This thesis advances the field of bioprinting and tissue modeling by addressing the limitations of current processing protocols for dECM-based bioresins. It explores effective enzymes for thermogelling dECM pre-gels, investigates the grafting of different methacryloylated groups on dECM for vat 3D bioprinting, and uses 3D printing to fabricate advanced dynamic culture systems that apply mechanical shear stress on epithelial cells to develop advanced in vitro tissue models. Precise control over the physical, mechanical, and biochemical properties of dECM hydrogels after solubilization and gelation is crucial for their application. While animal-derived pepsin is traditionally used for solubilization of dECM, in our first study, we investigated various nonanimal-based alternatives, including plant-derived papain, bacterial-based collagenase, and α- amylase. Although α-amylase-digested dECM exhibited the highest content of growth factors and cytokines, both α-amylase and collagenase treatments disrupted collagen self-assembly of dECM digests and compromised the protein structural integrity. Moreover, their insufficient digestion efficiency resulted in significantly reduced yields of dECM digests (∼ 7 – 10 %). In contrast, papain preserved the physical gel formation capacity of digested dECM and demonstrated comparable yields (∼79-86 %), thermo-gelation kinetics, and bulk mechanical properties to pepsin, along with increased amounts of retained growth factors and superior cell adhesion. As a conclusion, papain was found to be a cost-effective and reproducible alternative to pepsin for producing liver-derived dECM hydrogels, eliminating the risk for zoonotic disease transmission. The second study covered functionalization of papain-digested liver dECM with glycidyl methacrylate (dECM-GMA) and methacrylic anhydride (dECM-MA) into photoactive bioresins for vat photopolymerization. Both chemical modifications yielded a comparable degree of functionalization (DOF ∼ 81.5 – 83.5 %) without disrupting the dECM structure. Interestingly, dECM-GMA showed enhanced solubility and higher shear viscosity compared to dECM-MA due to the additional hydroxyl groups and the increased hydrogen bonding. When formulated into resins, both modifications yielded a similar degree of photocrosslinking (30 – 60 %, depending on the condition), enabling efficient on-demand crosslinking and good printing fidelity. The bulk stiffness of 3D printed dECM-GMA hydrogels was slightly softer (∼ 0.2 – 1.2 kPa) compared to dECM-MA (∼ 0.5 – 2 kPa), both being within the range of healthy human liver tissue (∼ 0.15 – 5.9 kPa), making them suitable candidates for bioprinting of HepaRG cells. dECM-GMA hydrogels exhibited enhanced hydrophilicity and cell compatibility and faster biodegradation compared to dECM-MA gels, highlighting the critical role of chemical functionalization in optimizing bioresins for vat bioprinting. In the third study, the papain digestion protocol established in the first study was applied to porcine intestinal dSIS, achieving characteristics similar to liver dECM and thereby demonstrating its versatility. Functionalized with methacrylic anhydride (MA) for vat 3D printing, dSIS produced biomimetic dSIS-MA hydrogels with tunable stiffness (3.7 ± 0.2 kPa), matching healthy human small intestinal tissue (∼ 1.3 – 4.0 kPa). Moreover, the functionalization enhanced biostability of the hydrogels when seeded with intestinal epithelial cells, essential for advanced intestinal in vitro tissue models. To enable dynamic cell culture conditions, a biocompatible 3D-printed millifluidic tissue chamber was designed to host cellular dSIS-MA hydrogel scaffolds while maintaining a laminar flow profile, as confirmed by computational fluid dynamics simulations. The millifluidic system enables advanced intestinal models with tunable stiffness and fluid shear stresses, offering a scalable and cost-effective solution for in vitro tissue modeling. As demonstrated by culturing HT29-MTX monolayers under low physiological shear stress (∼ 0.01 dyne/cm2), the system induced 3D tissue reorganization and enhanced differentiation and mucus production, particularly MUC5AC, when compared to static culture conditions as evidenced by immunofluorescent proteins staining. Experiments with human organoid-derived ileum ISCs showed that physiological shear stress combined with biomimetic hydrogel scaffolds decreased proliferation and stemness of the cells while promoting their multi-lineage differentiation. Changes in alkaline phosphatase (ALP) activity and secreted mucus demonstrated functional cell differentiation into enterocyte and goblet cell lineages. The studies in this thesis covered the critical steps in dECM bioprinting, from enzyme-based solubilization of dECM through their chemical functionalization for vat (bio)printing until the integration of advanced fluidic systems for tissue modeling. The studies in this thesis covered the critical steps in dECM bioprinting, from enzyme-based solubilization of dECM through their chemical functionalization for vat (bio)printing until the integration of advanced fluidic systems for tissue modeling. Each step advances the understanding and development of biomimical materials and strategies, enhancing the efficiency and functionality of bioengineered tissues. This paves the way for future applications in regenerative medicine and tissue engineering.
