体外裸鼠内成骨模型实验步骤.docVIP

Category by field:Stem cell Adult stem cell Mesenchymal stem cellCategory by organism:Mammalia Murine Circulatory Cell-based analysis In Vivo Heterotopic Bone Formation Assay Using Mouse or Human Mesenchymal Stem Cells Li Chen* and Nicholas Ditzel Molecular Endocrinology Laboratory (KMEB), Department of Endocrinology, Odense University Hospital, DK-5000 Odense, Denmark *corresponding author: Li Chen, lchen@health.sdu.dk [Abstract] Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types: osteoblasts (bone cells), chondrocytes (cartilage cells), and adipocytes (fat cells). Seeding exogenous (human or mouse) MSCs in HA scaffold and transplant subcutaneously in immune-deficient mice, the cells can finally form bone tissues in the in vivo environment. Detection and calculation the new bone formation ratios can check the bone formation ability of MSCs. This protocol describes how to get heterotopic bone formation in mice by mesenchymal stem cells (MSCs) in hydroxyapatite (HA) scaffolds. This is a simple and robust approach to detect the bone formation by tissue engineering approaches in vivo, and it also fits for examining the roles of different factors in bone formation with MSCs. Materials and Reagents Human or mouse mesenchymal stem cells (cultured, treated or transfected). Note: If using the primary bone marrow stem cells (we took the attached cells from bone marrow culturing), it’s better to use cells in low passages (not over passage 3); if using MSC cell lines, it’s also better to use low passages than high passages. Mice [NOD.CB17-Prkdcscid/J mice (NOD/SCID), around 8 weeks old] (The Jackson Laboratory) Hydroxyapatite/tricalcium phosphate (HA/TCP) sca 4% paraformaldehyde (Sigma, any from local companies should be fine, too) Hematoxylin/Eosin (H E) staining solution (Sigma) Formic acid solution (see Recipes) Equipment 1 ml syringes Cotton sticks (Pro-Ophtha) Racks (for fixing the syringes in vertica