Department of Cell Biology, Institute of Basic Medical Sciences, Beijing, China.
Nat Protoc. 2010 Mar;5(3):550-60. doi: 10.1038/nprot.2009.238. Epub 2010 Feb 25.
Unlike humans, mouse bone marrow-derived mesenchymal stem cells (MSCs) cannot be easily harvested by adherence to plastic owing to the contamination of cultures by hematopoietic cells. The design of the protocol described here is based on the phenomenon that compact bones abound in MSCs and hematopoietic cells exist in the marrow cavities and the inner interfaces of the bones. The procedure includes flushing bone marrow out of the long bones, digesting the bone chips with collagenase type II, deprivation of the released cells and culturing the digested bone fragments, out of which fibroblast-like cells migrate and grow in the defined medium. The entire technique requires 5 d before the adherent cells are readily passaged. Further identification assays confirm that these cells are MSCs. We provide an easy and reproducible method to harvest mouse MSCs that does not require depletion of hematopoietic cells by sorting or immunomagnetic techniques.
与人类不同，由于骨髓细胞的污染，鼠骨髓间充质干细胞（MSCs）不能简单地通过贴壁培养的方法获得。本实验方案的设计基于以下现象：致密骨中富含 MSCs，而造血细胞存在于骨髓腔和骨内界面。操作过程包括从长骨中冲洗骨髓，用 II 型胶原酶消化骨片，去除释放的细胞，培养消化后的骨碎片，其中成纤维样细胞在特定的培养基中迁移和生长。整个技术需要 5 天时间，然后才能容易地传代贴壁细胞。进一步的鉴定实验证实这些细胞是间充质干细胞。我们提供了一种简单且可重复的方法来获取鼠 MSCs，而不需要通过分选或免疫磁珠技术去除造血细胞。
