Introduction

Serum-Free, Xeno-Free Medium

StemPro MSC SFM XenoFree (Chase et al., 2013) has been developed for the growth and expansion of human mesenchymal stem cells (MSCs) and Adipose-derived Stem Cells (ADSCs) under completely serum-free and xeno-free conditions. Using StemPro MSC SFM XenoFree, human MSCs or ADSCs can be expanded for multiple passages while maintaining their multipotent phenotype (i.e., ability to differentiate into osteogenic, chondrogenic and adipogenic lineages) (see Appendix A).

Due to the low frequency of human MSCs in primary tissue, expansion of this stem cell population is critical and helps enable basic biological studies and clinical research. In addition, human MSCs can only be propagated a limited number of times, thereafter exhibiting reduced proliferation and differentiation potential. Expansion of human MSCs and adipose-derived stem cells (ADSCs) (Lindroos et al., 2009, Sugii, et al., 2010) in StemPro MSC SFM XenoFree is comparable to classical medium (DMEM + 10% MSC-Qualified FBS) in terms of morphology and growth characteristics (Figure 1).

Human-MSC-expansion-under-xeno-free-conditions

Figure 1. Human MSC expansion under xeno-free conditions. Human bone marrow–derived MSCs expanded in DMEM (low glucose) + 10% MSC-Qualified FBS, or in StemPro MSC SFM XenoFree + CTS™ CELLstart™ substrate–coated plates, revealed a similar expansion rate. (A) Morphology of expanded (passage 3) human MSCs (10x objective). (B) Net expansion of human MSCs. Input human MSCs = passage 5, 4-donor pool. Passage frequency = every 4–6 days. Seed density = 5–7 x 103 cells/cm2. Harvest enzyme = TrypLE™ Express. Counting method = Countess Automated Cell Counter.

StemPro MSC SFM XenoFree offers a completely xeno–free system when used in conjunction with CTS CELLstart substrate; thus cells are grown in a physiological environment that allows for clinically relevant results.

Serum-Free Medium

CTS™ StemPro MSC SFM is a serum-free medium specially formulated for the growth and expansion of human mesenchymal stem cells (MSC) or adipose-derived stem cells (ADSC). Using CTS™ StemPro MSC SFM, human MSC or ADSC can be expanded for multiple passages while maintaining their trilineage mesoderm potential (i.e., ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages) (see Appendix A). CTS™ StemPro MSC SFM is intended for human ex vivo tissue and cell culture processing applications.

StemPro MSC SFM (Chase et al., 2010, Agata et al., 2009, Ng et al., 2008) provides superior efficiency of human MSC expansion (Figure 2) at high cell densities, requiring less medium, surface area, and time compared with classical medium (DMEM (low glucose) + 10% FBS). While human MSCs grown in classical medium have a flattened cell morphology and reach confluency at 1.0–3.0 x 104 cells/cm2, human MSCs grown in StemPro MSC SFM have a much smaller, spindle-shaped morphology and can reach densities of >1.0 x 105 cells/cm2 (see Appendix A).

hMSCs

Figure 2. hMSCs grown on CTS CELLstart substrate–coated dishes in StemPro MSC SFM exhibit a 166% improvement in expansion over 10 passages, compared to classical medium. Average net total cell number per T25 flask was calculated for human MSCs growing in StemPro MSC SFM or classical medium (n = 3). The culture had a seed density of 1 x 104 cells/cm2, a split frequency of 3 days, and a medium change every 2 days.

