The native extracellular matrix (ECM) biomaterial derived from small intestinal submucosa (SIS) is widely applied in tissue engineering for tissue repair and regeneration. SIS ECM is obtained through physical and chemical methods to remove the intrinsic cells, which would otherwise cause adverse immune reactions when the SIS ECM is implanted into the host body. Several research teams have reported diverse SIS decellularization methods. However, there was no consensus on the criteria to be used for the decellularization methods for SIS and further research on the mechanism action of SIS is needed for comprehensive detection of the biological composition. In this present study, we used three reported methods to prepare SIS and compared their effects on decellularization and the remaining biological components, microstructure and cytocompatibility. SIS prepared by the three kinds of decellularization methods all achieved the recommended criteria, had good biocompatibility and retained most active components. Nevertheless, regardless of which decellularization method was used, the microstructure and bioactive components of the prepared SIS were damaged in varying degrees. We recommend that researchers need to select a decellularization method that would be appropriate to use according to their research purposes. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 689–697, 2019.

Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.