key: cord-0741979-uvxxnk8h
authors: de Melo, Bruna A.G.; Benincasa, Julia C.; Cruz, Elisa M.; Maricato, Juliana Terzi; Porcionatto, Marimelia A.
title: 3D Culture Models to Study SARS-CoV-2 Infectivity and Antiviral Candidates: From Spheroids to Bioprinting
date: 2020-11-21
journal: Biomed J
DOI: 10.1016/j.bj.2020.11.009
sha: eee8eea242d9e559276a36ae131a9c479c657ec4
doc_id: 741979
cord_uid: uvxxnk8h

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.

Despite its importance, 2D cell culture models fail to recapitulate the complexity of living organisms and often acquire phenotypes that differ significantly from native tissues, which leads to poor prediction of results [7] . Therefore, the use of platforms that provide increased similarity to the in vivo physiology and pathology can contribute to advances in the treatments of COVID-19.

In the past years, the three-dimensional (3D) approach has been widely used in cell culture studies, due to their increased capacity of simulating with greater fidelity the cellular microenvironment, as compared to the 2D cell culture, leading to improved cell responses regarding morphology, proliferation capacity, and gene expression profiles [8] . With the advances of tissue engineering, novel technologies have emerged and been used as more realistic in vitro models, allowing the construction of complex cytoarchitecture, with better representation of cell heterogeneity, extracellular matrix (ECM) composition, and functionality of native tissues [9] . 3D in vitro models consist of scaffold-free (spheroids and organoids) or scaffold-based (3D scaffolding and 3D bioprinting) systems used to study infectivity, replication kinetics, and host-viral interactions of many types of viruses, such as influenza [10, 11] , syncytial [12] , adenovirus [13] , norovirus [14] , Zika [15] , and more recently, SARS-CoV-2 [16] , showing increased physiological relevance as compared to 2D models.

Due to the severity and the speed of spread of the pandemic, strategies that contribute to the development of vaccines and drugs, as well as repositioning of currently used drugs, are urgently needed. In this work, we aimed to review the engineered 3D models used as in vitro platforms to study infections, host-virus interactions, and drug screening. We believe that these engineered models have great potential to shed light on the mechanisms of SARS-CoV-2 infection and to aid development of vaccines and screen antivirals to treat COVID- 19. J o u r n a l P r e -p r o o f SARS-CoV-2, such as the heart, causing myocarditis [24] , and the central nervous system, causing headache, anosmia, ageusia, encephalitis and vascular events [25, 26] .

Although the mechanisms of SARS-CoV-2 infection in the lung and other organs have not been fully elucidated, the literature reports that the virus likely has tropism for different cell types and, therefore, multiple infection routes. The angiotensin-converting enzyme 2 (ACE2) was described as the key receptor for SARS-CoV-2 infections by fusion glycoprotein spike (S) binding and entering the host cell via endocytosis [3, 27] . As ACE2 is highly expressed in mammalian organs, it may lead to high invasiveness and multiple tissue damage [28] . It was reported that ACE2 presents a higher affinity with the new coronavirus than with SARS-CoV, which may explain the increased infection rate of SARS-CoV-2 as compared to the previous virus [29] . It is important to highlight the role of the transmembrane serine protease 2 (TMPRSS2), a cellular protease which cleaves SARS-CoV-2 S glycoprotein protein enabling rapid viral internalization and accelerating SARS-CoV-2 replication kinetics in TMPRSS2expressing cells [30] . More recently, a new route of infection was described by Wang and collaborators, where S protein binds the transmembrane glycoprotein CD147, allowing the virus to enter into the host cell [31] . We emphasize that the work still needs peer review and should be interpreted with caution. Nevertheless, CD147-S route may represent a potential alternative for the development of specific antiviral drugs [32] .

