3D culture models to study SARS-CoV-2 infectivity and antiviral candidates: From spheroids to bioprinting

Abstract

The pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is receiving worldwide attention, due to the severity of the disease (COVID-19) that resulted in more than a million global deaths so far. The urgent need for vaccines and antiviral drugs is mobilizing the scientific community to develop strategies for studying the mechanisms of SARS-CoV-2 infection, replication kinetics, pathogenesis, host-virus interaction, and infection inhibition. In this work, we review the strategies of tissue engineering in the fabrication of three-dimensional (3D) models used in virology studies, which presented many advantages over conventional cell cultures, such as complex cytoarchitecture and a more physiological microenvironment. Scaffold-free (spheroids and organoids) and scaffold-based (3D scaffolding and 3D bioprinting) approach allow the biofabrication of more realistic models relevant to the pandemic, to be used as in vitro platforms for the development of new vaccines and therapies against COVID-19.

Keywords: 3D bioprinting; COVID-19; Organoids; SARS-CoV-2; Tissue engineering.

Fig. 1

Tissue engineering strategies to produce 3D in vitro models for studying SARS-CoV-2 infection of different organs and tissues, host–virus interaction, replication kinetics and drug screening.

Fig. 2

Fabrication of spheroids using a rotating wall vessel bioreactor. (A) Scheme illustrating the spheroids assembly and formation in the rotating wall vessel bioreactor. (B) Image of the bioreactor system. (C) Scanning electron microscopy of human neural progenitor spheroids used in SARS-CoV infection studies. Reproduced with permission from Elsevier [60].

Fig. 3

SARS-CoV-2 infecting intestinal organoids. (A) Immunofluorescent images showing the progressive SARS-CoV-2 infection, being possible to observe the infection clusters spreading through the organoid after 60 days. NP = Nucleoprotein. (B) Immunofluorescent images of dividing cells (KI67-positive) and post-mitotic enterocytes identified by Apolipoprotein A1 (APOA1) (pointed arrows). (C) ACE2 staining in intestinal organoids in expansion (EXP) and differentiated (DIF). Reproduced with permission from AAAS [42].

Fig. 4

Comparison between 2D and 3D models after infection with Cowpox virus (CPXV) for 48 h and treated with different concentrations of gefitinib. Immunofluorescence images showed that gefitinib strongly inhibited infection in 3D model when treated with the lowest concentration of the inhibitor, while cells in the 2D model remained infected even at 25 μM of gefitinib. Infected cells were visualized with GFP (green) and cellular nuclei was visualized with DAPI (blue). Scale bar = 100 μm. Reproduced with permission from Elsevier [52].

Fig. 5

3D bioprinting lung model for influenza A infection. (A) 3D bioprinted structures with different shapes (i) and the schematic illustration of the bioprinting process, in which alginate, gelatin, and Matrigel™ are extruded, followed by the alginate crosslinking with calcium ions (Ca²⁺), and the removal of gelatin during incubation at 37 °C. (B) Immunohistochemistry of the 3D bioprinted construct and 2D monolayer infected with H3N2 strain for 24 h, and stained with anti-nucleoprotein antibody, showing the widespread distribution of the virus in the bioprinted model. Reproduced from Nature Research [93]. http://creativecommons.org/licenses/by/4.0/.