Injectable hydrogels based on extracellular matrix-derived polymers show much promise in the field of tissue engineering and regenerative medicine. However, the hydrogels reported to date have at least one characteristic that limits their potential for clinical use, such as excessive swelling, complicated and potentially toxic cross-linking process, or lack of shear thinning and self-healing properties. We hypothesized that a collagen hydrogel cross-linked using thiol-Michael addition click reaction would be able to overcome these limitations. To this end, collagen was modified to introduce thiol groups, and hydrogels were prepared by crosslinking with 8-arm polyethylene glycol-maleimide. Rheological measurements on the hydrogels revealed excellent shear-thinning and self-healing properties. Additionally, only minimal swelling (6%) was observed over a period of 1 month in an aqueous buffer solution. Finally, tests using mesenchymal stromal cells and endothelial cells showed that the hydrogels are cell-compatible and suitable for cell encapsulation and delivery. Thus, the reported thiolatedcollagen hydrogel cross-linked using thiol-Michael addition click reaction overcomes most of the challenges in the injectable hydrogel design and is an excellent candidate for cell delivery in regenerative medicine and tissue engineering applications. The hydrogel reported here is the first example of a self-healing hydrogel containing covalent cross-links.

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a fibrotic environment actively supporting and driving hepatocarcinogenesis. Insight into hepatocarcinogenesis in terms of the interplay between the tumor stroma microenvironment and tumor cells is thus of considerable importance. Three-dimensional (3D) cell culture models are proposed as the missing link between current in vitro 2D cell culture models and in vivo animal models. Our aim was to design a novel 3D biomimetic HCC model with accompanying fibrotic stromal compartment and vasculature. Physiologically relevant hydrogels such as collagen and fibrinogen were incorporated to mimic the bio-physical properties of the tumor ECM. In this model LX2 and HepG2 cells embedded in a hydrogel matrix were seeded onto the inverted transmembrane insert. HUVEC cells were then seeded onto the opposite side of the membrane. Three formulations consisting of ECM-hydrogels embedded with cells were prepared and the bio-physical properties were determined by rheology. Cell viability was determined by a cell viability assay over 21 days. The effect of the chemotherapeutic drug doxorubicin was evaluated in both 2D co-culture and our 3D model for a period of 72h. Rheology results show that bio-physical properties of a fibrotic, cirrhotic and HCC liver can be successfully mimicked. Overall, results indicate that this 3D model is more representative of the in vivo situation compared to traditional 2D cultures. Our 3D tumor model showed a decreased response to chemotherapeutics, mimicking drug resistance typically seen in HCC patients.

The tumor micro-environment (TME) of hepatocellular carcinoma (HCC) consists out of cirrhotic liver tissue and is characterized by an extensive deposition of extracellular matrix proteins (ECM). The evolution from a reversible fibrotic state to end-stage of liver disease, namely cirrhosis, is characterized by an increased deposition of ECM, as well as changes in the exact ECM composition, which both contribute to an increased liver stiffness and can alter tumor phenotype. The goal of this study was to assess how changes in matrix composition and stiffness influence tumor behavior. HCC-cell lines were grown in a biomimetic hydrogel model resembling the stiffness and composition of a fibrotic or cirrhotic liver. When HCC-cells were grown in a matrix resembling a cirrhotic liver, they increased proliferation and protein content, compared to those grown in a fibrotic environment. Tumour nodules spontaneously formed outside the gels, which appeared earlier in cirrhotic conditions and were significantly larger compared to those found outside fibrotic gels. These tumor nodules had an increased expression of markers related to epithelial-to-mesenchymal transition (EMT), when comparing cirrhotic to fibrotic gels. HCC-cells grown in cirrhotic gels were also more resistant to doxorubicin compared with those grown in fibrotic gels or in 2D. Therefore, altering ECM composition affects tumor behavior, for instance by increasing pro-metastatic potential, inducing EMT and reducing response to chemotherapy.

