Collagenase type 2 digestion

Degeneration of the articular cartilage surface was achieved by collagenase type II digestion (Worthington Biochemical Corporation, Lakewood, NJ, USA, activity: 215 u/mg). The osteochondral plugs were immersed in collagenase dissolved in PBS for five minutes at 37 °C. To obtain different states of degeneration, we applied different collagenase concentrations: 5 µg/mL, 50 µg/mL, 100 µg/mL and 500 µg/mL. After the five minutes, the collagenase solution was discarded, the cartilage was rinsed with PBS and subsequently immersed in PBS supplemented with Complete Protease Cocktail Inhibitor (Roche AG, Basel, Switzerland). The protease inhibitor has been added to block any remaining collagenase activity on the cartilage. Based on preliminary studies using various combinations of collagenase concentration and digestion duration, the time of 5 min was chosen to not severely disrupt the collagen network of the cartilage and to limit ECM degradation to the superficial zone, thereby mimicking the very initial phase of OA.

Primary proximal tubular cells (PCT) were isolated from male C57/BL6 mice (7 to 14 weeks) as described previously^{56 (link)}. Briefly, renal cortices were sliced in dissection solution (HBSS with 15 mM HEPES, 10 mM D-glucose, 5 mM glycine, 1 mM L-alanine buffered to pH 7.4 and osmolality of 325 mosmol/kgH₂O) into pieces of approximately 1 mm² followed by collagenase type II digestion (Worthington Biochemical, Lakewood, NJ) for 30 min at 37 °C in presence of soybean trypsin inhibitor (Sigma, St. Louis, MO). Then they were transferred onto a sieve tower with two pore sizes (250 µm and 80 µm) (Retsch, Haan, Germany). The proximal tubule fragments on the lower sieve were collected by flushing the sieve in the reverse direction with dissection solution containing 1% BSA. Upon centrifugation the fragments of two kidneys were resuspended in culture medium (1:1 DMEM/F12 without phenol red and supplemented with 15 mM HEPES, 0.55 mM Na-pyruvate, 100 × non-essential amino acids 10 ml/L, and REGM SingleQuot Kit Supplements & Growth Factors (Lonza, Verviers, Belgium) and distributed onto one 24 well plate and left unstirred for 72 h at 37 °C and 95% air-5% CO₂ in a standard humidified incubator. After 3 days the medium was replaced and on day 7 the cells reached confluence and were used for further experiments.