Bovine chondroitin sulphate a

After 21 days in culture, the wet weight of pellets was measured. Media was collected at each feeding during the culture period and included in the GAG measurements. Triplicate pellets from each group were then digested with 150 µg/mL papain in PBS, pH 6.0, containing 8 mM sodium EDTA, 6 mM L-cysteine (all Sigma-Aldrich). Sulfated glycosaminoglycan (GAG) and DNA content were quantified using 1,9-dimethylene blue (DMMB) and Picogreen (Quant-iT dsDNA; Invitrogen, Carlsbad, CA, USA) assays, respectively. Digested pellet GAG and collected supernatant were quantified against a standard curve generated using bovine chondroitin sulphate A (Sigma-Aldrich) diluted in either DMEM or papain buffer as standard in serial dilution. DMMB dye (18 µg/mL in 0.5% ethanol, 0.2% formic acid, 30 mM sodium formate, pH 3) was added to standards and samples and absorbance measured at 575 nm (Tecan, Crailsheim, Germany). DNA content in pellet digests was quantified using Quant-iT dsDNA assay according to manufacturers’ instructions.

After 21 days in culture, the wet weight of pellets was measured using a balance. Macroscopic images of pellets were photographed with an optical microscope (PL2000, Optech, Germany). Triplicate pellets from each group were digested with 150 µg/mL papain in PBS, pH 6.0, containing 8 mM sodium EDTA, 6 mM L-cysteine (all Sigma-Aldrich). Digested pellet GAGs were quantified against a standard curve generated using bovine chondroitin sulphate A (Sigma-Aldrich) diluted in papain buffer as standard in serial dilution. DMMB dye (18 µg/mL in 0.5% ethanol, 0.2% formic acid, 30 mM sodium formate, pH 3) was added to standards and samples and absorbance measured at 575 nm (Tecan, Crailsheim, Germany).