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Millipore - Mesenchymal stem cell differentiation and Osteogenesis differentiation protocol
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Mesenchymal Stem Cell Differentiation Protocols

Stem cell technology, particularly embryonic stem cells and/or mesenchymal stem cells, offer attractive sources of adipocytes and osteoblasts for tissue culture studies and for the biochemical dissection of the earliest steps involved in cell determination. Mesenchymal stem cells are multipotent progenitor cells that have the capacity to differentiate into several mesenchymal cell lineages, including bone, cartilage, and fat.

Osteogenesis Differentiation Protocol

This protocol utilizes Millipore's Mesenchymal Stem Cell Osteogenesis Kit. Using this protocol, you will typically obtain >50% mature osteocytes from rat bone marrow-derived Mesenchymal stem cells. Efficiency of osteogenic differentiation may vary, depending upon the quality of the mesenchymal stem cells and if variations to the protocol are introduced.

Kit Components
  • Dexamethasone Solution: One vial containing 100 µL of 10 mM dexamethasone in ethanol.
  • Ascorbic Acid 2-Phosphate Solution: One vial containing 500 µL of 100 mM ascorbic acid 2-phosphate in water.
  • Glycerol 2- Phosphate Solution: Three vials containing 1 mL of 1 M glycerol 2-phosphate in water.
  • Collagen, Type I: One vial containing 150 µg collagen Type I.
  • Vitronectin: One vial containing 150 µg vitronectin.
  • Alizarin Red Solution: One bottle containing 50 mL Alizarin Red Solution.
Additional Materials Required
  • Human or rat mesenchymal stem cell and cell culture reagents
  • Mesenchymal Stem Cell Expansion Media — Millipore cat. no. SCM015
  • 24-well tissue culture plates
  • Phosphate-Buffered Saline (1x PBS) — Millipore cat. no. BSS-1005-B
  • Accutase — Millipore cat. no. SCR005
  • Fixative (e.g. 4% Paraformaldehyde in 1x PBS)
  • Hemacytometer
  • Microscope

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Preparation of Osteogenesis Induction Media

Osteogenesis Induction Media should be made fresh for each use or medium change. Thaw and then heat inactivate Fetal Bovine Serum (Millipore cat. no. ES-009-D) by incubating at 55°C for 30 minutes.

The recommended amount of medium for a 24-well plate is 1 mL/well. Make up a 1 mM stock solution of Dexamethasone by adding 900 µL ethanol to 100 µL of 10 mM Dexamethasone Solution. Store stock at –20°C. Mix the following sterile ingredients to make 10 mL of medium. Scale up according to experimental design.

ComponentStock ConcentrationAmountFinal Concentration
DMEM-low glucose
8.7 mL
Fetal Bovine Serum, heat inactivated
(Catalog No. ES-009-D)
l mL
Dexamethasone Solution
1 µL
0.1 µM
Ascorbic Acid 2-Phosphate Solution
20 µL
0.2 mM
Glycerol 2-Phosphate Solution
100 µL
10 mM
L-Glutamine (Catalog No. TMS-002-C)
100 µL
Penicillin and Streptomycin (Catalog No. TMS-AB2-C)
100 µL

Thawing of Cells
  1. Do not thaw the cells until proper media and plasticware are on hand.
  2. Remove the vial of mesenchymal stem cells (either rat or human) from liquid nitrogen and incubate in a 37°C water bath. Closely monitor until the cells are completely thawed. Maximum cell viability is dependent on the rapid and complete thawing of frozen cells.
    Important: Do not vortex the cells.
  3. As soon as the cells are completely thawed, disinfect the outside of the vial with 70% ethanol. Proceed immediately to the next step.
  4. In a laminar flow hood, use a 1 or 2 mL pipette to transfer the cells to a sterile 15 mL conical tube. Be careful to not introduce any bubbles during the transfer process.
  5. Using a 10 mL pipette, slowly add drop-wise 9 mL of Mesenchymal Stem Cell Expansion Medium (pre-warmed to 37°C) to the 15 mL conical tube.
    Important: Do not add the whole volume of medium at once to the cells. This may result in decreased cell viability due to osmotic shock.
  6. Gently mix the cell suspension by slow pipetting up and down twice. Be careful to not introduce any bubbles.
    Important: Do not vortex the cells.
  7. Centrifuge the tube at 300 xg for 3–5 minutes to pellet the cells.
  8. Decant as much of the supernatant as possible. Steps 5–8 are necessary to remove residual cryopreservative (DMSO). 9. Resuspend the cells into a suitable volume of Mesenchymal Stem Cell Expansion Medium (pre-warmed to 37°C). For a 10 cm tissue culture plate or T75 tissue culture flask, use 10–12 mL volume. For a 6 cm tissue culture plate, use 5 mL volume.
    Important: Do not vortex the Cells.
  9. Incubate the cells at 37°C in a 5% CO2 humidified incubator.
  10. Change to fresh Mesenchymal Stem Cell Expansion Medium (prewarmed to 37°C) the next day and every three to four days thereafter.
  11. When the cells are 80–90% confluent, they can be dissociated with Accutase (Millipore cat. no. SCR005) and subcultured or alternatively frozen for later use.

