Pepsin

Pepsin (Sigma-Aldrich) was dissolved in phosphate-buffered saline (pH 3.0) and then incubated with frozen left ventricular tissue sample (40 μg Pepsin/mg tissue) to digest the heart samples at 37C° for 30 min with gentle shaking. The digestion was then stopped by adding 2% SDS, 0.6M β-mercaptoethanol solution to each tissue suspension. This was followed by a 30-min sonication step to enhance the release of the soluble portion into the solution. Soluble protein fraction was then separated from the insoluble fraction by centrifuging at 10000xg for 90 min at 4C°. The soluble fraction (the supernatant) was then separated from the insoluble protein fraction (pellet) into prelabeled glass tubes. The pellets were then resuspended in DDI water, and both sets of samples were completely dried by heating at 100C° overnight. Hydroxyproline concentration in each sample was then quantified by a colorimetric assay as described below.

The gastric simulation methodology of Bilek and Bayram [30 (link)] was used with some modifications. First, 50 mL of the beverage was mixed with 6.5 mg of pepsin (15,750 units; Sigma-Aldrich; Munich, Germany). The pH of the sample-pepsin solution was adjusted to 2.0 with 1 M HCl and incubated at 37 °C for 2 h. Subsequently, 10 mL were taken and transferred to a dialysis membrane (2 KDa) and mixed with 2.5 mL of a bile acid-pancreatin (Sigma-Aldrich; Munich, Germany). The pH was adjusted to 7.5 with 0.5 M NaHCO₃. Along with this experiment, a buffer solution was prepared in a centrifuge tube with 10 mL of deionized water, the same amount of 0.5 M NaHCO₃, and adjusted to pH 5.0 with 1 M HCl. The dialysis membrane with the sample-pepsin solution was placed inside the centrifuge tube with a buffer and incubated at 37 °C for 2 h. The sample inside the membrane was subjected to hydroxyproline analysis to determine the amount of collagen present after gastric simulation. The percentage of bioavailability was calculated by using Equation (4): $\%\mathrm{Bioavailability}=\frac{\mathrm{Collagen}\mathrm{in}\mathrm{dialysate}\left(\mathrm{g}\right)}{\mathrm{Collagen}\mathrm{in}\mathrm{sample}}\times 100.$

For dECM gelation, the decellularized extracellular matrices of the three cardiovascular tissues were lyophilized and milled to a fine powder. The resulting dECM powder was digested with pepsin (#P6887; >3,200 IU; Sigma-Aldrich, St. Louis, USA) in hydrochloric acid solution (20 mg/mL dECM, 2 mg/mL pepsin, 0.01 M HCl) for 6 h at RT and constant stirring. After digestion, the solution pH was raised with sodium hydroxide (1/10 of solution volume, 0.1 M NaOH) and the electrolytes equilibrated with PBS (1/10 of solution volume, 10x PBS). The resulting solution was stored in liquid form (pre-gel) at 4°C or transformed into hydrogel by warming to 37°C for 1 h.

Lyophilized brain tissue was solubilized following the method of Freytes et al. [34 (link)]. Briefly, B-ECM was powdered with a mill (Wiley Mini Mill, 3383-L10, Thomas Scientific, USA) over a 60 mesh (equal to 250 μm particle size). Then, a desired volume of 0.1 M HCl solution diluted in H₂O was prepared, and pepsin (Sigma, USA) and B-ECM powder added at a ratio of 1:10 (e.g. 1 mg/mL pepsin together with 10 mg/mL B-ECM). After 72h incubation at room temperature with a magnetic stirrer, the solution was centrifuged at 13,000 rpm for 10 min, and the supernatant consisting of the solubilized B-ECM was collected. The solubilized B-ECM was stored at 4°C until the solution was cooled down. The B-ECM solution was then kept on ice and cold 10x PBS was added to make up a volume of 1/10^th of the total volume, resulting in a concentration of 1x PBS. While still on ice, the pH of the solution was adjusted to 7.2–7.8 with cold NaOH, and the solution was quickly frozen and later lyophilized. Then, the lyophilized tissue was powdered once more over a 60 mesh and stored until use. Directly before use, the neutralized B-ECM powder was dissolved in cold H₂O at the desired concentration and strongly vortexed, which led to hydrogel formation at 37°C. In this study, analysis on decellularized B-ECM are labeled as “B-ECM”, and analysis on fully formed B-ECM hydrogels are labeled as “B-ECM hydrogel”.