Procedure for Serum-Free 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 CD105⁺, CD90⁺, CD73⁺, CD44⁺, CD34^-, CD45^-, and CD11b^- cells. This protocol describes the expansion of human MSCs using the StemXVivo™ Serum-Free Human MSC Expansion Media (R&D Systems, Catalog # CCM014).

Please read the protocol in its entirety before starting.

Supplies Required

Reagents

Bone marrow derived human MSCs
StemXVivo Serum-Free Human Mesenchymal Stem Cell Expansion Media (R&D Systems, Catalog # CCM014)
Recombinant Human Fibronectin (R&D Systems, Catalog # 4305-FNB)
Penicillin-Streptomycin (100X)
TrypLE™ Express (Invitrogen^®, Catalog # 12604-013 or equivalent)
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 Serum-Free Human Mesenchymal Stem Cell (MSC) Expansion Media - Thaw the StemXVivo Serum-Free Human 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.
Note: If needed, Penicillin-Streptomycin can be added to StemXVivo Serum-Free Human MSC Expansion Media at a 1:100 dilution.

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.

Before plating the cells, coat plates with recombinant human Fibronectin. Gently dilute the Fibronectin in PBS to a final concentration of 5 µg/mL. Coat the T75 flask by adding 6 mL of Fibronectin solution to the flask, and incubate overnight at 2 - 8° C or for 3 hours at room temperature.
Note: Mesenchymal stem cells grown in StemXVivo Serum-Free Human MSC Expansion Media must be on extracellular matrix (ECM) protein coated plates. Types and amounts of ECM protein are dependent on the experimental design of each individual investigator.
Pre-warm the required amount of StemXVivo Serum-Free Human MSC Expansion Media at room temperature. This procedure uses 20 mL for each T75 flask used.
Resuspend 4.5 - 5.0 x 10⁵ cells in 20 mL of the pre-warmed media.
Note: If using a different size tissue culture vessel, seed cells at approximately 6000 cells/cm²/0.2 - 0.3 mL of media.
Gently pipette off Fibronectin solution from the flask. Slowly add the cell suspension to a T75 flask. Be careful to avoid scraping the coated surface.
Incubate the cells at 37° C and 5% CO₂ in a humidified atmosphere. Every 2 - 3 days remove and discard spent media and replace with 20 mL of pre-warmed StemXVivo Serum-Free Human 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

Pre-coat the plates with 5 µg/mL of recombinant human Fibronectin in PBS overnight at 2 - 8° C or for 3 hours at room temperature.
At room temperature, pre-warm 30 mL of StemXVivo Serum-Free Human MSC Expansion Media and 3 mL of TrypLE Express for each T75 flask used.
Remove and discard the media from the flasks. Wash the cells once with 10 mL of PBS for each T75 flask.
Note: Do not dispense the PBS directly onto the cells during washing so as not to disrupt the cells.
Add enough TrypLE Express to just cover the cells. Gently rock the flask to disperse the TrypLE Express evenly over the cells; 3 mL for each T75 flask.
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.
Add 5 mL of StemXVivo Serum-Free Human MSC Expansion Media to the flask. 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. Aspirate off the liquid.
Resuspend the cell pellet in a small amount of pre-warmed StemXVivo Serum-Free Human MSC Expansion Media and count the cells with a hemacytometer.
Resuspend 4.5 - 5.0 x 10⁵ cells into 20 mL of the pre-warmed StemXVivo Serum-Free Human MSC Expansion Media for each Fibronectin coated T75 flask.
Gently pipette off Fibronectin solution from the flask, and slowly add the cell suspension to a T75 flask. Be careful to avoid scraping the coated surface. Incubate the cells at 37° C and 5% CO₂ in a humidified atmosphere.

Results

	Figure 1. Morphology of human MSCs cultured with StemXVivo Serum-Free Human Mesenchymal Stem Cell Expansion Media.






	Figure 2. Phenotypic analysis of human MSCs expanded in StemXVivo Serum-Free Human Mesenchymal Stem Cell Expansion Media for 3 passages. Cells were stained with PE-conjugated Mouse Anti-Human CD105 Monoclonal Antibody (Catalog # FAB10971P), PE-conjugated Mouse Anti-Human CD90 Monoclonal Antibody (Catalog # FAB2067P), PE-conjugated Mouse Anti-Human CD73 Monoclonal Antibody (Catalog # FAB5795P), PE-conjugated Mouse Anti-Human CD44 Pan Specific Monoclonal Antibody (Catalog # FAB4948P), PE-conjugated Mouse Anti-Human CD45 Monoclonal Antibody (Catalog # FAB1430P), PE-conjugated Mouse Anti-Human CD11b Monoclonal Antibody (Catalog # FAB16991P), and CD34 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.



CCM004 Serum Containing Medium	CCM014 Serum-Free Media
Adipocytes stained with FABP-4	Adipocytes stained with FABP-4
Osteoblasts stained with Osteocalcin	Osteoblasts stained with Osteocalcin
Chondrocytes stained with Aggrecan	Chondrocytes stained with Aggrecan

Figure 3. Differentiation of human MSCs expanded in StemXVivo Serum-Free Human Mesenchymal Stem Cell Expansion Media. Cells grown in serum-containing StemXVivo Mesenchymal Stem Cell Expansion Media (R&D Systems, Catalog # CCM004) or StemXVivo Serum-free Human Mesenchymal Stem Cell Expansion Media (R&D Systems, Catalog # CCM014) were differentiated using the Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Catalog # SC006). Adipocytes were stained with a Goat Anti-Mouse FABP-4 Antigen Affinity-purified Polyclonal Antibody (R&D Systems, Catalog # AF1443), osteocytes were stained with a Mouse Anti-Human Osteocalcin Monoclonal Antibody (R&D Systems, Catalog # MAB1419), and chondrocytes were stained with a Goat Anti-Human Aggrecan Antigen Affinity-purified Polyclonal Antibody (R&D Systems, Catalog # AF1220). Cells were then stained with NorthernLights™ 557-conjugated Goat IgG or NorthernLights^TM 557-conjugated Mouse IgG Secondary Antibodies (R&D Systems, Catalog # NL001 and NL007) and counterstained with DAPI (blue).