GAG content in the culture medium and in tissues was quantified using 1,9-dimethyl-methylene blue (DMMB, Sigma-Aldrich) assay, as previously described.20 (link),21 (link),30 (link) Microtissues, and amalgamated microtissues, were digested overnight at 60°C in 1 mL solution containing 1.6 U/mL papain and L-cysteine (both from Sigma-Aldrich). Twenty-five microliter of tissue digest or the medium collected during medium exchanges was combined with 200 µL of DMMB dye in 96 well plates (Nunc), and absorbance was measured at 590 nm in a plate reader (ThermoFisher’s Multiscan GO). A standard curve was generated using dilutions of chondroitin sulfate sodium salt extracted from shark cartilage (Sigma-Aldrich). DNA content in the tissues was estimated using the PicoGreen assay, as per the manufacturer’s instructions (ThermoFisher). PicoGreen solution was added to the tissue digests in half volume black 96 well plates (Costar) and samples read at 480 nm excitation and 520 nm emission on a plate reader (OMEGA FLUOstar multi-plate reader, BMG Labtech). A DNA standard curve was generated using λ-DNA (Quant-iT™ PicoGreen™ dsDNA Assay Kit, ThermoFisher).