Chondrogenic Differentiation of Human/Mouse Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are functionally defined by their capacity to self renew and their ability to differentiate into multiple cell types including adipocytes, chondrocytes and osteocytes. This protocol describes the chondrogenic differentiation of MSCs using the StemXVivo^TM Human/Mouse Chondrogenic Base Media (Catalog # CCM005) and StemXVivo Human/Mouse Chondrogenic Supplement (Catalog # CCM006).

Figure 2. Chondrogenic Differentiation of MSCs. Mouse mesenchymal stem cell pellet differentiated in vitro for 21 days using the StemXVivoTM Human/Mouse Chondrogenic Base Media (Catalog # CCM005) and StemXVivo Human/Mouse Chondrogenic Supplement (Catalog # CCM006) was sectioned and stained for the chondrogenic lineage marker, Collagen II using a Sheep Anti-Mouse Collagen II Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3615) and a NorthernLights 557-conjugated Anti-Sheep Secondary Antibody (Catalog # NL010).

Please read the protocol in its entirety before starting.

Supplies Required

Reagents

• Bone marrow derived MSCs
• StemXVivo Human/Mouse Chondrogenic Base Media (Catalog # CCM005)
• StemXVivo Human/Mouse Chondrogenic Supplement (Catalog # CCM006)
• Penicillin-Streptomycin (100x)

Materials

• 15 mL centrifuge tubes
• Serological pipettes
• Pipettes and pipette tips
   

Equipment

• 37° C, 5% CO₂ humidified incubator
• Centrifuge
• Hemocytometer
• Water bath

Reagent & Media Preparation

Note: Sterile technique is required when handling the reagents.

• StemXVivo Chondrogenic Base Media - Thaw the StemXVivo Chondrogenic Base Media at 2 - 8° C or room temperature. Aliquot any unused thawed media and store at ≤ 20° C in a manual defrost freezer. Thawed media may be stored in the dark at 2 - 8° C for up to 1 month.
• Completed StemXVivo Chondrogenic Base Media - Add Penicillin-Streptomycin to the StemXVivo Chondrogenic Base Media at a 1:100 dilution.
  Note: If Penicillin-Streptomycin is not needed for the experiment, it can be omitted.
• Completed StemXVivo Chondrogenic Differentiation Media - Add StemXVivo Chondrogenic Supplement to the completed StemXVivo Chondrogenic Base Media at a 1:100 dilution.
  
  Note: This procedure will use 0.5 mL of completed StemXVivo Chondrogenic Differentiation Media for each 15 mL centrifuge tube.

Procedure (Figure 1)

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

1. Pre-warm 5 mL of the completed StemXVivo Chondrogenic Base Media and 0.5 mL of completed StemXVivo Chondrogenic Differentiation Media in a 37° C water bath.
2. Resuspend 2.5 x 10⁵ MSCs (for mouse, 1.25 x 10⁵ MSCs) in 5 mL of the pre-warmed completed StemXVivo Chondrogenic Base Media.
3. Centrifuge the cells at 200 x g for 5 minutes at room temperature. Remove the media and resuspend the cells with 0.5 mL of pre-warmed completed StemXVivo Chondrogenic Differentiation Media.
4. Centrifuge the cells at 200 x g for 5 minutes at room temperature. Do not remove the media. Loosen the cap of the tube to allow gas exchange and incubate upright at 37° C and 5% CO₂.
5. After 1 - 2 days the cell pellet will form a round ball approximately 1 - 2 mm in diameter. This pellet will remain about the same size for the entire culturing time.
6. Every 2 - 3 days remove and discard the spent media and replace with 0.5 mL of pre-warmed completed StemXVivo Chondrogenic Differentiation Media.
   Note: Use caution when removing the media to avoid aspirating the pellet.
7. Chondrogenic pellets can be harvested after 14 - 21 days in culture and used for the desired analysis.