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Cells from W++ mouse bone marrow were cloned by limiting dilution to obtain the W-20-17 cell line. RefThies RS. Recombinant human bone morphogenetic protein-2 induces osteoblastic differentiation in W-20-17 stromal cells. Endocrinology 130: 1318-1324, 1992. PubMed: 1311236

Cells used in the W-20-17 assay should not be carried in continuous culture. A working cell bank should be prepared with approximately 5?x 10⁵ cell/mL. Cells should be thawed before each assay.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) increased alkaline phosphatase activity in W-20-17 cells in a dose-responsive manner without an effect on proliferation.?RefThies RS. Recombinant human bone morphogenetic protein-2 induces osteoblastic differentiation in W-20-17 stromal cells. Endocrinology 130: 1318-1324, 1992. PubMed: 1311236
Bone morphogenetic protein-3 (BMP-3) was unable to induce an osteogenic response in W-20-17 cells. RefThies RS. Recombinant human bone morphogenetic protein-2 induces osteoblastic differentiation in W-20-17 stromal cells. Endocrinology 130: 1318-1324, 1992. PubMed: 1311236?

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-053mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.

Subcultivation Ratio: 1:4 to 1:6
Medium Renewal: At subculture.

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a Manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.

Wang W, et al. Ski represses bone morphogenic protein signaling in Xenopus and mammalian cells. Proc. Natl. Acad. Sci. USA 97: 14394-14399, 2000. PubMed: 11121043

Thies RS. Recombinant human bone morphogenetic protein-2 induces osteoblastic differentiation in W-20-17 stromal cells. Endocrinology 130: 1318-1324, 1992. PubMed: 1311236

Alden TD, et al. In vivo endochondral bone formation using a bone morphogenetic protein 2 adenoviral vector. Hum. Gene Ther. 10: 2245-2253, 1999. PubMed: 10498255

Bahamonde ME, Lyons KM. BMP3: to be or not to be a BMP.. J. Bone Joint Surg. Am. 83-A: S56-S62, 2001. PubMed: 11263666

Standard Test Method for In Vitro Biological Activity of Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) Using the W-20 Mouse Stromal Cell Line. West Conshohocken, PA:ASTM International;ASTM Standard Test Method F 2131-02 (Reapproved 2007)e1.

Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.

Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.