**2.3 ADSC 3D culture in RAD/heparin composite scaffold**

 RAD16-I (PuraMatrix™, 354250, Corning) and composites RAD/Hep were prepared at a final concentration of 0.3% (w/v) RAD16-I. The composites were prepared by combining 95 μL of 0.5% (w/v) RAD16-I and 5 μL of heparin sodium salt solution (H3149, Sigma) in a concentration range between 0.01% and 1% (w/v). The mixture was then diluted with 10% sucrose (S0389, Sigma) to a final concentration of 0.3% (w/v) RAD16-I. To obtain RAD and RAD/Hep 3D cultures, ADSC were harvested by trypsinization and resuspended to 4 × 106 cells/mL in 10% sucrose. The 0.3% (w/v) RAD16-I peptide solution was mixed with the cell suspension (1:1) to obtain a final concentration of 0.15% (w/v) RAD16-I and 2 × 106 cells/mL. Then, 80 μL of the cell-peptide mixture (160,000 cells) was loaded into individual wells of a 48-well culture containing 150 μL of medium, which induced the self-assembly of the peptide. The plate was placed in the incubator for 20 min

*Cartilage Tissue Engineering Using Self-Assembling Peptides Composite Scaffolds DOI: http://dx.doi.org/10.5772/intechopen.83716* 

at 37°C and 5% CO2, and then 650 μL of fresh medium was added to the 3D cell cultures. ADSC 3D cultures were maintained during 2 days under control medium. Cultures for chondrogenic differentiation were induced at day 2 with chondrogenic medium.
