Small intestinal submucosa (SIS) has been successfully used to treat a variety of damaged or diseased tissues in human patients. As a biologic scaffold, SIS stimulates repair of damaged or diseased tissues and organs with tissue that is similar in structure and function to the material it was meant to replace. To meet clinical safety requirements, biologic materials from animal tissues must undergo processing treatments to minimize host immune response and to eliminate the possibility of disease transmission. The effect of peracetic acid disinfection, lyophilization, and ethylene oxide sterilization on the in vitro bioactivity of the processed SIS was therefore examined in murine fibroblasts and pheochromocytoma (PC12) cells. Specifically, the ability of processed SIS to support fibroblast attachment, to stimulate PC12 cell differentiation, and to upregulate fibroblast VEGF secretion was examined. Fibroblasts attach to the sterilized SIS, remain viable, and more than double their secretion of VEGF as a result of interacting with the SIS matrix components. Additionally, PC12 cells exhibit increased neurite outgrowth following stimulation by SIS matrix proteins versus controls. We conclude that a biologic scaffold can be prepared for human use and still retain significant bioactivity.