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  1. Culture the cells in a T75 flask in Mesenchymal Stem Cell Expansion Medium until they are 80-90% confluent.
  2. Aspirate the media
  3. Wash the flask twice with 5-10 mL of 1x PBS (Millipore cat. no. BSS-1005-B). Aspirate after each wash.
  4. Apply 5–7 mL of Accutase and incubate in a 37°C incubator for 5–7 minutes.
  5. Inspect the plate and ensure the complete detachment of cells by gently tapping the side of the plate with the palm of your hand.
  6. Apply 10 mL of Mesenchymal Stem Cell Expansion Medium (prewarmed to 37°C) to the flask.
  7. Transfer the dissociated cell suspension into a 15 mL conical tube.
  8. Centrifuge the tube at 300 xg for 3–5 minutes to pellet the cells.
  9. Aspirate and discard the supernatant.
  10. Apply 2 mL of Mesenchymal Stem Cell Expansion Medium to the tube and resuspend the cells thoroughly.
    Important: Do not vortex the Cells.
  11. Count the number of cells using a hemcytometer.
  12. Plate the cells at athe desired cell density into appropriate flasks, plates or wells. Do not exceed a plating ratio of 1:7.
Preparation of Coated 24-Well Tissue Culture Plates

Studies indicate that coating tissue culture plastic-ware with vitronectin and collagen I promote osteogenic differentiation over non-coated plates (Salasznyk, 2004). Vitronectin and collagen I are provided in the kit and we recommend coating tissue culture plastic- or glasswares with these two ECM molecules to aid in the differentiation of mesenchymal stem cells to osteoblasts.

  1. Dilute vitronectin and collagen with 1x PBS to yield final concentrations of 12 µg/mL for each ECM molecule.
  2. Add 0.5 mL of the vitronectin/collagen mixture to each well of a 24–well plate. Incubate overnight at room temperature.
  3. The next day, remove the vitronectin/collagen mixture from the wells and rinse the wells once with 1X PBS. Aspirate just before using.
Cell Plating
  1. Follow steps 1–11 of the protocol outlined in the "Subculturing" section.
  2. Plate the cell suspension in Mesenchymal Stem Cell Expansion Medium at a density of 60,000 cells per well in the vitronectin/collagen coated 24-well culture dish with 1 mL volume per well.
  3. Incubate the cells at 37°C in a 5% CO2 humidified incubator overnight.

    Note: Cells should be attached and 100% confluent after overnight incubation. If they are not confluent, replace medium every three to four days until the cells are confluent. It is important that the cells be 100% confluent before initiating osteogenesis differentiation.

  4. When the cells are 100% confluent, carefully aspirate the medium from each well and add 1 mL Osteogenesis Induction Medium. This medium change corresponds to differentiation day 1.
  5. Replace with fresh Osteogenesis Induction Medium every 2–3 days for 14–17 days.
  6. After 14–17 days of differentiation, osteocytes can be fixed and stained with Alizarin Red Solution.
Alizarin Red S Staining Protocol

  1. Carefully aspirate the medium from each well. Be careful to not aspirate the cells.
  2. Fix osteocytes by incubating in iced cold 70% ethanol for 1 hour at room temperature.
  3. Carefully aspirate the alcohol and rinse twice (5–10 minutes each) with water.
  4. Aspirate the water and add enough Alizarin Red Solution to cover the wells (500 µL to 1 mL per well in a 24-well plate).
  5. Incubate at room temperature for 30 minutes.
  6. After 30 minutes, remove the Alizarin Red Solution and wash the wells four times with 1 mL water. Aspirate after each wash.
  7. Add 1–1.5 mL water to each well to prevent the cells from drying. The plate is now ready for visual inspection and/or image acquisition

    Note: Osteocytes containing calcium deposits will be stained orange-red by the Alizarin Red Solution.

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