Sodium cholate c1254

Cloned gene of amyloid beta (pCNS-Aβ-cDNA), Cat # KU000776) was obtained from the Human Gene Bank of Korea (Daejeon, Korea). The pET30a(+) expression vector and E.coli BL21 (DE3) were purchased from Novagen (Madison, WI, USA). The restriction enzymes were acquired from New England BioLabs (Beverly, MA, USA). Palmitoyloleoyl phosphatidylcholine (POPC, #850457) was supplied by Avanti Polar Lipids (Alabaster, AL, USA). Sodium cholate (#C1254) was procured from Sigma (St. Louis, MO, USA).

Porcine SIS was decellularized with an optimized protocol, which included mechanical agitation and the use of protease inhibitors, alternating with hypotonic and hypertonic solutions. We incubated tissue with a non-ionic detergent (Tergitol, 15S9, Sigma-Aldrich, Saint Louis, MO, USA) and an ionic one (Sodium Cholate, C1254, Sigma-Aldrich, Saint Louis, MO, USA) with intermediate washing cycles in PBS or saline solution, with a subsequential alcohol-based solution for tissue decontamination and delipidization, and endonuclease (Benzonase^®, E1014, Sigma-Aldrich, Saint Louis, MO, USA) treatment to remove the residual DNA fragments.
The resulting acellular tissue was treated with two different protocols (the first based on 70% ethanol/water and the second based on an antibiotic/antimycotic plus peracetic acid solution) to sterilize it in preparation for cytocompatibility tests.

Jejunum was collected from an abattoir and treated within 3 hrs. after animal sacrifice. The abattoir’s protocols followed EC guidelines 1099/2009 concerning animal health and protection at the time of sacrifice, was supervised by the Italian government, and was approved by the associated legal authorities of animal welfare (Food and Consumer Product Safety Authority). SIS isolation was obtained accordingly to an already described procedure [32 (link),40 (link),41 (link)] by removing the two external muscular layers and serosa and the internal tunica mucosa. Thereafter, SIS was rinsed in phosphate-buffered saline (PBS) and decellularized following the optimized procedure described in [32 (link)]. Decellularization is based on the use of protease inhibitors, hypo- and hyper-tonic solutions, Tergitol 15S9 (Sigma-Aldrich, Saint Louis, MO, USA) and Sodium Cholate C1254 (Sigma-Aldrich, Saint Louis, MO, USA), with a subsequent alcohol-based solution. Finally, an endonuclease (Benzonase^®, E1014, Sigma-Aldrich, Saint Louis, MO, USA) was used to remove DNA residues.