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Chondrocyte Differentiation Tool

PCS-500-051

The Chondrocyte Differentiation Tool (ATCC PCS-500-051) is a complete differentiation medium designed to induce chondrogenesis in actively proliferating Adipose-Derived Mesenchymal Stem Cells (ATCC PCS-500-011) with high efficiency.
Product category
Reagents
Components
*The Osteocyte Differentiation Tool provides enough medium for differentiation of ~ 1 million cells when plated at a recommended density of 18,000 viable cells/cm2 in a 6-well tissue culture format.
Formulation
Proprietary
Comments
The Chondrocyte Differentiation Tool is a complete differentiation medium designed to induce chondrogenesis in actively proliferating Adipose-Derived Mesenchymal Stem Cells with high efficiency.

The Chondrocyte Differentiation Tool provides enough medium to differentiate up to ~ 13.5 x 107 cells, sufficient to seed 20 wells of a 48-well tissue culture plate when using alginate encapsulation.

Chondrocyte differentiation requires that the cells be grown in a three-dimensional aggregate cell culture. Micromass culture can be used, but for the best results, ATCC recommends the use of a matrix, such as alginate, to provide a scaffold for the deposition of proteoglycans. Detailed instructions for the formation of chondrogenic microbeads are provided.
Applications
Stem cell research
Cell growth and viability
Volume
100 mL
Product format
Frozen
Storage conditions
-20°C or colder, -70°C for long-term storage
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Documentation

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Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

Detailed product information

Handling information

Handling procedure

Antimicrobials and phenol red are not required but may be added to the Chondrocyte Differentiation Tool if desired prior to use. The recommended volume of each optional component to be added to Chondrocyte Differentiation Tool is summarized in Table 1.

 

Table 1. OptionalAddition of Antimicrobials/Antimycotics and Phenol Red per 100 mL of Medium

Component

Volume

Final Concentration

Gentamicin-Amphotericin B Solution

0.1 mL

Gentamicin: 10 µg/mL

Amphotericin B: 0.25 µg/mL

Penicillin-Streptomycin-Amphotericin B Solution

0.1 mL

Penicillin: 10 Units/mL

Streptomycin: 10 µg/mL

Amphotericin B: 25 ng/mL

Phenol Red

0.1 mL

33 µM


Additional Materials Needed for Alginate Encapsulation (not provided):

  1. Sodium alginate (e.g., Sigma-Aldrich #71238)
  2. 3 mL syringe
  3.  27-gauge needle
  4.  Steriflip® Filter Unit, Millipore Corporation (Vacuum-driven 50 mL filtration system)
  5. Wide bore pipette tip
  6. 150 mM NaCl solution
  7. 100 mM CaCl2 solution (sterile)
  8. Small magnetic stir bar (sterile)
  9. 250 mL Beaker (sterile)
  10. Forceps or stir bar extractor (sterile)
  11. Magnetic stir plate 
Chondrocyte Differentiation

Chondrocyte differentiation requires that the cells be grown in a three-dimensional aggregate cell culture. Micromass culture can be used; but, for the best results, ATCC recommends the use of a matrix, such as alginate, to provide a scaffold for the deposition of proteoglycans. The following procedure demonstrates differentiation of ~2.7 x 107 cells seeded to four wells of a 48-well tissue culture plate, using 25 mL of the Chondrocyte Differentiation Tool and alginate encapsulation.

Preparation of a 1.5% (w/v) Alginate Solution

  1. Add 0.15 g of alginate to 10 mL of 150 mM NaCl while stirring rapidly or vortexing to minimize clumping.
  2. Agitate the solution on a rocker for at least 2 hr and up to 16 hr at room temperature to completely solubilize the alginate.
  3. Sterilize the solution using a 0.22 mm filter. Store at 4°C to 8°C for up to week.

Preparation of Differentiation Medium

  1. Pre-warm the Chondrocyte Differentiation Tool to 37°C in a water bath.
  2. Once thawed, store the remaining Chondrocyte Differentiation Tool in the dark at 2°C-8°C for later use. When stored under these conditions, the differentiation media is stable for up to three weeks.

Note: For procedures using less than the full 100 mL volume of the Chondrocyte Differentiation Tool, the medium can be dispensed into appropriate aliquots (e.g., for use with this protocol, we recommend dispensing into 25 mL aliquots). Unused portions can be refrozen once without loss of efficiency; however, multiple freeze/thaw cycles are NOT recommended.