Table 1. Choosing the right MSC culture system for your research needs

MSC Culture system  Reduced-serum*Serum-free*Xeno-free, serum-free*
Supports MSC derivation from primary tissueyesyesyes**
Cell or tissue sourcebone marrow, adipose, cord blood, placentabone marrow, adipose, cord blood, placentabone marrow, adipose, cord blood, placenta
Specieshuman, mouse, rat, sheep, goat, pig, horsehumanhuman
Maintains MSC phenotypeyesyesyes
Supports trilineage differentiationyesyesyes
Offers enhanced chondrogenesisyesyesyes
Produced under cGMP-manufacturing standardsyesyesyes
Offers lot-to-lot consistencyyesyesyes
Free of animal components  yes
Basal mediaMesenPRO RS Basal MediumCTS StemPro MSC SFM Basal MediumStemPro MSC SFM Basal Medium
SupplementsMesenPRO RS Growth Supplement
CTS GlutaMAX-I Supplement [2 mM L-glutamine]		CTS StemPro MSC SFM Supplement
CTS GlutaMAX-I Supplement [2 mM L-glutamine]		StemPro MSC SFM XenoFree Supplement
CTS GlutaMAX-I Supplement [2 mM L-glutamine]
Extracellular matricesNot requiredCTS CELLstart Substrate, FibronectinCTS CELLstart Substrate, Fibronectin
Supports growth at high cell densityyesyesyes
Seeding density3–5 x 103 cells/cm2≥ 5 x 103 cells/cm2≥ 1 x 104 cells/cm2
Feeding schedule3–4 days2-3 days2-3 days
Passage schedule4-7 days3-4 days3-4 days
PassagingTrypLE, trypsin, StemPro AccutaseTrypLE, trypsin, StemPro AccutaseTrypLE
Cryopreservation MediumMesenPRO RS Medium + 10% DMSOCTS StemPro MSC Serum-Free Medium + 10% DMSO or CTS Synth-a-Freeze MediumStemPro MSC SFM XenoFree Medium + 10% DMSO or CTS Synth-a-Freeze Medium

Materials Needed

For Either Culture and Isolation from Bone Marrow

  • Human MSCs (GMP) (StemPro BM Mesenchymal Stem Cells (Cat. No. A15652) or human ADSCs (StemPro Human Adipose-Derived Stem Cells (Cat. no. R7788-115)
  • L–Glutamine-200mM (100X), Liquid (Cat. nos. 25030-149, 25030-164) or CTS™ GlutaMAX™-I Supplement (Cat. no. A12860-01)
  • 37°C water bath
  • Appropriate tissue culture vessels and supplies
  • Cryogenic storage vessels and supplies
  • Countess™ II or Countess™ II FL Automated Cell Counter
  • CTS™ CELLstart™ Substrate (Cat. no. A10142-01)
  • Dimethyl sulfoxide (DMSO) (Cat. no. D12345)
  • CTS™ DPBS, calcium, magnesium (Cat. no. A12858-01)
  • CTS™ (Cell Therapy Systems) DPBS, without calcium chloride, without magnesium chloride (Cat. no A12856-01)
  • Gentamicin Reagent Solution (10 mg/mL) (Cat. no. 15710-072)
  • CTS™ TrypLE™ Select Enzyme (Cat. no. A12859-01)
  • CTS™ Synth-a-Freeze Medium (Cat. No. A13713-01)

For Serum-Free, Xeno-Free Cultures

  • StemPro MSC SFM XenoFree (Cat. no. A10675-01) consists of StemPro MSC SFM Basal Medium and
  • StemPro MSC SFM XenoFree Supplement
  • Gentamicin (50 mg/mL), liquid (Cat. no. 15750-060)

For Serum-Free Cultures

  • CTS™ StemPro MSC SFM (Cat. no. A10332-01) consists of CTS™ StemPro MSC SFM Basal Medium and CTS™ StemPro MSC SFM Supplement

For Isolating MSCs from Bone Marrow

  • Human bone marrow aspirate
  • HBSS, calcium, magnesium, no phenol red (Cat. no. 14025-134)
  • Heat-inactivated and pooled AB-Human Serum (Cat. no. 34005-100)
  • ACK Lysing Buffer (Cat. no. A10492-01)
  • Gradient density media (e.g., Ficoll-Paque® PREMIUM 1.073 from GE Healthcare
  • Trypan Blue Solution, 0.4% (Cat. no. 15250-061)

Preparing Media and Materials

StemPro MSC SFM XenoFree Complete Medium for Serum-Free, Xeno-Free Cultures (500 mL of complete medium)

