Responsive polymer‐assisted 3D cryogel supports Huh7.5 as in vitro hepatitis C virus model and ectopic human hepatic tissue in athymic mice

Abstract

The three‐dimensional (3D) cell culture models serve as the interface between conventional two‐dimensional (2D) monolayer culture and animal models. 3D culture offers the best possible model system to understand the pathophysiology of human pathogens such as hepatitis C virus (HCV), which lacks a small animal model, due to narrow host tropism and non‐permissiveness of murine hepatocytes. In this study, functionally robust spheroids of HCV permissive Huh7.5 cells were generated, assisted by the temperature or pH‐responsive polymers PNIPAAm and Eudragit respectively, followed by the long‐term growth of the multilayered 3D aggregates in poly(ethylene glycol) (PEG)–alginate–gelatin (PAG) cryogel. The human serum albumin (HSA), marker of hepatic viability was detected up to 600 ng/ml on 24th day of culture. The 3D spheroid culture exhibited a distinct morphology and transcript levels with the upregulation of hepato‐specific transcripts, nuclear factor 4α (HNF4α), transthyretin (TTr), albumin (Alb), phase I and phase II drug‐metabolizing genes. The two most important phase I enzymes CYP3A4 and CYP2D6, together responsible for 90% metabolism of drugs exhibited up to 9‐ and 12‐fold increment, respectively in transcripts. The 3D culture was highly permissive to HCV infection and supported higher multiplicity of infection compared to monolayer Huh7.5 culture. Quantitation of high levels of HSA (500–200 ng/ml) in circulation in mice for 32 days asserted integration with host vasculature and in vivo establishment of 3D culture implants as an ectopic human hepatic tissue in mice. The study demonstrates the 3D spheroid Huh7.5 culture as a model for HCV studies and screening potential for anti‐HCV drug candidates.

Bioengineered Liver Cell Models of Hepatotropic Infections

“…Despite the wide use of Matrigel™ and other basement membrane mimetic hydrogels, their animal origin and inconsistency between lots makes them less favorable than fully synthetic polymers, such as Mebiolgel [ 105 ] and PAG (poly(ethylene glycol)- alginate-gelatine) [ 106 ]. Mebiolgel is a synthetic polymer with thermo-reversible gelation, composed of thermo-responsive and hydrophilic polymer blocks.…”

“…The macroporous PAG cryogel, produced from smart polymer responsive to pH and temperature, has recently been employed to cultivate Huh-7.5 spheroids for up to 30 days. These cultures were explored to assess the potential of monoclonal antibodies in blocking the entry of HCVcc [ 106 ]. While PEG provides hydrophilicity to the scaffold, the biopolymers alginate and gelatine supply support and adherence moieties, effectively promoting cell–cell contact and cell–ECM interactions underlying the formation of hepatic cell spheroids [ 106 ].…”

“…These cultures were explored to assess the potential of monoclonal antibodies in blocking the entry of HCVcc [ 106 ]. While PEG provides hydrophilicity to the scaffold, the biopolymers alginate and gelatine supply support and adherence moieties, effectively promoting cell–cell contact and cell–ECM interactions underlying the formation of hepatic cell spheroids [ 106 ]. Mebiolgel [ 105 ] and the PAG cryogel [ 106 ] had a modulatory effect on the spheroids, limiting their size to up to 150 µm, while providing enough surface area for cell migration and nutrient and oxygen bulk flow, hindering the formation of necrotic cores.…”

Hydrophilic cryogels as potential 3D cell culture materials: Synthesis and characterization of 2‐(methacrylamido) glucopyranose‐based polymer scaffolds

“…Besides the use in the cryopreservation of cells, [17][18][19][20][21][22] there are lots of examples of cryogels used for cell culture applications. A variety of synthetic polymers like poly(acrylonitrile), 23 poly(N-isopropylacrylamide), 23,24 poly(acrylamide), 23 poly(vinyl alcohol), 25 poly(2-hydroxypropylmethacrylamide), 26 poly(2-hydroxyethyl methacrylate) (HEMA), [27][28][29] or poly(ethylene glycol) [30][31][32] have been used for cryogel preparation separately or in combination with biopolymers like dextran, 27,28 gelatin, 24,29,32,33 heparin, 30 alginate, 33 or hyaluronan. 17 Since highly hydrophilic materials are better mimics of aqueous in vivo environments, 1 cryogels with an increasing amount of the water-binding sugar-based building block 2-(methacrylamido) glucopyranose (MAG) were prepared in this study.…”