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.

The luminal surface of the intestinal epithelium is protected by a vital mucus layer, which is essential for lubrication, hydration, and fostering symbiotic bacterial relationships. Replicating and studying this complex mucus structure in vitro presents considerable challenges. To address this, we developed a hydrogel-integrated millifluidic tissue chamber capable of applying precise apical shear stress to intestinal models cultured on flat or 3D structured hydrogel scaffolds with adjustable stiffness. The chamber is designed to accommodate nine hydrogel scaffolds, 3D-printed as flat disks with a storage modulus matching the physiological range of intestinal tissue stiffness (~3.7 kPa) from bioactive decellularized and methacrylated small intestinal submucosa (dSIS-MA). Computational fluid dynamics simulations were conducted to confirm a laminar flow profile for both flat and 3D villi-comprising scaffolds in the physiologically relevant regime. The system was initially validated with HT29-MTX seeded hydrogel scaffolds, demonstrating accelerated differentiation, increased mucus production, and enhanced 3D organization under shear stress. These characteristic intestinal tissue features are essential for advanced in vitro models as they critically contribute to a functional barrier. Subsequently, the chamber was challenged with human intestinal stem cells (ISCs) from the terminal ileum. Our findings indicate that biomimicking hydrogel scaffolds, in combination with physiological shear stress, promote multi-lineage differentiation, as evidenced by a gene and protein expression analysis of basic markers and the 3D structural organization of ISCs in the absence of chemical differentiation triggers. The quantitative analysis of the alkaline phosphatase (ALP) activity and secreted mucus demonstrates the functional differentiation of the cells into enterocyte and goblet cell lineages. The millifluidic system, which has been developed and optimized for performance and cost efficiency, enables the creation and modulation of advanced intestinal models under biomimicking conditions, including tunable matrix stiffness and varying fluid shear stresses. Moreover, the readily accessible and scalable mucus-producing cellular tissue models permit comprehensive mucus analysis and the investigation of pathogen interactions and penetration, thereby offering the potential to advance our understanding of intestinal mucus in health and disease.