Dmem with 4.5 g l glucose

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.

This study used the KB-3-1 human cervical carcinoma cell line (a subline of HeLa), and its cisplatin-resistant sublines KB-CP.5 and KB-CP20. Originally, KB-CP.5 and KB-CP20 cells were selected in a single step in 0.5 μg/mL (1.6 μM) and in multiple steps up to 20 μg/mL (0.103 mM) cisplatin, respectively, in our laboratory as described previously [30 (link)]. All cell lines were thawed immediately before experimentation, and cell lines are characterized by NCI using short tandem repeat profiling. The cisplatin stock solutions used for culturing CP.5 and CP20 cells were prepared in PBS. The cisplatin-resistant cells were maintained in the presence of cisplatin, which was removed from growth medium 3 days prior to all experiments. All cell lines were grown as monolayer cultures at 37°C in humidified 5% CO₂, using DMEM with 4.5 g/L glucose (from Invitrogen), supplemented with L-glutamine, penicillin, streptomycin, and 10% FBS (BioWhittaker).