  1. Thaw StemPro MSC SFM XenoFree Supplement at 2–8°C overnight (thawed supplement will have a slightly cloudy appearance). Do not thaw at 37°C. Use thawed material immediately or aliquot (i.e., 1 mL) unused material and store at –20°C to –5°C. Avoid additional freeze-thaw cycles.
    Note: When stored frozen at –20°C to –5°C, the StemPro MSC SFM XenoFree Supplement is stable for 12 months.
  2. To prepare 500 mL of complete StemPro MSC SFM XenoFree Medium, aseptically mix the following components:

     

    StemPro MSC SFM Basal Medium 490 mL
    StemPro MSC SFM XenoFree Supplement 5 mL
    CTS™ GlutaMAX™-I Supplement or 200 mM L-Glutamine 5 mL (2 mM final concentration)
    Optional: Gentamicin (50 mg/mL), Liquid 50 µL (5 µg/mL final concentration)
  3. Complete StemPro MSC SFM XenoFree Medium can be stored in the dark 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.
     

Primary Isolation Medium: StemPro MSC SFM XenoFree Complete Medium with 2.5% human AB Serum for Isolation of MSCs from Bone Marrow (500 mL of complete medium)

  1. Thaw StemPro MSC SFM XenoFree Supplement at 2–8°C overnight (thawed supplement will have a slightly cloudy appearance). Do not thaw at 37°C. Use thawed material immediately or aliquot (i.e., 1 mL) unused material and store at –20°C to –5°C. Avoid additional freeze-thaw cycles.
    Note: When stored frozen at –20°C to –5°C, the StemPro MSC SFM XenoFree Supplement is stable for 12 months.
  2. To prepare 500 mL of complete StemPro MSC SFM XenoFree Medium, aseptically mix the following components:

    StemPro MSC SFM Basal Medium 477.5 mL
    StemPro MSC SFM XenoFree Supplement 5 mL
    AB-Human Serum 12.5 mL (2.5% final concentration)
    CTS™ GlutaMAX™-I Supplement or 200 mM L-Glutamine 5 mL (2 mM final concentration)
    Optional: Gentamicin (50 mg/mL), Liquid 50 µL (5 µg/mL final concentration)

    Note: Filtering the AB-Human Serum through a 0.22 µm filter is recommended before use.  

  3. Complete StemPro MSC SFM XenoFree Medium with 2.5% human AB serum can be stored in the dark 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.

StemPro MSC SFM CTS™ Complete Medium for Serum-Free Cultures (500 mL of complete medium)

  1. Thaw CTS™ StemPro MSC SFM Supplement at 2–8°C overnight. Do not thaw at 37°C. Use thawed material immediately or aliquot (i.e., 15 mL) unused material and store at –20°C to –5°C protected from light. Avoid additional freeze-thaw cycles.
    Note: When stored frozen at –20°C to –5°C in the dark, the CTS™ StemPro MSC SFM Supplement is stable for 12 months.
  2. To prepare 500 mL of complete StemPro MSC SFM XenoFree Medium, aseptically mix the following components:

    CTS™ StemPro MSC SFM Basal Medium420 mL
    CTS™ StemPro MSC SFM Supplement75 mL
    CTS™ GlutaMAX™-I Supplement or 200 mM L-Glutamine5 mL (2 mM final concentration)
    Optional: 10 mg/mL Gentamicin Reagent Solution50 µL  (5 µg/mL final concentration)
  3. CTS™ StemPro MSC SFM Medium can be stored in the dark at 2–8°C for up to 4 weeks, within the expiration date of all three components.
    Note: Before use, warm complete medium required for that day at room temperature until it is no longer cool to the touch.

