8 marzo 2018

Elasticity-based development of functionally enhanced multicellular 3D liver encapsulated in hybrid hydrogel

Current in vitro liver models provide 3D microenvironments perform more accurate in vivo mimicry than 2D models. Lee HJ et al. from Korea Research Institute of Bioscience and Biotechnology (KRIBB), prepared hybrid hydrogels of varying elasticity and compared them with a normal liver, to develop a more mature liver model that preserves liver properties in vitro. Their 3D liver organoid represents critical progress in developing a biomimetic liver system and provides a versatile platform in drug development and disease modeling, ranging from physiology to pathology.

Current in vitro liver models combined with tissue engineering technologies provide various 3D microenvironments, which can be expected to mimic in vivo conditions more accurately than the 2D models in drug efficacy and toxicity tests for preclinical drug development (Malinen MM et al., 2014; S.C. Ramaiahgari et al., 2014; Y. Takahashi et al., 2015; C.C. Bell et al., 2016). However, a more physiologically relevant and functionally improved in vitro liver system is still lacking. Primary human hepatocytes (PHH) are regarded as the gold standard in vitro model to evaluate hepatic metabolism (P. Gunness et al., 2013). However, limited availability and the absence of proliferative capacity are significant obstacles when primary hepatocytes are used in an in vitro liver model (Godoy P et al., 2009). HepaRG, a liver progenitor cell line, is a useful alternative source of human primary hepatocytes, which proliferates and then differentiates to hepatocytes and biliary cells possessing stable phenotype and functional capacity of Phase I and II xenobiotic metabolizing enzymes and transporters over other hepatic cell lines (Gripon P et al., 2002; Andersson TB et al., 2012; Gerets et al., 2012). HepaRG cells also enhance metabolic functions in a 3D culture format compared with conventional, 2D monolayer cultures (M. Rimann & U. Graf-Hausner; 2012). Therefore, Lee HJ and colleagues from Korea Research Institute of Bioscience and Biotechnology (KRIBB) co-encapsulated HepaRG cells with stromal cells [human sinusoidal endothelial cells (ECs) fibroblasts] in malleable substrates such as hydrogels with varying stiffness in order to emulate the hepatic microenvironment.

In their study, scientists prepared various concentrations of this hybrid hydrogel, with lower, similar, or higher starting stiffness values than a normal liver. Undifferentiated HepaRG, which is a hepatic progenitor cell, was differentiated into hepatocytes and biliary cells within a selected composition of semi-IPNs (semi-interpenetrating networks, hydrogel) and co-cultured with endothelial and mesenchymal lineages under the presence of a differentiation agent (dimethyl sulfoxide; DMSO) for 2 weeks. The elastic modulus of the 3D liver changed dynamically during culture by virtue of prolonged degradation of the hydrogel combined with formation of extracellular matrix provided by the supporting cells. This resulted in both, phenotypic and functional maturation of the 3D liver, including enhancements of the hepatic gene expression, albumin (Alb) secretion, cytochrome p450-3A4 (CYP3A4) enzyme activity, and drug metabolism. Next, embryonic stem cell-derived hepatocytes (ESC-Hep) and PHH were tested in the developed 3D liver model to validate its feasibility and expandability. ESC-Hep also showed mechanical compliance for albumin production similar to the results with HepaRG cells, although the absolute amount of albumin production was relatively low compared to HepaRG. Importantly, PHH showed high viability and functionality in a soft substrate, and this soft 3D liver supported prolonged cell survival, CYP3A4 activity, and albumin production along with tissue remodeling over 28 days in culture. These results suggest that the integrative processes of co-cultures with supporting cells and dynamic changes of elasticity over the in vitro culture period are critical for the 3D liver function, and recapitulation of the in vivo mimic stiffness could improve hepatic function or mimic diseased liver state. Therefore, this 3D model can be considered a critical advancement in developing a biomimetic liver system to simulate liver tissue remodeling either in the normal state or in pathophysiological model systems such as fibrosis and cirrhosis.

Fig. 1 from Lee HJ et al., 2017. Brief outline of the 3D cell culture scheme and macroscopic representative images of HepaRG cells either (A) encapsulated alone (HepaRG) or (B) with ECs and fibroblasts (co-culture) in semi-IPNs at 4%, 6%, and 8% w/w for PEGdA and 0.24%, 0.36%, and 0.48% w/w for HA, respectively (Lee HJ et al., 2017).

Fig. 7 from Lee HJ et al., 2017. Schematic illustration of our findings of the 3D cell culture in PEGdA/HA semi-IPNs with varying elastic modulus. Along with the formation of 3D supporting extracellular matrix from the co-cultured cells and with the dynamic changes in the elasticity during the culture period, the 3D microenvironment in the specific elastic modulus range—approximately from 2.8 to 6.17 kPa as measured in this study, which is close to the normal liver stiffness—enhanced the hepatocyte differentiation and eventually liver function. Scale bars = 50 µm.



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