Chondrocyte Differentiation Tool
PCS-500-051 ™
PCS-500-051 ™
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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):
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
Preparation of Differentiation Medium
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
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.
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.
Alginate Encapsulation of Cells: Formation of Chondrogenic Microbeads
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