Coating Culture Vessels with CTS™ CELLstart™ Substrate

  1. Dilute CTS™ CELLstart™ 1:100 in Dulbecco’s Phosphate Buffered Saline (DPBS) CTS™ with calcium and magnesium (i.e., 100 µL CTS™ CELLstart™ Substrate into 10 mL of CTS™ DPBS) or in primary isolation medium if you are isolating MSCs from bone marrow (i.e., 100 µL CTS™ CELLstart™ Substrate into 10 mL of primary isolation medium). Mix by gentle pipetting or inversion, do not vortex.  Add 10 mL of the CTS™ CELLstart™ Substrate solution to each T-75 flask and ensure complete surface coverage. Note: Do not store diluted CTS™ CELLstart™ Substrate solution; prepare fresh before each use.
  2. Incubate at 36°C to 38°C in a humidified atmosphere of 4–6% CO2 in air for 60–120 minutes. After incubation, remove flasks from the incubator and temporarily place them in a laminar flow hood until use. Immediately before use, remove all CTS™ CELLstart™ Substrate solution and replace with complete medium. Do not rinse coated plates before use. Note: If CTS™ CELLstart™ Substrate coated plates (with coating solution in the plate) are wrapped tightly with Parafilm sealing film, they can be stored at 2°C to 8°C for 2 weeks. DO NOT REMOVE COATING SOLUTION PRIOR TO STORAGE.

Recovering Cryopreserved Human MSCs

Note: These instructions can be used for either serum-free and xeno-free cultures or for just serum-free cultures by adding the appropriate complete medium in steps 3, 5, 6, and 8. For serum-free, xeno-free cultures, use StemPro MSC SFM XenoFree Complete Medium and for serum-free cultures, use CTS™ StemPro MSC SFM Complete Medium.

  1. Rapidly thaw (<1 minute) a frozen vial of human MSCs in a 37°C water bath until a small amount of ice remains.
  2. Pipet the entire contents of the cryovial into a sterile 50-mL conical tube.
  3. Carefully add 5–10 mL of pre-warmed (37°C) complete medium to the conical tube at a rate of approximately 1 drop every 2 seconds and gently swirl after every addition to ensure homogeneity of the cell suspension.
  4. Centrifuge the tubes at 100–200 × g for 5 minutes at room temperature. Aspirate and discard supernatant, being careful not to disturb the cell pellet.
  5. Resuspend the cell pellet in a minimum volume of pre-warmed (37°C) complete medium for cell counting. Determine total viable cell density with a Countess Automated Cell Counter (alternative automated or manual procedures may be used). Calculate the volume of cell suspension required to seed cells at a density of ≥ 5 × 103 cells/cm2.
  6. Add the cell suspension to an appropriate CTS™ CELLstart™ substrate-coated flask containing 10–15 mL pre-warmed complete medium (see Coating Culture Vessels with CTS™ CELLstart™ Substrate).
  7. Incubate at 36°C to 38˚C in a humidified atmosphere of 4% to 6% CO2 in air.
  8. Replace the medium in the flasks every 2–3 days with fresh pre-warmed complete medium.

Isolating MSCs from bone marrow

Note: For the initial isolation of MSCs, supplementation of the serum-free, xeno-free complete medium with 2.5% human AB serum facilitates cell attachment and growth. For subsequent passages, human AB serum is not required.

