Generation of Sheffield (Shef) human embryonic stem cell lines using a microdrop culture system

被引:33
作者
Aflatoonian, Behrouz [1 ]
Ruban, Ludmila [1 ]
Shamsuddin, Shamsul [1 ]
Baker, Duncan [1 ]
Andrews, Peter [1 ]
Moore, Harry [1 ]
机构
[1] Univ Sheffield, Ctr Stem Cell Biol, Sheffield S10 2TN, S Yorkshire, England
基金
英国医学研究理事会;
关键词
Blastocyst; Derivation; Differentiation; Inner cell mass; Microdrop system; Open system; Human embryonic stem cells (hESCs); BLASTOCYST CULTURE; MASS ISOLATION; DERIVATION; EFFICIENCY;
D O I
10.1007/s11626-010-9294-2
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
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 mu l 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.
引用
收藏
页码:236 / 241
页数:6
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