Development of 3D Hepatic Constructs Within Polysaccharide-Based Scaffolds with Tunable Properties

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.

Recent Progress in Polysaccharide Aerogels: Their Synthesis, Application, and Future Outlook

“…After the freezing process, the solvent generally forms a dry powder, which is removed by vacuum or hydrothermal treatment. In addition, the effects of the temperature, time, and direction of ice growth during freezing will affect the physical and mechanical properties of the aerogel [ 101 ]. For instance, pore structures with different diameters are formed under high- or low-pressure freeze drying.…”

In Vitro Strategies to Vascularize 3D Physiologically Relevant Models

“…However, due to the heterogeneous composition and immunogenic potential of currently used matrices, an alternative ECM-mimicking source should be considered. [13] Alternatively, biomaterial-based 3D scaffolds have been employed to mimic the components of the ECM while providing structural support and external cues to guide cell-cell and cell-matrix interactions, leading to functional and vascularized spheroids. [147,[152][153][154] While these scaffolds can www.advancedsciencenews.com www.advancedscience.com www.advancedsciencenews.com www.advancedscience.com provide mechanical and biochemical cues for cell growth within the 3D structures, lack of access to adequate supply of oxygen and nutrients to the center of the structure often results to necrotic core and premature growth in the outer layer of organoids, when missing an adequate vascularization of the 3D constructs.…”

“…Spheroids are established from simple clusters of cells, ranging from immortalized cell lines, primary cells, or fragments of human tissue. [ 13 , 111 ] Spheroid technology was developed based on the ability of cells to self‐organize during embryonic development. This self‐assembly process takes place in vitro when cells cannot attach to their biomaterial surface, hence aggregate into spherical 3D structures, namely, spheroids.…”

“…In parallel, new biomaterials have been developed to mimic the cell niche, with advancements from 2D culture on extracellular matrix (ECM) gels (i.e., Matrigel) to 3D scaffolds with tunable physical–chemical and mechanical properties. [ 12 , 13 , 14 ] These systems have been extensively used as in vitro models consisting of multiple cell types and the combination with bioreactors has allowed researchers to provide the cells with physiological‐like biochemical and mechanical cues. Recently, these in vitro models have often adopted the emerging strategy of 3D bioprinting to engineer more complex systems, eventually replacing the conventional fabrication methods.…”

Polymeric Scaffolds: Design, Processing, and Biomedical Application

“…A very promising method of producing scaffolds mimicking the extracellular matrix with bioactive functional motifs is proposed by Pugliese and Gelain [ 13 ], who designed self-assembled peptides exhibiting self-healing properties. The last article shows an original approach to the production of hepatic organoids based on pullan and dextran matrices [ 14 ]. Organoid technology is more and more often used as an in vitro tool for testing novel drugs.…”