Pet 1009030

Chondrocytes were applied to a custom double-diffusion biochamber with porous (10 µm pore diameter) polyester membrane (PET 1009030; Sterlitech, Kent, WA), which had been precoated with 5 µg/ml fibronectin (BD Bioscience, San Jose, CA), as previously described (11 (link)), at a density of 3.13 × 10⁶ cells/cm². The cells were cultured under the same O₂ condition as in expansion with bioreactor medium (serum-free DMEM with 4.5 g/L glucose (Invitrogen, Grand Island, NY), containing 1% ITS-premix (BD Bioscience, San Jose, CA), 100 nM dexamethasone (Sigma-Aldrich), 37.5 µg/mL L-ascorbate-2-phosphate (Wako chemicals, Osaka, Japan), and 1% sodium pyruvate, 1% non-essential amino acid, 1% glutamax, and 1% Penicillin/Streptomycin (all from Invitrogen, Grand Island, NY)). The medium was changed every other day. After 3 weeks, cartilage sheets were removed from the biochamber and allowed to free float until 6.5 weeks. Punches (5 mm in diameter) were taken for the mechanical testing from each sheet. Combined GAG/DNA analysis was made in triplicate (3 mm diameter punches), as was collagen/collagen cross-link analysis. Histology was performed on a single neutral buffered formalin fixed 3 mm punch from each sheet. The sheets were made in duplicate from 4 donors.

Cartilage sheets were fabricated as previously described (9 (link)), with minor modifications. Briefly, chondrocytes from second passage were typsinized and plated at 1.9 × 10⁶ cells/cm² into custom stainless steel biochambers consisting of a 4.0 × 4.0 cm upper chamber with a porous (10 μm) base consisting of a porous polyester membrane (PET1009030; Sterlitech, Kent, WA) coated with human fibronectin (BD Biosciences, San Jose, CA), which suspended the cells above the lower chamber which was either a 100 mm tissue culture plate or a custom cylindrical chamber with sufficient capacity to allow the addition of sufficient medium volume to totally immerse the biochamber (~300 ml). The large container allowed the cells in the upper chamber to be exposed to much more medium, thus avoiding medium depletion, as evinced by yellowing medium.