  1. Mix human bone marrow aspirate 1:2 with HBSS (i.e. 20 mL aspirate + 20 mL HBSS).
  2. Pipet 15 mL gradient density media into a 50-ml centrifuge tube.
  3. Slowly layer 20 mL of dilute bone marrow aspirate on top of the gradient density media, taking care not to mix cells and the media.
  4. Centrifuge (with brake off) tubes at 400 x g for 35 minutes at 20°C.
  5. Carefully remove the mononuclear cell (MNC) layer with a sterile transfer pipette or 1-mL serological pipette.
    Note: For successful human MSC isolation, it is critical to avoid red blood cell (RBC) contamination that will affect MSC adherence and proliferation.
  6. Add 30 mL HBSS to the tube containing harvested MNCs and pipet to dissociate the cell pellet.
  7. Centrifuge at 400–500 x g for 10 minutes. Remove supernatant.
  8. Repeat step 7.
  9. Resuspend cells in a minimal volume of primary isolation medium (StemPro MSC SFM XenoFree Complete Medium with 2.5% human AB serum).
  10. Transfer 25 μL of the cell solution to a microfuge tube and add 50 μL of ACK Lysing Buffer for RBC lysis.
  11. Pipet cell solution to mix and incubate at room temperature for 5 minutes.
  12. Dilute cell lysis solution 1:2 with Trypan Blue Solution.
  13. Count the cell number using the Countess automated cell counter or other preferred method (e.g., a hemacytometer).
  14. Seed viable MNCs at a density of 2.67 x 105 cells/cm2 on CTS™ CELLstart substrate-coated flasks, using primary isolation medium to dilute the substrate.
  15. After 3–4 days, harvest the supernatant and centrifuge at 300 x g for 5 minutes.
  16. Add pre-warmed primary isolation medium to the flask.
  17. Change complete primary isolation medium every two days.
    Note: A supernatant cell harvest is NOT necessary after the initial medium change.
  18. Upon reaching 70–80% confluence, propagate cells under serum-free conditions as noted below.

Subculturing MSCs

General Guidelines

For xeno-free, serum-free cultures:

  • StemPro MSC SFM XenoFree has been developed for the multi-passage expansion of human bone marrow-derived MSCs and Adipose-derived Stem Cells (ADSCs) at greater than clonal densities (≥ 5 × 103 cells/cm2). Reduced seeding densities may result in suboptimal cell expansion, although optimal growth conditions must be determined for each application.
  • When subculturing human MSCs in StemPro MSC SFM XenoFree, input cell confluence should be 60–90%, cell viability should be at least 90%, and the growth rate should be in mid-logarithmic phase prior to subculture. Initiating cultures under suboptimal conditions may affect product performance. Transitioning MSCs or ADSCs from serum-containing medium to StemPro MSC SFM XenoFree does not require an adaptation protocol.
  • For optimal performance, re-feed the cultures every 2 days with StemPro MSC SFM XenoFree complete medium.

Note: Procedures detailed below are for cultures maintained in a T-75 culture flask (75 cm2). Adjust the volumes accordingly for desired vessel size (see Table 1: Reagent Volumes).

 

For serum-free cultures:

  • CTS™ StemPro MSC SFM has been developed for the primary isolation and multi-passage expansion of human bone marrow-derived MSC and ADSC at greater than clonal densities, with optimal cell expansion observed at ≥ 5 × 103 cells/cm2.
  • Reduced seeding densities may result in suboptimal cell expansion. Optimal growth conditions must be determined for each application.
  • It is recommended that human MSC in CTS™ StemPro MSC SFM be subcultured when cell confluency reaches 60–80%, cells are in mid-logarithmic phase of growth and cell viability is at least 90%. Initiating cultures under suboptimal conditions may affect product performance. Transitioning MSC or ADSC from serum–containing medium to Complete CTS™ StemPro MSC SFM does not require an adaptation protocol.
  • For optimal performance and cell growth, cultures should be re-fed every 2 days with fresh complete CTS™ StemPro MSC SFM.

Note: Procedures detailed below are for cultures maintained in a T-75 culture flask (75 cm2). Adjust the volumes accordingly for desired vessel size (see Table 1: Reagent Volumes).

Table 2 Reagent Volumes

 

Culture vessel

Surface area (cm2)

CTS™ TrypLE reagent (mL)

Complete medium (mL)CTS™ CELLstart™ Solution (1:100) (mL)
Dishes35-mm9.620.5–12–30.5–1
 60-mm28.271–23–52–3
 100-mm78.542–310–157–10
 150-mm176.713–515–2015–20
Plates6-well9.620.5–12–32.0
 12-well4.010.2–0.51–21
 24-well2.000.1–0.20.5–10.5
FlasksT-2525.001–24–52–3
 T-7575.002–312–157–10
 T-175175.003–535–4015–20
 T-225225.005–740–4520–25

