Papain p3125

After chondrogenic differentiation, the GAG and DNA content of the hydrogels was determined to evaluate cartilage-like matrix formation. After 1 and 28 days of culture, the GelMA discs were cut in half. One half of each disc was weighed, freeze-dried and weighed again prior to digestion using 200 µL papain digestion buffer (0.2 M NaH₂PO₄ + 0.01 M EDTA, pH = 6.0, supplemented with 250 µg/mL papain (P3125, Sigma-Aldrich) and 1.57 mg/mL Cysteine HCL (C9768, Sigma-Aldrich) overnight at 60 °C.
For determination of GAG concentration, papain-digested samples were diluted in PBS-EDTA and 46 µM 1,9-Dimethyl-Methylene Blue (DMMB, Sigma-Aldrich) solution was added to the samples. Absorption was measured at 525 and 595 nm on a spectrophotometer [66 (link)] and the ratio was used to calculate the concentration with a chondroitin sulphate C (Sigma-Aldrich) standard curve.
DNA concentration was measured in the papain digests with Picogreen (Quant-iT, Thermo Fisher Scientific) according to the manufacturers’ instructions. In short, samples were diluted 1:20 in TE-buffer (0.5 M EDTA in 1 M Tris (1:5), pH 8) and 100 µL sample was mixed with 100 µL Picogreen reagent, incubated 5 min in the dark and fluorescence was measured with an excitation of 485 nm and emission of 520 nm. Known concentrations of λDNA were used as a reference.

Primary neuronal cultures were obtained from P1-P2 C57BL/6J or Thy1-YFP pups as described in [61 (link)], with minor modification. In brief, after brain dissection, hippocampi were incubated with 200 U of papain (P3125, Sigma-Aldrich, St. Louis, MO, USA) (30 min, 34 °C), with trypsin inhibitor (T-9253, Sigma-Aldrich, St. Louis, MO, USA) (45 min, RT), and subsequently mechanically dissociated. Neurons were plated onto ibidi chamber slides (Ibidi GmbH, Germany) (300,000 cells/mL). Plating medium was B27/neurobasal-A (Gibco, Waltham, MA, USA) supplemented with 0.5 mM glutamine (Gibco, Waltham, MA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, Waltham, MA, USA). We did not treat the cultures with any compound used to eliminate other contaminating cells, such as astrocytes.

Primary neuronal cultures were obtained from P1-P2 C57bl6 pups as described in Sclip et al., 2013 [32 (link)], with minor modification. In brief, after dissection, hippocampi were incubated with 200 U of papain (P3125, Sigma Aldrich, St. Louis, MO, USA) (30 min, 34 °C), with trypsin inhibitor (T-9253, Sigma Aldrich, St. Louis, MO, USA) (45 min, RT) and subsequently mechanically dissociated. Neurons were seeded on the astrocyte layer both on ibidi flat chamber slides (300,000 cells/mL) and on the Nichoid substrate (1,500,000 cells/mL). The plating medium was B27/neurobasal-A (Gibco-Invitrogen) supplemented with 0.5 mM glutamine (Gibco-Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco-Invitrogen).