A Biomimetic Model for Liver Cancer to Study Tumor-Stroma Interactions in a 3D Environment with Tunable Bio-Physical Properties

Abstract

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 Material World of 3D‐Bioprinted and Microfluidic‐Chip Models of Human Liver Fibrosis

“…While maintaining the collagen concentration constant (2 mg mL −1 ), the addition of 10, 30, or 40 mg mL −1 of fibrinogen rendered the hydrogels with stiffnesses of 3, 6, and 10 kPa, respectively, correspondent to mechanical properties similar to those on the onset of fibrosis, cirrhosis and HCC. [ 92 ]…”

“…While maintaining the collagen concentration constant (2 mg mL −1 ), the addition of 10, 30, or 40 mg mL −1 of fibrinogen rendered the hydrogels with stiffnesses of 3, 6, and 10 kPa, respectively, correspondent to mechanical properties similar to those on the onset of fibrosis, cirrhosis and HCC. [92] Collagen matrices have also been used for modeling nonalcoholic steatohepatitis (NASH) in vitro. Hepatocytes, HSCs, endothelial cells and Kupffer cells were co-cultured in a collagen matrix and NASH was induced by the addition of free fatty acids and tumor necrosis factor-𝛼 (TNF-𝛼) to promote steatosis and inflammation, respectively.…”

“…For instance, a collagen matrix embedding cells was seeded on both sides of a Transwell insert, replicating the fibrotic compartment on one side of the membrane and a vascular compartment on the opposite side. [ 92 ] The inclusion of fibrinogen in the collagen matrix allowed the tuning of its stiffness to mimic different stages of liver disease. While maintaining the collagen concentration constant (2 mg mL −1 ), the addition of 10, 30, or 40 mg mL −1 of fibrinogen rendered the hydrogels with stiffnesses of 3, 6, and 10 kPa, respectively, correspondent to mechanical properties similar to those on the onset of fibrosis, cirrhosis and HCC.…”

Uniformity of spheroids-on-a-chip by surface treatment of PDMS microfluidic platforms

“…3 Nevertheless, tumor cells respond to chemotherapy and environmental cues differently when they are cultured in a 3D microenvironment. 1,4 3D cell culture has gained much attention over the past decades with its evident advantages of more physiologically relevant data and more predictive responses in drug discovery and disease modeling. 5 Among various 3D cell culture models, spheroids are cell aggregations formed by self-assembly which act as excellent physiologically relevant models to provide more reliable therapeutic readouts.…”