Procedure for the Expansion of Human Mesenchymal Stem Cells: R&D Systems

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 (1, 2). MSCs are phenotypically characterized as CD34^-, CD105⁺, and CD90⁺ cells. This protocol describes the expansion of human MSCs using the StemXVivo™ MSC Expansion Media (R&D Systems, Catalog # CCM004).

Please read the protocol in its entirety before starting.

Supplies Required

Reagents

Bone marrow derived MSCs
StemXVivo Mesenchymal Stem Cell Expansion Media (R&D Systems, Catalog # CCM004)
Penicillin-Streptomycin (100X)
Trypsin-EDTA (10X)
Phosphate-Buffered Saline (PBS)

Materials

75 cm² tissue culture flasks
15 mL centrifuge tubes
Serological pipettes
Pipettes and pipette tips

Equipment

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

Reagent & Media Preparation

Note: Sterile technique is required when handling the reagents.

StemXVivo Mesenchymal Stem Cell (MSC) Expansion Media - Thaw the StemXVivo MSC Expansion 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 MSC Expansion Media - Add Penicillin-Streptomycin to the StemXVivo MSC Expansion Media at a 1:100 dilution.
Note: If Penicillin-Streptomycin is not needed for the experiment, it can be omitted.

Trypsin-EDTA (1X) - Dilute 10X Trypsin-EDTA to 1X using PBS.

Procedure

Culturing of Mesenchymal Stem Cells

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

Pre-warm the Completed StemXVivo MSC Expansion Media in a 37° C water bath. This procedure uses 20 mL for each T75 flask used.
Resuspend 3.5 - 4.0 x 10⁵ cells in 20 mL of the pre-warmed Completed StemXVivo MSC Expansion Media.
Note: If using a different size tissue culture vessel, seed cells at approximately 5000 cells/cm²/0.2 - 0.3 mL of media.
Add this cell suspension to a T75 flask.
Every three days remove and discard spent media and replace with 20 mL of pre-warmed Completed StemXVivo MSC Expansion Media.
Note: Dispense media down the side of the flask so as not to disrupt cells.
Subculture when cells become 80 - 90% confluent. Do not let the cultures become totally confluent.

Subculturing of Mesenchymal Stem Cells

In a 37° C water bath, pre-warm 30 mL of Completed StemXVivo MSC Expansion Media and 2 mL of 1X Trypsin-EDTA for each T75 flask used.
Remove and discard the media from the flasks. Wash the cells twice with 10 mL of PBS.
Note: Do not dispense the PBS directly onto the cells during washing so as not to disrupt the cells.
Add enough 1X Trypsin-EDTA to just cover the cells. Gently rock the flask to disperse the Trypsin-EDTA solution evenly over the cells.
Incubate the flask at 37° C, monitoring periodically for cell detachment by observing the cells under the microscope. Cells will start to round and detach. Tap the side of the flask to aid the detachment of the cells. This process should take 5 - 10 minutes.
Add 5 mL of pre-warmed Completed StemXVivo MSC Expansion Media to the flask to neutralize the Trypsin-EDTA. Disperse the cells by pipetting the media over the entire growing surface of the flask.
Transfer the cells to a 15 mL conical tube and centrifuge at 400 x g for 5 minutes.
Resuspend the cell pellet in a small amount of pre-warmed Completed StemXVivo MSC Expansion Media and count the cells with a hemocytometer.
If further expansion is desired, resuspend 3.5 - 4.0 x 10⁵ cells into 20 mL of the pre-warmed Completed StemXVivo MSC Expansion Media for each T75 flask.

Results

	Figure 1. Morphology of human MSCs cultured with StemXVivo Mesenchymal Stem Cell Expansion Media at 90% confluency.

	Figure 2. Phenotypic analysis of human MSCs expanded in StemXVivo MSC Expansion Media. Cells were stained with PE-conjugated Mouse Anti-Human CD34 Monoclonal Antibody (Catalog # FAB7227P), PE-conjugated Mouse Anti-Human CD105 (Catalog # FAB10971P), and PE-conjugated Mouse Anti-Human CD90 Monoclonal Antibody (Catalog # FAB2067P) antibodies (open histogram). Cells were analyzed using a Becton Dickinson FACSCalibur™ flow cytometer. For each antibody, isotype-matched controls are shown with a filled histogram.