Collagenase cls 2

NP cells were isolated using a modified method as described before [26 (link)]. In brief, the tissue was digested with collagenase CLS II (333.3 U/mL) (Biochrom), collagenase P (1 U/mL) (Roche, Mannheim, Germany), and hyaluronidase (33.3 U; Roche) for 2–4 h under continuous stirring in a spinner flask at 37°C and 5% CO₂. After digestion, the cells were plated with a density of 10⁴ cells/cm². The cells were cultivated at 37°C and 5% CO₂ with medium supplemented either with or without 2 ng/mL basicFGF (Peprotech, Hamburg, Germany). The medium was changed every 2–3 days. When reaching about 80–90% confluence the cells were passaged using trypsin/ethylenediaminetetraacetic acid (EDTA; Biochrom). For subsequent passages, the seeding density was 5.000 cells/cm². For the tissue engineered grafts, cells were used at the end of passage 2. For investigation of growth kinetics, the NP cells were cultivated up to passage 7 with or without 2 ng/mL basicFGF supplementation with a seeding density of 8.000 cells/cm² at passage 1 and following passages. The volume of medium was 25 mL in each cell culture flask with a surface of 175 cm².

Enzyme solutions containing 1X PBS with 1.0 mg/mL collagenase CLS II (Biochrom, Berlin, Germany, sterile-filtered), 0.5 mg/mL collagenase P (Sigma-Aldrich^®, St. Louis, MO, USA, sterile-filtered), 0.1 mg/mL hyaluronidase (Sigma-Aldrich^®, St. Louis, MO, USA, sterile-filtered), 10% heat-inactivated fetal bovine serum (FBS, Biochrom, Berlin, Germany), 1% penicillin/streptomycin (Biochrom, Berlin, Germany) and 0.9% amphotericin (Sigma-Aldrich^®, St. Louis, MO, USA) were mixed directly before isolation of the cartilage and kept at 4 °C until usage. Transport solutions were discarded carefully before adding the enzymatic solution to each tube with the cartilage slices. The samples were incubated at 37 °C and shaken at 108 rpm (Heidolph^® unimax 1010, Incubator 1000, Schwabach, Germany) for 18 to 19 h. After digestion, the enzymatic solution was diluted with 1X PBS (37 °C), filtered through a 100 µm cell strainer and washed twice with 1X PBS at room temperature (Multifuge^® 3 S-R, Heraeus^®, Hanau, Germany, at 1200 rpm for 6 min) before suspending the chondrocytes in 2 mL of culture medium (37 °C).

AF and NP samples were washed with phosphate-buffered saline (PBS, Biochrom, Berlin, Germany), and wet weight was determined, separately. Samples were minced, and cells were released by enzymatically treating with 3333 U/mL collagenase CLS II (Biochrom), 1 U/mL collagenase P (Sigma-Aldrich, Taufkirchen, Germany) and 333 U/mL hyaluronidase (Sigma-Aldrich) in a spinner flask under gentle stirring for 4 h at 37 °C (5% CO_2, 95% humidity, normoxia) [16 (link)]. After cellular release, the cell solution was put through a 100 μm nylon cell strainer (BD Falcon, Franklin Lakes, NJ, USA), centrifuged at 600×g, and supernatant was discarded. The viable cell number was determined using trypan blue (Sigma-Aldrich) dye exclusion. From cellular release, three NP and five AF samples of all five donors had a sufficient cell yield to perform all experiments with all media and were thus included in this study.
Cells were cultured in 12-well plates with the different media in duplicates until passage P2 under consistent culture conditions (37 °C, 5% CO_2, 95% humidity, normoxia). Medium was replaced every second day. For subcultivation, the cells were detached with trypsin/ethylenediaminetetraacetic acid (Biochrom) at day 6 of passage P0 and day 3 of passage P1 and P2, respectively. Seeding density was always 9000 cells/cm².