Introduction

MSCs comprise a mere 0.01–0.0001% of total bone marrow nucleated cells and need in vitro cell culture expansion in order to get sufficient numbers for clinical applications. MesenPRO RS™ Medium is a reduced serum (2%) medium specifically formulated to support the growth of human mesenchymal stem cells (MSCs) in culture. MesenPRO RS™ Medium helps improve MSC expansion compared with conventional serum-supplemented media while maintaining a comparable gene expression profile. Using MesenPRO RS™ Medium, MSCs can be expanded for multiple passages while maintaining their multipotential phenotype (i.e., differentiation into osteogenic, chondrogenic, and adipogenic lineages).

Reducing the concentration of FBS to 2% in a highly consistent formula produced under cGMP conditions helps reduce the variability typically introduced by the 10–20% FBS used in classical media. There also are many unknown elements in FBS, such as signaling molecules, apoptotic factors, and nutrients. The variable concentrations of these components can cause lot-to-lot variation, which means that some FBS lots do not support MSC culture. When used to recover cryopreserved MSCs, our MSC-Qualified FBS (Ng et al., 2008, Kuçi, et al., 2010) minimizes the need to test multiple FBS lots to identify the optimal one for your use.

MesenPRO RS™ Medium (Sugii et al., 2010, Ng et al., 2008, Eibes et al., 2010, Tsigkou et al., 2010, Schraufstatter et al., 2009, Carreras et al., 2009) helps improve expansion of MSCs (Figure 1) compared with classical media (DMEM + 10% FBS) and maintains trilineage mesoderm differentiation potential (Figure 2).

Figure 1. MesenPRO RS™ Medium provides a 69–169% improvement in expansion over 6 days, compared to classical media. MSCs were isolated from normal human bone marrow mononuclear cells by standard techniques. Early-passage cells were plated into 6-well plates at either 3 × 103 or 5 × 103 cells/cm2 in DMEM (low glucose) containing 4 mM L-glutamine, 5 μg/mL gentamicin, and 10% MSC-Qualified FBS; or MesenPRO RS™ Medium containing 2 mM L-glutamine and 5 μg/mL gentamicin. Cells were incubated at 37°C with 5% CO2 in humidified air and fed on day 3. On day 6, cells were harvested using TrypLE™ reagent and counted. Data represent cell count averages from duplicate wells (p ≤ 0.007 and p ≤ 0.002, respectively, by Student’s t-test).

Figure 2. Human MSCs cultured in MesenPRO RS™ Medium retain trilineage differentiation potential. Human MSCs cultured in MesenPRO RS™ Medium were seeded into adipogenic, chondrogenic, or osteogenic differentiation medium for 14 days, revealing (A) adipocytes (oil red O lipid stain), (B) chondrocytes (alcian blue glycosaminoglycan stain), and (C) osteoblasts (alkaline phosphatase stain).

Visit www.lifetechnologies.com/mesenpro to learn more.

Materials Needed

  • MesenPRO RS™ Medium  (Cat. no. 12746-012) consists of MesenPRO RS™ Basal Medium and MesenPRO RS™ Growth Supplement. The medium is also available as MesenPRO RS™ (New Zealand origin) kit under Cat. no. E07-1000. (PDF)
  • L–Glutamine-200mM (100X), Liquid  (Cat. nos. 25030-149, 25030-164) or CTS™ GlutaMAX™-I Supplement  (Cat. no. A12860-01)
  • Human MSCs (GMP) (StemPro® BM Mesenchymal Stem Cells  (Cat. No. A15652) or human ADSCs (StemPro®Human Adipose-Derived Stem Cells  (Cat. no. R7788-115)
  • CTS™ (Cell Therapy Systems) DPBS, without calcium chloride, without magnesium chloride  (Cat. no. A12856-01)
  • Countess® Automated Cell Counter  (Cat. no. C10227)
  • 37°C water bath
  • Appropriate tissue culture vessels and supplies

For recovery only of cryopreserved MSCs:

