An Appropriate Selection of a 3D Alginate Culture Model for Hepatic Huh-7 Cell Line Encapsulation Intended for Viral Studies

Abstract

Three-dimensional (3D) culture systems have been introduced to provide cells with a biomimetic environment that is similar to in vivo conditions. Among the polymeric molecules available, sodium-alginate (Na-alg) salt is a material that is currently employed in different areas of drug delivery and tissue engineering, because it offers biocompatibility and optimal chemical properties, and its gelation with calcium chloride provides calcium-alginate (Ca-alg) scaffolds with mechanical stability and relative permeability. In this work, four different preparations of Ca-alg beads with varying Na-alg viscosity and concentration were used for a human hepatoma cell line (Huh-7) encapsulation. The effects of Ca-alg bead preparation on structural cell organization, liver-specific functions, and the expression of specific receptors implicated in hepatotropic virus permissivity were evaluated. Hepatic cells were cultured in 500 μm diameter Ca-alg beads for 7 days under dynamic conditions. For all culture systems, cell viability reached almost 100% at day 7. Cell proliferation was concomitantly followed by hepatocyte organization in aggregates, which adopted two different morphologies (spheroid aggregates or multicellular channel-like structures), depending on Ca-alg bead preparation. These cellular organizations established a real 3D hepatocyte architecture with cell polarity, cell junctions, and abundant bile canaliculi possessing microvillus-lined channels. The functionality of these 3D cultures was confirmed by the production of albumin and the exhibition of CYP1A activity over culture time, which were variable, according to Ca-alg bead condition. The expression of specific receptors of hepatitis C virus by Huh-7 cells suggests encouraging data for the further development of a new viral culture system in Ca-alg beads. In summary, this 3D hepatic cell culture represents a promising physiologically relevant system for further in vitro studies and demonstrates that an adequate encapsulation condition can be selected for each target application in liver tissue engineering, specifically in viral studies.

Impact of Alginate Composition: From Bead Mechanical Properties to Encapsulated HepG2/C3A Cell Activities for In Vivo Implantation

“…4). The same behaviors for cell reorganization have been observed in the literature for hepatic cell-lines encapsulated in alginate [46], [47]. Moreover, due to a better interaction between the cells and the biomaterial, HepG2/C3A cell proliferation was more important in the compositions where collagen was mixed to alginate.…”

Controlled Generation of Microspheres Incorporating Extracellular Matrix Fibrils for Three‐Dimensional Cell Culture

“…Other studies have shown that MVM and MVG do not significantly affect encapsulated cell viability. [36] …”

Alginate-embedded HuH-7 cells increase MMP-9 and reduce OCLN expression in vitro

“…Many different 3D models were developed for different approaches [10] and one of them is the use of alginate capsules [19]. Sodium alginate is one of the materials most commonly chosen because it form hydrogels with desirable characteristics such as lightweight, flexibility and mechanical stability [20].…”

“…In order to improve the model, an increment in time culture could be carried out to asses if a longer period induces different expression of other markers. It would also be important to asses different alginate concentrations to investigate the effect of this variable on EMT and metastatic progression markers in HuH-7 cells, according to studies performed for viral infections [19]. …”