StemXVivo™ Procedure for the Adipogenic 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 Adipogenic Supplement (R&D Systems, Catalog # CCM011)
• 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
• 37° C and 5% CO₂ humidified incubator
• Centrifuge
• Hemacytometer
• Inverted Microscope
• 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 MediaAdd 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 Adipogenic Differentiation Media - If a precipitate forms, warm the Adipogenic Supplement vial in a 37° C water bath for 5 minutes. Vortex until the precipitate dissolves. Add StemXVivo Adipogenic Supplement to the completed StemXVivo Osteogenic/Adipogenic Base Media at a 1:100 dilution.

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 1 x 10⁶ 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 2.1 x 10⁴ cells/cm².
3. Add this cell suspension to a 10 cm tissue culture dish. Incubate overnight in a 37° C and 5% CO₂ incubator. Cells should be 100% confluent after overnight incubation. If they are not confluent, replace medium every 2 - 3 days with Osteogenic/Adipogenic Base Media until 100% confluency is reached.
4. At 100% confluency, replace the media with 10 mL of pre-warmed completed StemXVivo Adipogenic Differentiation Media to induce adipogenesis.
5. Every 3 - 4 days remove and discard spent media and replace with 10 mL of freshly prepared, pre-warmed completed StemXVivo Adipogenic Differentiation Media.
   Note: Differentiation is complete after 7 - 21 days, at which time adipogenic induced cells will have morphological changes and lipid vacuoles (Figure 1).

Sample Data

Morphology of differentiated MSCs cultured with StemXVivo Osteogenic/Adipogenic Base Media with StemXVivo Adipogenic Supplement
Figure 1. MSCs differentiated to adipocytes for 21 days and stained with Oil Red. For staining protocol, please see reference 3.	Figure 2. MSCs differentiated to adipocytes for 21 days. FABP4 was detected using R&D Systems goat anti-mouse FABP4 polyclonal antibody (Catalog # AF1443). Cells were stained using Rhodamine Red-conjugated anti-goat secondary antibody.

References

1. Gronthos, S. et al. (1995) Blood 85:929.
2. Pittenger, M.F. et al. (1999) Science 284:143.
3. Colter, D.C. et al. (2001) Proc. Natl. Acad. Sci. USA 98:7841.