Propagating MSCs

  1. Observe the stock culture flask (cells growing in current medium formulation or in either complete medium with an inverted microscope and confirm that the cells are ready to be subpassaged (~60–90% confluent for xeno-free, serum-free medium and ~60–80% confluent for serum-free medium).
  2. Pre-warm CTS™ TrypLE™ Select reagent (Cat. no. 12859) and the appropriate complete medium before use.
  3. Add 10 mL of pre-warmed complete medium to a 50 mL conical tube for each flask being harvested.
  4. Aspirate spent medium from the T-75 flask and discard.
  5. Wash the cell monolayer surface with 5–10 mL of CTS™ DPBS without Ca2+ and Mg2+, aspirate, and discard.
  6. Add 3–5 mL of CTS™ TrypLE™ reagent to the T-75 flask, tilt the flask in all directions to ensure complete coverage of cell monolayer. Incubate the cells in CTS™ TrypLE™ reagent for 2 to 10 minutes at 37°C.
    Note: Cells coming out of serum-containing medium may require a longer incubation time (5 to 10 minutes), while cells growing under serum-free conditions should detach more readily (2 to 3 minutes).
  7. After incubation, check the flask with an inverted microscope for cell detachment. Firmly tap the flask as necessary to facilitate complete cell detachment.
  8. Upon detachment, add 5 mL of pre-warmed StemPro MSC SFM XenoFree (for xeno-free, serum-free cultures) or 5 mL pre-warmed CTS™ DPBS with calcium and magnesium (for serum-free cultures) to each flask to completely cover the surface area.  Transfer cell suspension to a sterile 50-mL conical tube containing the corresponding complete medium. Firmly tap the flask, re-wash with 5 mL of the appropriate complete medium, and collect.
    Note: The addition of complete medium to harvested cells is critical for preventing the cells from adhering to the wall of the conical tube during centrifugation.
  9. Centrifuge the tubes at 100–200 × g for 5 minutes at room temperature. Aspirate medium and discard being careful not to disturb cell pellet.
  10. Resuspend the cell pellet in a minimal volume of pre-warmed complete medium for cell counting, using a preferred counting method (e.g., Countess Automated Cell Counter).
  11. Remove the CTS™ CELLstart™ substrate solution from each coated flask and add 15 mL of complete medium.
  12. Add enough cell suspension to each flask to provide ≥ 5 × 103 cells/cm2 (i.e. 3.75 × 105 viable cells per T-75 flask). Gently mix or swirl the cell suspension to ensure even distribution.
  13. Place the culture flask(s) in the incubator at 37˚C with a humidified atmosphere of 4–6% CO2.

Replace the spent culture medium every 2–3 days with pre-warmed complete medium for optimal performance and cell growth.

Cryopreservation of MSCs

  1. Prepare a cryopreservation solution on the day of use according to the type of culture or use CTS™ Synth-a-Freeze Medium:

    a. For xeno-free, serum-free cultures: Prepare 10 mL of XFM cryopreservation solution (2X) by supplementing StemPro MSC SFM XenoFree complete medium with 20% Dimethyl Sulfoxide (DMSO). Store at 4°C until use.

    StemPro MSC SFM Basal Medium 7.9 mL
    StemPro MSC SFM XenoFree Supplement 0.1 mL  (1% final concentration)
    DMSO 2.0 mL  (20% final concentration)

    b. For serum-free cultures: Prepare 10 mL of SFM cryopreservation solution on day of use by supplementing CTS™ StemPro MSC SFM Basal Medium with 25% CTS™ StemPro MSC SFM Supplement and 10% Dimethyl Sulfoxide (DMSO). Store at 4°C until use.

