 46 个囊胚（早至扩张期）中，生成了 5 株 hESC 系（Shef3-7）。Shef3-6 是从 25 个囊胚的 MEF 中生成的。Shef7 是从另外 21 个囊胚的人胎儿性腺胚胎成纤维细胞中生成的。根据我们的经验，微滴技术比传统方法更有效地分离 hESC，并且更容易监测 hESC 的早期衍生。微滴方法非常适合 hESC 的良好生产规范分离。
