Abstract
The generation of hepatic spheroids is beneficial for a variety of potential applications, including drug development, disease modeling, transplantation, and regenerative medicine. Natural hydrogels are obtained from tissues and have been widely used to promote the growth, differentiation, and retention of specific functionalities of hepatocytes. However, relying on natural hydrogels for the generation of hepatic spheroids, which have batch to batch variations, may in turn limit the previously mentioned potential applications. For this reason, we researched a way to establish a three-dimensional (3D) culture system that more closely mimics the interaction between hepatocytes and their surrounding microenvironments, thereby potentially offering a more promising and suitable system for drug development, disease modeling, transplantation, and regenerative medicine. Here, we developed self-assembling and bioactive hybrid hydrogels to support the generation and growth of hepatic spheroids. Our hybrid hydrogels (PC4/Cultrex) inspired by the sandcastle worm, an Arg-Gly-Asp (RGD) cell adhesion sequence, and bioactive molecules derived from Cultrex BME (Basement Membrane Extract). By performing optimizations to the design, the PC4/Cultrex hybrid hydrogels can enhance HepG2 cells to form spheroids and express their molecular signatures (e.g., Cyp3A4, Cyp7a1, A1at, Afp, Ck7, Ck1, and E-cad). Our study demonstrated that this hybrid hydrogel system offers potential advantages for hepatocytes in proliferating, differentiating, and self-organizing to form hepatic spheroids in a more controllable and reproducible manner. In addition, it is a versatile and cost-effective method for 3D tissue cultures in mass quantities. Importantly, we demonstrate that it is feasible to adapt a bioinspired approach to design biomaterials for 3D culture systems, which accelerates the design of novel peptide structures and broadens our research choices on peptide-based hydrogels.
Highlights
Modeling physiologically relevant events using a traditional two-dimensional (2D)monolayer culture system for drug development, disease modeling, transplantation, and regenerative medicine can be challenging for hepatocytes [1–3]
When further examining the growth of spheroids in 3D conditions, we found that the number and size of spheroids were significantly higher in the hybrid hydrogel at 1:1 ratio of PC4/Cultrex as compared of spheroids were significantly higher in the hybrid hydrogel at 1:1 ratio of PC4/Cultrex as compared to other 3D conditions at day 4, 7, and 12 (Figure 5B)
This result indicates that the microenvironment created by hybrid hydrogel at 1:1 ratio of PC4/Cultrex is fa vorable for the proliferation and differentiation of HepG2 cells, which in turn lays a foun dation for further development of liver cell functions
Summary
Modeling physiologically relevant events using a traditional two-dimensional (2D). monolayer culture system for drug development, disease modeling, transplantation, and regenerative medicine can be challenging for hepatocytes [1–3]. The charged amino acid residues (i.e., Tyr and His) in the repetitive motif of pc glue proteins interact with other charged glue proteins (i.e., phosphor-Ser) to form an underwater adhesive via the formation of complex coacervates [19,20,23–25] These characteristics make the sandcastle worm glue protein an intriguing model for the development of adhesive materials that can be used in wet environments. Adhesive proteins from the sandcastle worm can fold into complex yet stable molecular architectures in response to various environmental cues (e.g., pH, divalent cations, salt concentrations) [21,23,25–27] Such properties of the adhesion proteins make them attractive targets for the design and development of 3D cell culture scaffolds. We report a hybrid hydrogel inspired by sandcastle worm glue protein pc combined with the ECM-based natural hydrogel Cultrex, which enhances the physiological relevance for generating functional hepatic spheroids. Our results suggest that these PC4/Cultrex hybrid hydro of 14 gels can provide a reliable performance, for 3D culture systems
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