Due to the rapid spread and contamination, severity of symptoms, mortality rate, and the unknown long-term effect of SARS-CoV-2 infection, the scientific community joined efforts to understand the mechanisms involved in COVID-19 pathogenicity, transmission, and viral infection of multiple organs, while working on the development of vaccines and new drugs. For these reasons, in vitro models using different cell lines J o u r n a l P r e -p r o o f became a very useful and powerful tool to accelerate SARS-CoV-2 studies and discoveries. These models are proven to efficiently mimic the physiology of native tissues, contributing to the studies of several types of diseases, including viral infections in a more physiologic manner [33, 34] .

Due to the seriousness of the pandemic situation caused by COVID-19, the rapid development of in vitro models to study SARS-CoV-2 infection was necessary in order to assist clinical approaches and treatments through the knowledge acquired in basic research, almost in real time. For these purposes, 2D monolayers have been extensively used to study SARS-CoV-2 life cycle and pathogenesis analysis, drug screening and preclinical evaluation of antiviral potential, and cytopathic effect of candidate molecules [35] .

Vero cells E6 cells, isolated from African green monkeys kidneys, are susceptible to many types of viruses, including the SARS-CoV [36] and SARS-CoV-2 [37] . They produce high viral titers, probably due to the expressive presence of ACE-2 in their apical region, and because these cells do not produce type I interferons (IFN) when infected by several viruses. This phenomenon is due to a deletion of ~ 9 Mbp deletion on chromosome 12, which when in homozygosis, results in a more permissive phenotype for viruses. Thus, the IFN deficiency allows SARS-CoV-2 to replicate sustainably in Vero cells [38] . This cell line was used in some important studies involving SARS-Cov-2, such as for identifying the ACE2 as the functional receptor of SARS-CoV, for demonstrating that anti-ACE2 acted as an inhibitor of viral replication J o u r n a l P r e -p r o o f in these cells [5] , for identifying other potential routes of infection [31] , and for testing the inhibition potential of antiviral candidates [6] . However, the highly permissive phenotype Vero cells have some limitations, as it does not accurately represent the pathogenesis of COVID-19, as its initial target organs are the air and pulmonary epithelia and the venous endothelium. Therefore, other cell types seem to serve as in vitro models that may better recapitulate the real physiology of the disease.

Primary Human Airway Epithelial Cells (HAE) are now commercially available and have been of great utility in studies involving coronaviruses. Besides being efficient for SARS-CoV-2 (and other similar viruses) isolation, these cells mimic infected human lung cells, and it is clearly possible to observe cytopathic effects about 96 hours after SARS-CoV-2 infection. However, an important disadvantage is that these cells have limited replication capacity, requiring constantly acquisition of new stocks [20, 39] .

Besides HAE, it was reported that primary human nasal epithelial, large airway epithelial (bronchi and large airway epithelial), lower airway epithelial (bronchiolar and small airway epithelial) [40] , type I and type II pneumocytes (AT1 and AT2) [41] , and primary enterocytes [42, 43] supports SARS-CoV-2 infection and replication, as well as primary neurons and neural stem cells (NSCs) [44, 45] . However, obtaining all these cell types is difficult and limited, besides the high costs.

To circumvent this problem, many studies have used the most diverse types of proliferating cells lineages, usually derived from tissue-specific carcinomas. Among them, we can highlight other cell lines recently used in studies involving SARS-CoV-2, such as the pulmonary cell lines BEAS-2B (human bronchial epithelium) and A549 (adenocarcinomic human alveolar basal epithelial cells). BEAS-2B, appears to have an accelerated viral replication kinetics, in addition to produce higher viral loads than A549, probably due to its higher ACE-2 and TMPRSS2 expression levels [30] . Calu-3 J o u r n a l P r e -p r o o f cells, another human pulmonary cell lineage, isolated from non-small cell lung cancer, has shown to have great permissiveness and increased viral load when infected with SARS-CoV-2, becoming a promising cell line to be used in COVID-19 in vitro studies [46] . Other cell lineages, mainly Caco-2 cells (human colon adenocarcinoma) and

HEK293T (human embryonic kidney (HEK) grown in tissue culture) are rising as alternative models for SARS-CoV-2 in vitro infection to study the viral tropism of human non-pulmonary cells [47, 48] . However, both cell lines present low levels of SARS-CoV-2 replication in culture.

