Development of liver microtissues with functional biliary ductular network

Abstract

Liver tissue engineering aims to create transplantable liver grafts that can serve as substitutes for donor's livers. One major challenge in creating a fully functional liver tissue has been to recreate the biliary drainage in an engineered liver construct through integration of bile canaliculi (BC) with the biliary ductular network that would enable the clearance of bile from the hepatocytes to the host duodenum. In this study, we show the formation of such a hepatic microtissue by coculturing rat primary hepatocytes with cholangiocytes and stromal cells. Our results indicate that within the spheroids, hepatocytes maintained viability and function for up to 7 days. Viable hepatocytes became polarized by forming BC with the presence of tight junctions. Morphologically, hepatocytes formed the core of the spheroids, while cholangiocytes resided at the periphery forming a monolayer microcysts and tubular structures extending outward. The spheroids were subsequently cultured in clusters to create a higher order ductular network resembling hepatic lobule. The cholangiocytes formed functional biliary ductular channels in between hepatic spheroids that were able to collect, transport, and secrete bile. Our results constitute the first step to recreate hepatic building blocks with biliary drainage for repopulating the whole liver scaffolds to be used as transplantable liver grafts.

Production of Multiple Cell‐Laden Microtissue Spheroids with a Biomimetic Hepatic‐Lobule‐Like Structure

“…In contrast, non‐structured microtissue spheroids showed transitory morphogenic shift by day 7 as budding and surface localization of endothelial cells occurred because of the self‐organization of hepatic multicellular spheroids, as previously reported. [ 40,41 ] The cross‐section of the structured microtissue spheroid showed a clear pattern and difference with the outer surface. As shown in Figure 3, the designed structure was implemented in microtissue spheroids and maintained for at least 10 days.…”

A Critical Perspective on 3D Liver Models for Drug Metabolism and Toxicology Studies

“…Non-parenchymal cells have a key role in liver injury. Thus, the incorporation of stellate cells, Kupffer cells, and sinusoidal endothelial cells in liver cell models has been attempted for improving the prediction of drug toxicity ( Messner et al, 2013 ; Bell et al, 2016 ; Leite et al, 2016 ; Proctor et al, 2017 ; Hafiz et al, 2020 ; Nudischer et al, 2020 ). Proctor et al (2017) demonstrated the higher predictive value of 3D human liver microtissues (multicellular spheroids), consisting of a co-culture of hpHep, Kupffer cells and liver endothelial cells, due to the increased sensitivity in identifying hepatotoxic drugs within a panel of 110 compounds, when compared to 2D-cultured hpHep ( Proctor et al, 2017 ).…”

Prospect of in vitro Bile Fluids Collection in Improving Cell-Based Assay of Liver Function

“…Spheroids increase cell density, cell-contact polarity, and culture plasticity, including coculture modulation and ECM inclusion (Soldatow et al, 2013;Ramaiahgari et al, 2014;Fatehullah et al, 2016). Unlike sandwich configurations, liver cultures in spheroid configurations allow multiple canalicular sites, thus maintaining superior phase I and II enzyme activities (Soldatow et al, 2013;Ramaiahgari et al, 2014) with a culture age Ni et al, 2016;Ahmed et al, 2017;Kizawa et al, 2017;Vorrink et al, 2017;Baze et al, 2018;Underhills and Khetani, 2018;Fiorotto et al, 2019 (Continued) Frontiers in Toxicology | www.frontiersin.org Swift et al, 2010;Marion et al, 2012;Fukuda et al, 2014;Bachour-El Azzi et al, 2015;Ni et al, 2016;Yang et al, 2016;Yan et al, 2017;Ying et al, 2018 Tanimizu et al, 2007Tanimizu et al, , 2012Kido et al, 2015;Sampaziotis et al, 2015;Miura et al, 2018;Rizki-Safitri et al, 2018Funfak et al, 2019;Du et al, 2020;Hafiz et al, 2021 Potentially high Potentially high Katsuda et al, 2013;Vyas et al, 2018;Wu et al, 2019 Integrated multiorgans Liver-intestine model, multiorganoid chip system Human High d -* Potentially high Maschmeyer et al, 2015;Chen et al, 2017;Choe et al, 2017;…”

“…Liver spheroids comprising hepatocytes, biliary cells, and fibroblasts have had biliary cyst structures on their periphery. The structures developed into duct-like structures that connected liver spheroids while possibly transporting the bile conjugate (Hafiz et al, 2021 ). A collagen culture insert has demonstrated the likelihood of transporting bile conjugate from the hepatocyte to the bile-duct structure (Rizki-Safitri et al, 2020 ).…”