Effect of Spheroidal Age on Sorafenib Diffusivity and Toxicity in a 3D HepG2 Spheroid Model

Abstract

The enhanced predictive power of 3D multi-cellular spheroids in comparison to conventional monolayer cultures makes them a promising drug screening tool. However, clinical translation for pharmacology and toxicology is lagging its technological progression. Even though spheroids show a biological complexity resembling native tissue, standardization and validation of drug screening protocols are influenced by continuously changing physiological parameters during spheroid formation. Such cellular heterogeneities impede the comparability of drug efficacy studies and toxicological screenings. In this paper, we demonstrated that aside from already well-established physiological parameters, spheroidal age is an additional critical parameter that impacts drug diffusivity and toxicity in 3D cell culture models. HepG2 spheroids were generated and maintained on a self-assembled ultra-low attachment nanobiointerface and characterized regarding time-dependent changes in morphology, functionality as well as anti-cancer drug resistance. We demonstrated that spheroidal aging directly influences drug response due to the evolution of spheroid micro-structure and organo-typic functions, that alter inward diffusion, thus drug uptake.

Ultrastructural Features of Gold Nanoparticles Interaction with HepG2 and HEK293 Cells in Monolayer and Spheroids

“…Analysis of the data obtained by TEM, SEM and light microscopy, clearly indicates that bile capillaries never come to the surface; they are "hidden" inside the spheroids. Correct identification of bile capillaries in HepG2 spheroids by TEM was reported in [ 14 ], and some published TEM studies of HEpG2 spheroids demonstrated pseudosinusoids instead bile capillaries [ 48 , 50 ].…”

“…Previously published studies showed changes in the shape and compactization of HepG2 spheroids during their development; early, middle and late stages of spheroid growth were identified, the duration of which varied in different works and mainly depended on the dose of cell seeding. Common recommendation was to work with HepG2 spheroids at middle stage (beginning from days 4–5 after cell seeding) which is characterized by active function of spheroid cells and absence of cell destruction [ 10 , 14 , 45 ]. Amount of published studies using HEK293 spheroids is incomparably less than studies with HepG2 spheroids; however, authors noted a very fast compactization of HEK293 cells on a non-adhesive surface and regular spherical shape of the spheroids [ 46 , 47 ].…”

“…Immunohistochemistry and fluorescence methods are increasingly introduced in recent years [ 11 , 12 , 13 , 14 , 15 , 45 ]. Published TEM data are scarce and usually present small portions of HepG2 cells [ 14 , 48 ]. Here we describe the ultrastructure of HepG2 spheroids with an emphasis on cell relationships and structure of spheroid outer surface and adjacent areas.…”

“…HepG2 cells cultured in 2D- and 3D-forms are used as experimental model in various studies related with hepatic functions and pathology, drug delivery and safety, toxicology and others [ 11 , 12 , 14 , 15 ]. Such a wide range of applications is due to the unique properties of this cell line, which reproduces structural and functional features of the liver, in particular, in 3D-form (spheroids), which is reviewed in [ 7 , 9 , 11 ].…”

“…Similar to other NPs, AuNPs are studied in cell cultures and in laboratory animals; and in last decade a new experimental model has been developed: multicellular spheroids or micro-tissues (cell cultures in 3D-form); the advantages of spheroids are described in details [ 7 , 8 , 9 , 10 ]. Spheroids that mimic the structure and functions of various tissues have shown their suitability for studies of different problems in modern biomedicine, including the effects of drugs, drug damage to the liver, toxicity of chemical compounds, and human hepatocarcinoma (HepG2) spheroids are considered in such studies as a practically adequate replacement of primary hepatocytes [ 11 , 12 , 13 , 14 , 15 ]. The advent of commercially available devices for cultivation of spheroids has transformed their obtaining from "high art" into affordable technology, which expanded the scope of their application.…”