*4.2. Cell Culture*

## 4.2.1. 2D Cell Culture

IVD tissue was diced to around 1 mm<sup>3</sup> pieces and treated with a mixture of 0.3% dispase (04942078001, Roche Diagnostics, Mannheim, Germany) and 0.2% collagenase (17454, SERVA Electrophoresis, Heidelberg, Germany) in PBS at 37 ◦C for 4–8 h to isolate the cells. After the incubation, the suspension was filtered through a 70 μm cell strainer (542070, Greiner Bio-One, Kremsmünster, Austria), centrifuged at 700× *g* for 5 min, and resuspended in DMEM/F12 (31330-038, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum (FCS, F7524, Merck, Darmstadt, Germany), 100 units/mL penicillin, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B (15240-062, Gibco, Carlsbad, CA, USA). The primary IVD cells were

expanded in monolayer in a standard cell culture incubator (5% CO2, 37 ◦C) up to passage 3 before being used in the experiments.
