Abstract
Organoids production is a key tool for in vitro studies of physiopathological conditions, drug-induced toxicity assays, and for a potential use in regenerative medicine. Hence, it prompted studies on hepatic organoids and liver regeneration. Numerous attempts to produce hepatic constructs had often limited success due to a lack of viability or functionality. Moreover, most products could not be translated for clinical studies. The aim of this study was to develop functional and viable hepatic constructs using a 3D porous scaffold with an adjustable structure, devoid of any animal component, that could also be used as an in vivo implantable system. We used a combination of pharmaceutical grade pullulan and dextran with different porogen formulations to form crosslinked scaffolds with macroporosity ranging from 30 µm to several hundreds of microns. Polysaccharide scaffolds were easy to prepare and to handle, and allowed confocal observations thanks to their transparency. A simple seeding method allowed a rapid impregnation of the scaffolds with HepG2 cells and a homogeneous cell distribution within the scaffolds. Cells were viable over seven days and form spheroids of various geometries and sizes. Cells in 3D express hepatic markers albumin, HNF4α and CYP3A4, start to polarize and were sensitive to acetaminophen in a concentration-dependant manner. Therefore, this study depicts a proof of concept for organoid production in 3D scaffolds that could be prepared under GMP conditions for reliable drug-induced toxicity studies and for liver tissue engineering.
Highlights
The liver is a vital organ with many crucial functions of detoxification, lipid and carbohydrate metabolism, homeostasis maintenance or blood protein synthesis
These functions can be impaired in many pathological conditions such hepatitis, cancer, genetic diseases or long term medication that induce liver fibrosis followed by cirrhosis which is irreversible and often leads to liver failure associated with poor prognosis
We describe for the first time the self-assembly of hepatic constructs in pullulan-dextran porous matrices
Summary
The liver is a vital organ with many crucial functions of detoxification, lipid and carbohydrate metabolism, homeostasis maintenance or blood protein synthesis These functions can be impaired in many pathological conditions such hepatitis, cancer, genetic diseases or long term medication that induce liver fibrosis followed by cirrhosis which is irreversible and often leads to liver failure associated with poor prognosis. Organoid biotechnologies emerged from the idea to recapitulate organogenesis and morphogenesis in order to create in vitro “organ on a chip” systems [6,7] This technology is based on cell self-assembly, differentiation and production of extracellular matrix to produce a system mimicking organ structures. This strategy represents a highly valuable tool in particular for disease modeling. As hepatocytes rapidly lose their phenotype and specific functions when cultured in 2D [8,9,10], this approach has been largely investigated to improve in vitro models for drug-toxicity and liver diseases [11] or for tissue engineering applications including extracorporeal [11,12,13,14] or implantable devices to sustain liver functions during acute phases of diseases [5,15]
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