Sodium desoxycholate

PBCECs were cultivated in rat tail collagen-coated 24 well culture plates and cultured according to the cultivation in Transwell^® filters. After 72 h incubation of the respective Hg species, PBCECs were washed twice with PBS (100 mM NaCl, 4.5 mM KCl, 7 mM Na₂HPO₄, 3 mM KH₂PO₄ (all Sigma Aldrich); pH 7.4) and incubated with 120 μL lysis buffer (RIPA-buffer; 0.01 M Tris, pH 7.6, 0.15 M NaCl, 0.001 M EDTA, 1% sodium desoxycholate, 0.1% (all Sigma Aldrich)) for 15 min on ice. After scrapping off and sonication, the suspension of lysed cells was centrifuged at 10 000 x g for 20 min at 4 °C. Total cellular Hg content was quantified by inductively coupled mass spectrometry (ICP-MS; Agilent 8800 ICP-QQQ, Agilent Technologies Deutschland GmbH, Boeblingen, Germany) in an aliquot of the supernatant. The Bradford assay was used to determine the cellular protein level.

Scaffolds were cut, clamped into 11 mm diameter cell crowns and afterwards sterilized by autoclavation. Hdf were seeded on top in a concentration of 30,000 cells per square centimeter. Cell culture medium consisted of dulbecco’s modified eagle medium (DMEM, Gibco^® Life Technologies, Carlsbad, CA, USA), containing 10% Fetal Calf Serum (FCS, LOT: BS 210601.5, Bio and SELL GmbH, Feucht, Germany), 1% penicillin/streptomycin (pen/strep, PAA, Coelbe, Germany) and either 100 or 500 µmol L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Asc) (Sigma-Aldrich, Schnelldorf, Germany) for proliferation and ECM synthesis stimulation. Medium was renewed three times a week. Cultivation lasted four weeks under standard cell culture conditions (37 °C, with an amount of 5% CO₂ and a relative humidity of 95%). The complete decellularization process was conducted under sterile conditions. Therefore, specimens were rinsed thrice with phosphate buffered saline (PBS^-) (Sigma-Aldrich) and incubated in decellularization solution containing 22.5 g/L sodium desoxycholate (Sigma-Aldrich, Schnelldorf, Germany) in deionized (DI) water. The procedure was repeated on the following day. Specimens were stored in pen/strep containing PBS- until further usage.

Protein extracts were prepared using a RIPA buffer (50 mM Tris/HCl (pH 8.0), 0.1% SDS, 0.5% sodium desoxycholate (all from Sigma-Aldrich), 150 mM NaCl (Carl Roth, Karlsruhe, Germany), and 1% Triton X-100 (Roche, Penzberg, Germany)) with 5% protease inhibitor (Sigma-Aldrich). Protein concentrations were determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Samples were diluted to equal concentrations with 1× Roti^®-Load 1 (Carl Roth). SDS-PAGE was performed following standard protocols. The Pierce™ Power Blotter (Thermo Fisher Scientific) was used for semi-dry protein transfer to PVDF membranes (Pall, Port Washington, NY, USA). Membranes were blocked with 5% non-fat dried milk (AppliChem, Darmstadt, Germany)/TBS-T (40 mM Tris/HCl (pH 7.6), 273 mM NaCl, 0.1% Tween^® 20 (Sigma-Aldrich)) for 1 h at room temperature. Incubation with primary antibodies was performed in blocking solution overnight at 4 °C, and incubation with secondary antibodies was performed in TBS-T for 1 h at room temperature. Immunoblots were developed with SuperSignal West Pico or Femto Chemiluminescent Substrates (Thermo Fisher Scientific), and signals were detected on a ChemiDoc Touch Imaging System (Bio-Rad). Bands were quantified using Image Lab v6 (Bio-Rad). The antibodies and the concentrations used are listed in Supplementary Table S2.