Although these cell lineages are mainly used in 2D cell culture approach, they are promising elements for the biofabrication of in vitro 3D models, which can better represent the hosts' physiological environment where SARS-CoV-2 infection naturally occurs.

Conventional 2D cell cultures have greatly contributed to the understanding of host cell-virus interactions, mechanisms of virus transmission, replication, and adaptation, as well as screening of antiviral drugs [6, 37] . However, this model have some limitations that rely on the difficulty of reconstituting the accurate and complex microenvironment found in living organisms. Cell-cell junctions, apical-basal polarity, and cell communication through gradients of endogenous growth factors, chemokines, and nutrients may be inadequate to guarantee the similarity with an in vivo system [7] .

These limitations exemplify the necessity to develop new platforms for in vitro modeling [7, 49] . A 3D cell culture approach represents a more realistic environment for cells, contributing to the cell adhesion, maintenance of cytoarchitecture, perception of J o u r n a l P r e -p r o o f the mechanical stimulus, and cell signaling, which in turn, regulate functional responses that differ from traditional 2D cultures [50] .

Many works in the literature reported comparative studies of monolayer cells and 3D cultures infected with different types of viruses, such as poxviruses [51, 52] , adenovirus [53] , hepatitis B and C [53, 54] , Zika [15, 55] and influenza [10] . These works showed that different approaches of 3D cell culture, such as spheroids, organoids, 3D scaffolds and bioprinted structures were able to mimic many types of tissues using different cell lines, presenting higher sensitivity in virus isolation and specificity of several antiviral compounds than conventional 2D models. Therefore, scaffold-free or scaffold-based 3D cell culture may serve as better in vitro models to study SARS-CoV-2 infection mechanisms, replication, pathophysiology of injured organs, and tissues, and to provide high-throughput drug screening strategies ( Figure 1 ). 

Spheroids are cellular aggregates that self-assemble when cultured on nonadhesive surfaces, preserving cell-cell interactions and tissue-specific phenotype [33] .

This cell culture method is widely used in studies of virus infection [12, 13, [55] [56] [57] , being fabricated by culturing cells in non-adherent cell culture flasks and wells [58] , non-adherent spheroid molds [59] , bioreactors [60] or by the hanging drop method [53] .

cells, among other cell lines, aiming to study the sensitivity and isolation of cytomegalovirus, adenovirus, and herpes simplex virus [53] . Results showed that 3D spheroids, infected by the three viruses, were more sensitive to infection than 2D monolayers of the same cell lines, in addition to express viral proteins faster, probably due to the increased number of viral receptors in the spheroids. Recently, Saleh et al.

fabricated A549 spheroids by seeding the cells in an ultra-low attachment 96-well plate to mimic the alveolar tissue and study respiratory syncytial virus pathogenesis [12] .

Results showed that the 3D model was permissive to the virus, leading to syncytia spread towards the nucleus and fast cell death at the core of the spheroids, suggesting the model is promising for studies of infections in vitro. Neurospheres, fabricated using neural stem cells cultured in a non-adherent surface, were used to study molecular mechanisms implicated in brain malformation due to Zika virus infection [15, 61] . In these studies, it was observed that the viral Although spheroids have shown an increased capacity to respond to virus infection over 2D monolayers, they still lack the biological complexity that may be found in other 3D models that possess increased complexity, such as organoids.

