StemXVivo™ Procedure for the Osteogenic Differentiation of Human/Mouse Mesenchymal Stem Cells

Introduction

Materials Required

• Bone marrow derived MSCs
• StemXVivo Human/Mouse Osteogenic/Adipogenic Base Media (R&D Systems, Catalog # CCM007)
• StemXVivo Human Osteogenic Supplement (R&D Systems, Catalog # CCM008) or StemXVivo Mouse Osteogenic Supplement (R&D Systems, Catalog # CCM009)
• Penicillin-Streptomycin 100X (Invitrogen, Catalog # 15070-063 or equivalent)
• 10 cm tissue culture dishes (Falcon, Catalog # 353003 or equivalent)
• 15 mL centrifuge tubes (Corning Costar, Catalog #430052 or equivalent)
• Serological pipettes
• Pipettes and pipette tips
• Water bath

Reagent Preparation

StemXVivo Osteogenic/Adipogenic Base Media - Thaw the StemXVivo Osteogenic/Adipogenic Base Media at 2 ‑ 8° C or room temperature. Thawed media may be stored in the dark at 2 ‑ 8° C for up to 1 month. Aliquot any unused thawed media and store at ≤ ‑20° C in a manual defrost freezer.

Completed StemXVivo Osteogenic/Adipogenic Base Media - Add Penicillin-Streptomycin to the StemXVivo Osteogenic/Adipogenic Base Media at a 1:100 dilution.
Note: If Penicillin-Streptomycin is not needed for the experiment, it can be omitted.

Completed StemXVivo Osteogenic Differentiation Media - Add StemXVivo Osteogenic Supplement to the completed StemXVivo Osteogenic/Adipogenic Base Media at a 1:20 dilution.
Note: The reagent and material preparation for mouse and human completed StemXVivo Osteogenic/Adipogenic Base Media and completed StemXVivo Osteogenic Differentiation Media is the same using either the mouse or the human Supplement.

Procedure

Note: When handling biohazard materials such as human cells, safe laboratory procedures should be followed and protective clothing should be worn.

1. Pre-warm the completed StemXVivo Osteogenic/Adipogenic Base Media in a 37° C water bath. This procedure uses 10 mL for each 10 cm tissue culture dish used.
2. Resuspend 2.3 - 2.5 x 10⁵ MSCs in 10 mL of the pre-warmed completed StemXVivo Osteogenic/Adipogenic Base Media.
   Note: If using another size tissue culture vessel, seed cells at approximately 4.2 x 10³ cells/cm²/0.2 - 0.3 mL media.
3. Add this cell suspension to a 10 cm tissue culture dish. The cells should be 50 - 70% confluent in 1 - 2 days.
4. At 50 - 70% confluency, replace the media with 10 mL of pre-warmed completed StemXVivo Osteogenic Differentiation Media to induce osteogenesis.
5. Every 3 - 4 days remove and discard spent media and replace with 10 mL of pre-warmed completed StemXVivo Osteogenic Differentiation Media.
   Note: Dispense media down the side of the dish so as not to disrupt cells.
6. After 2 - 3 weeks osteogenic induced cells will have morphological changes and calcium deposition (Figure 1).

Sample Data

Morphology of differentiated MSCs cultured with StemXVivo Osteogenic/Adipogenic Base Media with StemXVivo Osteogenic Supplement
Figure 1. MSCs differentiated to osteocytes for 21 days and stained with Alizarin Red.	Figure 2. MSCs differentiated to osteocytes for 21 days. Osteocalcin was detected using R&D Systems mouse anti-human Osteocalcin monoclonal antibody (Catalog # MAB1419). Cells were stained using Rhodamine Red-conjugated anti-mouse secondary antibody and counterstained using Fluoro Nissl (green).

References

1. Gronthos, S. et al. (1995) Blood 85:929.
2. Pittenger, M.F. et al. (1999) Science 284:143.