  • Dulbecco’s Modified Eagle Medium (DMEM) [DMEM/F-12, GlutaMAX™  (Cat. nos. 10565-018, 10565-042) or DMEM, low glucose, pyruvate, no glutamine, no phenol red  (Cat. no. 11054-001)] with 10% Fetal Bovine Serum, MSC-Qualified (USDA Approved)  (Cat. no. 12662-002). The MSC-qualified medium is also available as MSC-Qualified FBS Australia (500 mL, Cat. no. 12664-025), MSC-Qualified FBS New Zealand (100 mL, Cat. no. 12665-014) (PDF), and MSC-Qualified FBS New Zealand (500 mL, Cat. no. 12665-022) (PDF).

For passaging and cryopreservation of cells:

  • CTS™ TrypLE™ Select Enzyme  (Cat. no. A12859-01)
  • Optional: StemPro® Accutase® Cell Dissociation Reagent  (Cat. No. A1110501)
  • Dimethyl sulfoxide (DMSO)  (Cat. no. D12345)

Preparing Media and Materials

MesenPRO RS™ Medium for Recovering and Culturing MSCs (500 mL of complete medium)

  1. Thaw MesenPRO RS™ Growth Supplement at 2–8°C overnight, use immediately once thawed. Do not thaw at 37°C. Avoid repeated freeze/thaw cycles of MesenPRO RS™ Growth Supplement.
  2. To prepare 500 mL of complete MesenPRO RS™ Medium, aseptically mix the following components:
    • MesenPRO RS™ Basal Medium                 485 mL
    • MesenPRO RS™ Growth Supplement        10 mL
    • 200 mM L-glutamine or GlutaMAX™-I          5 mL    (2 mM final concentration)
  3. Complete, supplemented MesenPRO RS™ Medium protected from light can be stored at 2–8°C for up to 2 weeks.

Note: Before use, warm complete medium required for that day at room temperature until it is no longer cool to the touch. Do not warm the medium at 37°C.

Recovery of Cryopreserved MSCs

Note: We recommend that you recover MSCs into complete MesenPRO RS™ Medium from thaw*.

*You can use conventional medium for recovery of MSCs. To prepare 500 mL of complete serum-supplmented DMEM medium, thaw Fetal Bovine Serum, MSC-Qualified (USDA Approved) at 2–8°C

  1. Rapidly thaw (<1 minute) frozen cells in a 37°C water bath.
  2. Pipet the entire contents of the cryovial into a sterile 50-mL conical tube.
  3. Carefully, by dropwise addition (2–3 drops per 10 seconds), add 5–7 mL of prewarmed complete MesenPRO RS™ Medium while gently swirling the tube.
  4. Add a further 5 mL prewarmed complete MesenPRO RS™ Medium while gently swirling the tube.
  5. Transfer all the contents of the conical tube into an appropriate tissue culture flask.
  6. Incubate at 37°C in a humidified atmosphere of 5% CO2 in air.
  7. Exchange spent medium with fresh prewarmed complete MesenPRO RS™ Medium 24 hours post-thaw.
    Note: For recovery of MSCs, it is recommended to seed cells at ≥7 × 103 cells/cm2 for the initial recovery passage.

Subculturing MSCs

MesenPRO RS™ has been developed for the culturing of human mesenchymal stem cells that have been initiated using standard adherent isolation and growth conditions (e.g., DMEM + 10% FBS) and has not been tested for the initial expansion of MSCs directly from primary tissue sources. MesenPRO RS™ has been developed for culturing MSCs at greater than clonal densities (see step 9).

It is recommended to subculture human mesenchymal stem cells directly into MesenPRO RS™ complete medium. It is critical that cell confluency be 60–80%, cell viability be at least 90%, and the growth rate be in mid-logarithmic phase prior to sub culturing.

Note: The following procedures apply to adherent cultures in a T-75 culture flask (75 cm2). Volumes should be adjusted accordingly for desired vessel size.

