Papain-Based Solubilization of Decellularized Extracellular Matrix for the Preparation of Bioactive, Thermosensitive Pregels

Abstract

Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties was used as a decisive criterion for further application of the enzymes, with emphasis on minimal destruction of the protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests, and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAG content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogeneous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to those on pure rat tail collagen type I gels. Papain is not only highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human-tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided.

Rise of tissue- and species-specific 3D bioprinting based on decellularized extracellular matrix-derived bioinks and bioresins

“…It breaks down the α-chains of the collagen triple helix into small fragments. Consequently, dECM preparations solubilized with collagenase in our study exhibited significantly reduced viscosity and did not have the ability to form gels [28] . In another study, Jia et al.…”

“…In a recent study, we explored alternative enzymes to pepsin for solubilizing porcine liver for bioink formulation, including plant-derived papain and bacterial-derived α-amylase and collagenase [28] . Among the investigated enzymes, only pepsin- and papain were successful in digesting dECM to later yield stable hydrogels.…”

“…In our study, we found that the α-amylase digestion of porcine liver dECM preserved the sGAG content better than pepsin and papain digestion. However, the overall digestion efficiency was lower, and the resulting gelation properties were impaired [28] .…”

“… Oxidative and acidic residuals left in dECM that require careful purification. [ 28 , [45] , [46] , [47] ] Ethanol (60 % −80 %) Easy, simple safety requirements, and broadly accessible. Ineffective in killing all microorganisms.…”

Hydrogel-Integrated Millifluidic Systems: Advancing the Fabrication of Mucus-Producing Human Intestinal Models

“…All chemicals, buffers, and reagents utilized in this investigation were acquired and prepared as previously published [26,32,33]. The Supplementary Materials (SM) section provides a comprehensive listing of these materials.…”