The Tergitol-based decellularization protocol was modified from the TRICOL protocol previously reported [19 (link)].
Decellularization protocol was performed in an agitation system: treatment with protease inhibitors cocktail (1% v/v) and DMSO (supplier) (10%) at 4 °C (8 h) was followed by washing with a hypotonic solution (12 h). Subsequently, a second phase with protease inhibitors in combination with 1% Tergitol (12 h) (Sigma Aldrich, Saint Louis, MO, USA) was carried out at room temperature. After further washing, tissues were treated with Tergitol (0.1%) in a hypertonic solution (24 h in two cycles). Thereafter, they were treated for 20 h with sodium cholate (Sigma Aldrich, 4% v/v). Finally, tissue samples were treated with peracetic acid (Sigma Aldrich, 0.1%) and ethanol 4% (Carlo Erba, Cornaredo, MI, Italy) solutions (90′) for bioburden removal and primary decontamination.
Valves were cut into 8 mm patches with a biopsy puncher and treated with endonuclease enzyme (Benzonase 25 k U, Sigma Aldrich, E1014) at 37 °C for 48 h to complete the removal of nucleic acid residues. Several washing cycles with Phosphate Buffered Saline (PBS, Sigma Aldrich) were performed to remove residues of the enzyme. Samples (Native pulmonary wall (PW Native), Leaflets (LL Native) and Myocardia (MYO Native), Decellularized Pulmonary wall (PW DC), Leaflets (LL DC) and Myocardia (MYO DC))were then embedded in OCT (Tissue-Tek, 4583, Sakura Finetek, Torrance, CA, USA) and stored at −80 °C for histological and IF analysis, while the rest of the tissues were lyophilized for DNA and biochemical analysis. A subgroup of the decellularized valves (n = 3) was used for biomechanical tests.
Decellularization protocol was performed in an agitation system: treatment with protease inhibitors cocktail (1% v/v) and DMSO (supplier) (10%) at 4 °C (8 h) was followed by washing with a hypotonic solution (12 h). Subsequently, a second phase with protease inhibitors in combination with 1% Tergitol (12 h) (Sigma Aldrich, Saint Louis, MO, USA) was carried out at room temperature. After further washing, tissues were treated with Tergitol (0.1%) in a hypertonic solution (24 h in two cycles). Thereafter, they were treated for 20 h with sodium cholate (Sigma Aldrich, 4% v/v). Finally, tissue samples were treated with peracetic acid (Sigma Aldrich, 0.1%) and ethanol 4% (Carlo Erba, Cornaredo, MI, Italy) solutions (90′) for bioburden removal and primary decontamination.
Valves were cut into 8 mm patches with a biopsy puncher and treated with endonuclease enzyme (Benzonase 25 k U, Sigma Aldrich, E1014) at 37 °C for 48 h to complete the removal of nucleic acid residues. Several washing cycles with Phosphate Buffered Saline (PBS, Sigma Aldrich) were performed to remove residues of the enzyme. Samples (Native pulmonary wall (PW Native), Leaflets (LL Native) and Myocardia (MYO Native), Decellularized Pulmonary wall (PW DC), Leaflets (LL DC) and Myocardia (MYO DC))were then embedded in OCT (Tissue-Tek, 4583, Sakura Finetek, Torrance, CA, USA) and stored at −80 °C for histological and IF analysis, while the rest of the tissues were lyophilized for DNA and biochemical analysis. A subgroup of the decellularized valves (n = 3) was used for biomechanical tests.
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