Abstract
The integration of bile duct epithelial cells (cholangiocytes) in artificial liver culture systems is important in order to generate more physiologically relevant liver models. Understanding the role of the cellular microenvironment on differentiation, physiology, and organogenesis of cholangiocytes into functional biliary tubes is essential for the development of new liver therapies, notably in the field of cholangiophaties. In this study, we investigated the role of natural or synthetic scaffolds on cholangiocytes cyst growth, lumen formation and polarization. We demonstrated that cholangiocyte cyst formation efficiency can be similar between natural and synthetic matrices provided that the mechanical properties of the hydrogels are matched. When using synthetic matrices, we also tried to understand the impact of elasticity, matrix metalloprotease-mediated degradation and integrin ligand density on cyst morphogenesis. We demonstrated that hydrogel stiffness regulates cyst formation. We found that controlling integrin ligand density was key in the establishment of large polarized cysts of cholangiocytes. The mechanism of lumen formation was found to rely on cell self-organization and proliferation. The formed cholangiocyte organoids showed a good MDR1 (multi drug resistance protein) transport activity. Our study highlights the advantages of fully synthetic scaffold as a tool to develop bile duct models.
Highlights
Cellular microenvironment cues, including surface topography, substrate rigidity and biochemical signals determine to a large extent the outcome of many biological processes (Tan et al, 2003; Gobaa et al, 2011; Unadkat et al, 2011)
Based on observations made with other epithelial cell lines we first seeded Normal Rat Cholangiocytes (NRC) in both synthetic, fully defined polyethylene glycol (PEG) hydrogels and in natural extracellular matrix-based Matrigel (MT) (Figure 1A)
Over a culture period of 10 days, cultured NRCs self-organized into hollow spheres or cysts where a very thin layer of epithelial cells marked the boundary between an inner lumen and the extracellular milieu (Video S1)
Summary
Cellular microenvironment cues, including surface topography, substrate rigidity and biochemical signals determine to a large extent the outcome of many biological processes (Tan et al, 2003; Gobaa et al, 2011; Unadkat et al, 2011). The exposure of stem cells to mechanical cues allows steering the differentiation process (Engler et al, 2006). Approaches, based on the use of primary adult hepatic cells (Broutier et al, 2016) or on the controlled differentiation of induced Pluripotent Stem Cells (iPSC) (Takebe et al, 2014) have led to the derivation of new organotypic models. These advanced cell culture systems were developed in order to improve on the physiological relevance of standard 2D culture hepatic cell lines
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