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a fibrotic environment actively supporting and driving hepatocarcinogenesis. Insight into hepatocarcinogenesis in terms of the interplay between the tumor stroma microenvironment and tumor cells is thus of considerable importance. Three-dimensional (3D) cell culture models are proposed as the missing link between current in vitro 2D cell culture models and in vivo animal models. Our aim was to design a novel 3D biomimetic HCC model with accompanying fibrotic stromal compartment and vasculature. Physiologically relevant hydrogels such as collagen and fibrinogen were incorporated to mimic the bio-physical properties of the tumor ECM. In this model LX2 and HepG2 cells embedded in a hydrogel matrix were seeded onto the inverted transmembrane insert. HUVEC cells were then seeded onto the opposite side of the membrane. Three formulations consisting of ECM-hydrogels embedded with cells were prepared and the bio-physical properties were determined by rheology. Cell viability was determined by a cell viability assay over 21 days. The effect of the chemotherapeutic drug doxorubicin was evaluated in both 2D co-culture and our 3D model for a period of 72h. Rheology results show that bio-physical properties of a fibrotic, cirrhotic and HCC liver can be successfully mimicked. Overall, results indicate that this 3D model is more representative of the in vivo situation compared to traditional 2D cultures. Our 3D tumor model showed a decreased response to chemotherapeutics, mimicking drug resistance typically seen in HCC patients.

23 24 SUMMARY: 25 A protocol for a novel 3D biomimetic HCC model with accompanying fibrotic stromal 26 compartment and vasculature, to study endocrine and paracrine signaling in liver cancer. The 27 model uses physiological relevant hydrogels in ratios mimicking the bio-physical properties of the 28 stromal extracellular matrix, which is an active mediator of cellular interactions, tumor growth 29 and metastasis. 30 31 ABSTRACT: 32 Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver 33 disease. Chronic liver disease and inflammation leads to a fibrotic environment actively 34 supporting and driving hepatocarcinogenesis. Insight into hepatocarcinogenesis in terms of the 35 interplay between the tumor stroma micro-environment and tumor cells is thus of considerable 36 importance. Three-dimensional (3D) cell culture models are proposed as the missing link 37 between current in vitro 2D cell culture models and in vivo animal models. Our aim was to design 38 a novel 3D biomimetic HCC model with accompanying fibrotic stromal compartment and 39 vasculature. Physiologically relevant hydrogels such as collagen and fibrinogen were 40 incorporated to mimicking the bio-physical properties of the tumor ECM. In our model LX2 and 41HepG2 cells embedded in a hydrogel matrix were seeded onto the inverted insert membrane of 42 a Transwell™ system. HUVEC cells were then seeded onto the opposite side of the membrane. 43 Three formulations consisting of ECM-hydrogels embedded with cells were prepared and the bio-44 physical properties determined by rheology. Cell viability was determined by the AlamarBlue® 45 assay over 21-days. The effect of the chemotherapeutic drug doxorubicin was evaluated in both 46 a 2D co-culture and our 3D model for a period of 72h. We show that this model is viable for 25-47 129 premalignant and tumor microenvironment by incorporating some of the key players in the 130 development of HCC. These include endothelial cells, hepatic stellate cells and malignant 131 hepatocytes, grown in a microenvironment composed of physiologically relevant hydrogels. With : Collagen gels 4 mg/mL containing 1.0 x 10 5 cells/mL seeded onto inserts in varying 442 volumes. (A) Insert on the left 100 µL, middle 150 µL and right 200 µL, directly after seeding. 443 Bubbles in the gels are still present. (B) Insert on the left 200 µL, middle 150 µL and right 100 µL, 444 after 60 min crosslinking. All gels regardless of volume still appear flat. 445 446 Combination collagen and Fibrinogen 447Based on the results from the collagen and fibrinogen concentration ranges the effect of 448 combining the collagen and fibrinogen was evaluated. Three inserts were setup with the 449 following, 4 mg/mL collagen, 20 mg/mL fibrinogen and a 1:1 ration of collagen (4 mg/mL) and 450 fibrinogen (20 mg/mL), see figure 6. Directly after seeding the hydrogels, we observed that the 451 insert with collagen alone still had a flat appearance combined with the occurrence of bubbles 452 within the gel. The inserts ...