  1. Observe culture flask on inverted microscope and confirm that the cells are ready to be subcultured (60–80% confluent).
  2. Aspirate medium and floating cells from a confluent monolayer and discard.
  3. Wash cells with 5–10 mL Dulbecco's Phosphate Buffered Saline (DPBS), without calcium, magnesium, or phenol red.
  4. Remove DPBS and add 5–7 mL of prewarmed CTS™ TrypLE™ Select Enzyme to the culture flask. Incubate at 37˚C until cells have fully detached (approximately 3–5 minutes).
  5. Observe cell monolayer using an inverted microscope to ensure complete detachment from the surface of the flask.
  6. Stop cell dissociation by adding 10 mL of prewarmed complete MesenPRO RS™ Medium to flask. Gently pipet up and down to disperse cells into a single-cell suspension.
  7. Transfer cell suspension into a sterile conical tube. Wash flask with an additional 5 mL complete MesenPRO RS™ Medium and combine into the conical tube.
  8. Centrifuge cell suspension at 100 × g for 5–10 minutes.
  9. Aspirate supernatant and resuspend the pellet in an appropriate volume of prewarmed complete MesenPRO RS™ Medium.
  10. Determine total viable cell density using a Countess® Automated Cell Counter (alternative automated or manual methods may be used). methods may be used).
  11. Inoculate flask at 3–5 × 103 viable cells/cm2 and return to incubator.

Note: For optimal performance and cell growth, cultures should be re-fed every 3–4 days with fresh complete medium.

Cryopreservation of MSCs

  1. Prepare a 2X cryopreservation solution on the day of use:

    a). Prepare 10 mL of 2X cryopreservation solution by supplementing MesenPRO RS™ complete medium with 20% Dimethyl Sulfoxide (DMSO). Store at 4°C until use.

    • MesenPRO RS™ complete medium       8 mL
    • DMSO      2 mL    (20% final concentration)

  2. Harvest cells for cryopreservation using the MesenPRO RS™ complete medium according to steps 1 through 9 of Subculturing MSCs.
  3. Reconstitute the harvested cell pellet to twice the desired final cell concentration (i.e., 2 × 106 cells/mL) in 0.5 mL of pre-warmed MesenPRO RS™ complete medium.
  4. Slowly add 0.5 mL of the 2X cryopreservation solution to the cell suspension, and gently mix to ensure even cell distribution.
  5. Immediately add the desired volume of cell suspension (i.e., 1 mL) to pre-chilled (2°C to 8°C) cryovials.
  6. Place the cryovials at –70°C in a cryogenic freezing container (e.g., Mr. Frosty® (1°C) Freezing Container).
  7. After 24 hours, transfer the frozen cells to liquid nitrogen (vapor phase); storage at –200°C to 125°C is recommended.

References

  1. Sugii S et al. (2010) Human and mouse adipose-derived cells support feeder-independent induction of pluripotent stem cells. Proc Natl Acad Sci U S A 107(8):3558–3563.
  2. Ng F et al. (2008) PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages. Blood 112(2):295–307.
  3. Eibes G et al. (2010) Maximizing the ex vivo expansion of human mesenchymal stem cells using a microcarrier-based stirred culture system. J Biotechnol 146(4):194–197.
  4. Tsigkou O et al. (2010) Engineered vascularized bone grafts. Proc Natl Acad Sci U S A 107(8):3311–3316.
  5. Schraufstatter IU et al. (2009) C3a and C5a are chemotactic factors for human mesenchymal stem cells, which cause prolonged ERK1/2 phosphorylation. J Immunol 182(6):3827–3836.
  6. Kuçi S et al. (2010) CD271 antigen defines a subset of multipotent stromal cells with immunosuppressive and lymphohematopoietic engraftment-promoting properties. Haematologica 95(4):651–659.
  7. Carreras A et al. (2009) Obstructive apneas induce early release of mesenchymal stem cells into circulating blood. Sleep 32(1):117–119.

 

Last update: 27 Feb 14