A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone

• Published in: Nature protocols Vol. 5; no. 3; pp. 550 - 560
• Main Authors: Zhu, Heng, Guo, Zi-Kuan, Jiang, Xiao-Xia, Li, Hong, Wang, Xiao-Yan, Yao, Hui-Yu, Zhang, Yi, Mao, Ning
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.03.2010; Nature Publishing Group
• Subjects: 631/1647/1407/651; 631/1647/2230; 631/1647/334/1874/345; 631/532/2074; Analytical Chemistry; Animals; Biological Techniques; Biomedical and Life Sciences; Bone marrow; Bone Marrow Cells - cytology; Cell Adhesion; Cell Culture Techniques - methods; Cell Differentiation; Cell Separation - methods; Cells, Cultured; Collagenases; Compact bone; Computational Biology/Bioinformatics; Female; Flushing; Hematopoietic Stem Cells - cytology; Identification and classification; Life Sciences; Male; Mesenchymal Stromal Cells - cytology; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Microarrays; Multipotent Stem Cells - cytology; Organic Chemistry; Physiological aspects; protocol; Stem cells; China
• ISSN: 1754-2189; 1750-2799; 1750-2799
• DOI: 10.1038/nprot.2009.238

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.