Alginate hydrogel

The two hit compounds obtained from HTS were validated in alginate-cultured chondrocytes^{24 (link)}. In brief, chondrocytes were encapsulated in 1.2% alginate hydrogel (Sigma, US) at a seeding density of 2 × 10⁶ cells/mL (approximately 4 × 10⁴ cells in each bead) and maintained in complete DMEM supplemented with 50 μg/mL ascorbic acid for 7 days. The culture was treated  with the two compounds for another three days. Alginate cultured cells were further incubated with MTT for 3 h. MTT formazan were then released by papain digestion, precipitated by high-speed centrifuge and further dissolved by DMSO. Absorbance at 490 nm was measured. GAGs in the supernatant were precipitated by DMMB reagent (1×) and were further released in dissociation buffer (38.1 g of guanidine hydrochloride and 0.68 g of sodium acetate trihydrate in 100 mL of dH₂O containing 10% propan-1-ol). Next, GAGs were purified by ethanol precipitation and digested with chondroitinase ABC (100 mU/mL, from Proteus vulgaris, Seikagaku, JP). Chondroitin sulfate (CS) disaccharides were tagged with fluorescent 2-aminoacridone (dAMAC), fractionated in 25% polyacrylamide gels and quantified using the gel densitometry function of ImageJ (Version 1.46r, National Institutes of Health, US).

To demonstrate the spatial position capabilities of our DVDOD bioprinter, we used two dispensing units loaded with bioink composed of low viscosity (≤12 cP) alginate hydrogel (Sigma-Aldrich, St. Louis, MO, USA) mixed with FITC and Texas Red labeled dextran (Invitrogen, Carlsbad, CA, USA), respectively. Different patterns of droplets were used. To prevent liquid drying during the assay, 1 µL droplet was used to dispense onto the surface of a Petri dish. Images were captured using an epifluorescence microscope (Nikon, Minato City, Tokyo, Japan).

Mouse follicle isolation, encapsulation, and culture were carried out as previously described (33 (link), 34 (link), 70 (link), 71 (link)). In short, morphologically normal multilayered secondary follicles (150 to 180 μm in diameter) were mechanically isolated and selected from 16-d-old CD-1 female mice in L15 media (Invitrogen) containing 1% FBS (Invitrogen). Follicles were then incubated in the maintenance media (50% minimal essential medium [αMEM GlutaMAX; Invitrogen] and 50% nutrient mixture [F-12 with GlutaMAX]) with 1% FBS at 37 °C, 5% CO₂ in air for 2 h. Afterward, individual follicles were encapsulated in 5 μL 0.5% (weight/volume) alginate hydrogel (Sigma-Aldrich), immediately immersed in 50 mM CaCl₂ and 140 mM NaCl for 2 min to allow cross-linking, and incubated in the maintenance media to recover for 2 h. Individual follicles were then cultured in 96-well plates for 7 d in the follicle-culture media (maintenance media + 3 mg/mL bovine serum albumin + 10 mIU/mL human recombinant follicle-stimulating hormone [FSH; from A. F. Parlow, National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD] + 1 mg/mL bovine fetuin [Sigma-Aldrich] + ITS [5 μg/mL insulin, 5 μg/mL transferrin, and 5 ng/mL selenium; Sigma-Aldrich]). Half of the follicle-culture media was replaced every other day.