Department of Medical Biochemistry and Biophysics, Division of Matrix Biology, Karolinska Institute, Stockholm, Sweden.
Department of Clinical Sciences, Division of Obstetrics and Gynecology, Intervention and Technology, Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden.
Nat Protoc. 2014 Oct;9(10):2354-68. doi: 10.1038/nprot.2014.159. Epub 2014 Sep 11.
A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here, we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform, under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm², where they attach, migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521, in combination with E-cadherin, allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities.
一种在化学定义和无动物源条件下培养人类多能干细胞（hPS）的稳健方法是干细胞研究和再生医学发展的重要工具。在这里，我们描述了一种在无动物源和化学定义条件下，使用特定层粘连蛋白-521（LN-521）异构体单层培养 Oct-4 阳性 hPS 细胞的方案。将细胞分散成单细胞悬液，然后以高于每平方厘米 5,000 个细胞的密度接种到 LN-521 异构体上，在那里它们通过形成小的单层细胞群附着、迁移和存活。细胞会旺盛分裂和扩展，直到整个培养皿被铺满。LN-521 与 E-钙黏蛋白结合，可在无 rho 相关蛋白激酶（ROCK）抑制剂或任何其他抗失巢凋亡抑制剂的情况下，将单个 hPS 细胞克隆到 96 孔板的单独孔中。对培养数月的细胞进行表征显示，其具有多能性，遗传异常程度最小。