Preparing Cells for Chondrocyte Differentiation

  1. Follow the instructions for the growth of Adipose-Derived Mesenchymal Stem Cells (ATCC PCS-500-011) using Mesenchymal Stem Cell Basal Medium (ATCC PCS-500-030) supplemented with Mesenchymal Stem Cell Growth Kit – Low Serum (ATCC PCS-500-040) components.
  2. Expand the cells as needed for experimental design, but do not passage more than four (4) times prior to initiating chondrocyte differentiation.

Note: It will take approximately 10 to 15 T-75 flasks at 70%-80% confluence to obtain 2.7 x 107 cells, which is enough to seed four wells of a 48-well tissue culture plate using the alginate encapsulation method described below.

  1. Cells should be collected and counted when the culture is 70%-80% confluent and actively proliferating.
  2. After centrifugation at 150 x g for 3-5 minutes, discard the supernatant and resuspend the cell pellet (2.5 x 107 cells) in 800 µL of the 1.5% (w/v) Alginate Solution. The final volume should be ~1.0 mL.  

Note: The Alginate Solution must not be diluted lower than 1.2% (w/v) by the addition of the cells. If you have fewer cells, adjust the volume of the prepared 1.5% Alginate Solution used in order to maintain this ratio. Likewise, if you have more cells, scale proportionally.

  1. Gently mix the cell-alginate suspension by pipetting up and down; taking care not to introduce bubbles into the solution.
  2. Proceed to Step 1 (below) for alginate encapsulation of cells.

Alginate Encapsulation of Cells: Formation of Chondrogenic Microbeads

  1. Transfer 75 mL of a 100 mM sterile CaCl2 solution to a sterile 250 mL beaker containing a sterile stir bar.
  2. Create a gentle funnel in the CaCl2 solution at room temperature on a stir plate.
  3. Transfer the alginate-cell suspension with a 3 mL syringe fitted with a 27 gauge needle.
  4. Rapidly dispense the alginate-cell suspension into the CaCl2 solution to form chondrogenic microbeads.
  5. Allow the chondrogenic microbeads to stir for 10 minutes to solidify (cure) the alginate.
  6. Remove the beaker from the stir plate and allow the chondrogenic microbeads to settle on the bottom.
  7. Transfer the chondrogenic microbead solution into a 50 mL conical tube and attach to the vacuum-driven Steriflip® Filter Unit. Immediately break the vacuum as soon as the liquid is removed to prevent damage to the beads.
  8. Resuspend the chondrogenic microbeads in 2 mL of pre-warmed Chondrocyte Differentiation Tool.
  9. Aseptically transfer enough chondrogenic microbeads to cover the bottom surfaces of four wells of a 48-well plate (about 0.5 mL per well). This set up will yield ~6.75 x 106 encapsulated cells/well.
  10. Allow the chondrogenic microbeads to settle to the bottom of the wells. To remove residual CaCl2, wash the chondrogenic microbeads by replacing the medium in each well twice with 0.5 mL Chondrocyte Differentiation Tool.  Add 0.5 mL Chondrocyte Differentiation Tool to each well after the last wash.
  11. Incubate the cells at 37°C with 5% CO2 for 2-3 days before renewing the medium.
  12. When ready to renew the medium, retrieve the Chondrocyte Differentiation Tool from storage and transfer the required volume to a sterile tube. (For 4 wells in a 48-well plate, this volume would be 2 mL).
  13. Warm the aliquot of Chondrocyte Differentiation Tool to 37°C in a water bath.
  14. Carefully remove the spent medium, taking great care  not to disturb or aspirate the chondrogenic microbeads.
  15. Add 0.5 mL of fresh, pre-warmed Chondrocyte Differentiation Tool to each well.
  16. Incubate the cells at 37°C with 5% CO2 for 2-3 days before renewing the medium.
  17. Repeat steps 12 through 16 every 2-3 days until the cells have been exposed to the Chondrocyte Differentiation Tool for a total of 21 days.
  18. Cells can be used at any phase of chondrocyte differentiation as predicated upon experimental design. To confirm calcium accumulation, cells can be fixed and stained with Alcian Blue (not provided).

Quality control specifications

Bacterial and fungal testing
Not detected
Mycoplasma contamination
Not detected
Functional tests
Differentiation of cells into chondrocytes as demonstrated by Alcian Blue staining.
A Certificate of Analysis (COA) is available upon request for each lot of the Chondrocyte Differentiation Tool.

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Warranty

The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

Permits & Restrictions

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

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References

Frequently Asked Questions

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