    CTS™ StemPro MSC SFM Basal Medium 6.5 mL
    CTS™ StemPro MSC SFM Supplement 2.5 mL (25% final concentration)
    DMSO 1 mL (10% final concentration)
  2. Harvest cells for cryopreservation using the appropriate complete medium according to steps 1 through 8 of Propagating 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 complete medium. Slowly add 0.5 mL of cold XFM or SFM cryopreservation solution (2X) to the cell suspension, and gently mix to ensure even cell distribution. If you use CTS™ Synth-a-Freeze Medium, resuspend the cell pellet to the desired final concentration (1 x 106 cells/mL) and follow steps 4–6 below.
  4. Immediately add the desired volume of cell suspension (i.e., 1 mL) to cryovials.
  5. Use an automated or manually controlled rate freezing apparatus following standard procedures (1°C decrease per minute) and place the cryovials at –70°C in a cryogenic freezing container (e.g., Mr. Frosty® (1°C) Freezing Container).
  6. After 24 hours, transfer the frozen cells to liquid nitrogen (vapor phase); storage at –200°C to –125°C is recommended.

Appendix A. Expected results

Maintaining trilineage potential under xeno-free conditions

Using StemPro MSC SFM XenoFree, human MSCs or ADSCs can be expanded for multiple passages while maintaining their multipotent phenotype (i.e., ability to differentiate into osteogenic, chondrogenic and adipogenic lineages) (Figure 3).

Figure 3. Characterization of human MSCs grown under xeno-free conditions. Human bone marrow–derived MSCs expanded in (A) DMEM (low glucose) + 10% MSC-Qualified FBS, or (B) StemPro MSC SFM XenoFree + CTS™  CELLstart™ substrate–coated plates, exhibited trilineage mesoderm differentiation potential as shown through oil red O staining (adipocyte), alcian blue staining (chondrocyte), and alkaline phosphatase staining (osteoblast). Data shown = passage 3 (input human MSCs = passage 5, 4-donor pool, 10x objective). Differentiation reagents = StemPro Differentiation Kits (adipogenesis, chondrogenesis, osteogenesis). (C) Passage 5 human MSCs analyzed using multiplex flow cytometry revealed a retained characteristic human MSC surface antigen profile after expansion in classical 10% FBS-containing medium or StemPro MSC SFM XenoFree. NEG = multiplex analysis of CD14, CD19, CD45, and HLA-DR.

 

Maintaining trilineage potential under serum-free and xeno-free conditions

Using CTS™ StemPro MSC SFM, human MSC or ADSC can be expanded for multiple passages while maintaining their trilineage mesoderm potential (i.e., ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages) (Figure 4). 

Figure 4. hMSCs cultured in StemPro MSC SFM retain trilineage differentiation potential through long-term passaging. hMSCs cultured in StemPro MSC SFM (after passage 5) were seeded into adipogenic, chondrogenic, or osteogenic differentiation medium for 14 days, revealing adipocytes (oil red O lipid stain), chondrocytes (alcian blue glycosaminoglycan stain), and osteoblasts (alkaline phosphatase stain).

Morphology of hMSCs grown under serum-free and xeno-free conditions

While human MSCs grown in classical medium have a flattened cell morphology and reach confluency at 1.0–3.0 x 104 cells/cm2, human MSCs grown in StemPro MSC SFM have a much smaller, spindle-shaped morphology and can reach densities of >1.0 x 105 cells/cm2 (Figure 5).

Reference

  1. Lindroos B et al. (2009) Serum-free, xeno-free culture media maintain the proliferation rate and multipotentiality of adipose stem cells in vitro. Cytotherapy 11(7):958–972.
  2. 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.
  3. Chase LG, et al. (2013) Development and characterization of a clinically compliant xeno-free culture medium in good manufacturing practice for human multipotent mesenchymal stem cells. Stem Cells Transl Med 1:750–758.
  4. Chase L et al. (2010) A novel serum-free medium for the expansion of human mesenchymal stem cells. Stem Cell Res Ther 1:8 doi:10.1186/scrt8.
  5. Agata H et al. (2009) Feasibility and efficacy of bone tissue engineering using human bone marrow stromal cells cultivated in serum-free conditions. Biochem Biophys Res Commun 382(2):353–358.
  6. 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.

Last update: 17 Mar 14

For Research Use Only. Not for use in diagnostic procedures.