Isolation and Characterization of Engineered DIAS Cells

Human DIAS cells isolated from foreskin were selected based on in vivo results. Foreskin-derived hDIAS cells were grown in ARC in which cells were seeded at 7.5x10⁶ cells/mL in petri dishes coated with 1% agarose and cultured in CHG containing 10 ng/mL TGF-β1, 100 ng/mL GDF-5 (Propotech), and 100 ng/mL BMP-2 at 37°C for 7 days on an orbital shaker at 60 rpm [37 (link)]. Aggregates were digested with 0.05% trypsin-EDTA for 20 minutes followed by 0.2% collagenase solution containing 3% FBS for 1 hour with agitation every 15 minutes to release cells. The resulting cells were filtered through 70 μm cell strainers and counted. Human DIAS cell constructs using ARC and non-aggregate cultured (control) hDIAS cells were formed via the self-assembling process as described above. For biochemical analysis, lyophilized samples were digested using papain as previously described [38 (link)]. Sulfated GAG content was assayed using the Blyscan Glycosaminoglycan Assay kit (Biocolor, Westbury, NY). Total collagen content was quantified using a hydroxyproline assay (Biocolor) [39 (link)]. DNA content was determined using the PicoGreen® dsDNA Assay Kit (Life Technologies) [40 (link)]. Mechanical testing was performed as described above.