Porcine pepsin p7000

APCS was prepared from the native porcine cornea (NPC), as described previously [19 (link)]. In short, NPC was freeze dried (Labconco, USA), powdered by a freezer-mill (SPEX, USA), and filtered through a 40-mesh screen. Comminuted acellular corneal stoma powder ranging from 10–30 mg was separately enzymatically digested in 8 mg/ml porcine pepsin (P7000, ≥250 unites/mg, Sigma) at 0.01N HCl with shaking for 48 h at 25 °C. The obtained APCS digest solutions were frozen until use. Gelation was induced by neutralizing the pH and salt concentration at 4°C followed by warming to 37 °C. In detail, pH neutralization was accomplished by adding one-tenth of the digest volume of 0.1 N NaOH, and the salt concentration was adjusted by one-ninth the volume of 10x PBS. After that, the neutralized digest (APCS-sol) was placed in a non-humidified incubator heated to 37 °C until the acellular porcine corneal stromal hydrogel (APCS-gel) was formed. As a control to compare the characteristics of the APCS-gels, fibrin gels were prepared with final concentrations of 7.4 mg/ml fibrinogen and 5.4 U/ml thrombin as previously reported [25 (link), 26 (link)]. Additionally, a commercial fibrin gel, Tisseel (Baxter Healthcare Corporation, Deerfield, IL, USA) was also included which was produced by fibrinogen (67–106 mg/ml) and thrombin (400–625 IU/ml) according to the manufacturer’s instructions.