Organoids are self-organizing structures established from organ-specific cell types [iPSCs or multipotent adult tissue stem cells (ASCs)] that retain multicellular diversity, cytoarchitecture at early development, and functional hallmarks to their counterpart organs and tissues in vivo [63] . These complex 3D culture systems have been used to mimic multiple types of tissues, such as brain, lung, intestine, and liver to study host-virus interactions for important viruses, such as the Zika [15, 64] , influenza viruses [11] , noroviruses [14] , hepatitis C [65] , and MERS-CoV [66] . In all cases, results showed that the organoids simulated the native tissues morphologically and functionally, indicating they are suitable to be used as in vitro models to study infectivity.

Since the COVID-19 outbreak, many studies involving SARS-CoV-2-host interactions using organoids have been reported, with results indicating their potential to contribute to the development of treatments and drug candidates [67] .

The respiratory tract is the first target for SARS-CoV-2, and respiratory symptoms correspond to the main clinical presentation of COVID-19. In a preliminary study, human bronchial organoids fabricated using commercial human bronchial epithelial cells presented high expression of ACE2 and were successfully infected by J o u r n a l P r e -p r o o f SARS-CoV-2, which was also replicated in this model [68] . In addition, camostat, a therapeutic candidate against COVID-19 acting through transmembrane serine protease 2 (TMPRSS2) inhibition, was tested and results showed that, after the treatment of infected organoids, SARS-CoV-2 viral genome reduced to 2%. Lung organoids were also used to high throughput screen the FDA-approved drugs imatinib, mycophenolic acid, quinacrine dihydrochloride, and chloroquine, and evaluate their capacity to inhibit SARS-CoV-2 entry [69] . Reproduced with permission from AAAS [42] .

Liver abnormalities have become a common hallmark in COVID-19 patients [73, 74] . Recently, it was reported that cholangiocytes, epithelial cells of the bile ducts that express both ACE2 and TMPRSS2, are subject to the SARS-CoV-2 infection [30, 75] . Brain organoids have also been biofabricate using human iPSCs to study brain cells behavior in the presence of SARS-CoV-2. For instance, Jacob et al. studied SARS-CoV-2 neurotropism using human iPSC-derived brain organoids as an in vitro infection platform [77] . In this study, organoids of specific brain regions, such as cerebral cortex, hippocampus, hypothalamus, and midibrain were exposed to SARS-CoV-2 for 8 h.

Results showed an increased number of neurons infected in all organoids, as compared to other neural cell types, and a stabilization of the number of infected cells, indicating that the virus may not spread among neurons. Another preliminary study reported the fabrication of human iPSCs-derived brain organoids to study the neurodegenerative effects of SARS-CoV-2 on the central nervous system [78] . In this work, the virus also preferably targeted human cortical neurons instead of neural stem cells within the brain Moreover, they demonstrated that the in vitro infection was able to induce metabolic changes in these cells [45] .

As aerosol transmission is the main mechanism of spreading the virus, Makovoz et al. aimed to study the tropism of SARS-CoV-2 for ocular cell types in eye organoids, as a potential entry route [79] . The organoid produced from human pluripotent stem cell enabled the researchers to observe SARS-CoV-2 RNA present in several eye regions, such as cornea, sclera, limbus iris, retinal pigment epithelium, and choroid, with low replication in the central cornea. In addition, due to the high expression of ACE2 and TMPRSS2, ocular surface ectoderm was also permissive to virus infection, suggesting the risk of contamination by SARS-CoV-2 regardless wearing facemasks.

Some of the challenges in culturing organoids are related to the lack of vascularization, neuronal circuit, immune system, reproducibility, and preclinical validation [80] [81] [82] . However, the results discussed above indicate that organoids not only are capable of recapitulate the native tissue morphology, physiology, and functionality, but also may substantially contribute to studies of SARS-CoV-2 infection mechanisms, drug screening and disease research.

J o u r n a l P r e -p r o o f

In tissue engineering, 3D scaffold-based models present some advantages over spheroids and organoids, especially due to the presence of biomaterials that simulate the ECM microenvironment. This system can be fabricated using many types of biocompatible materials, naturally present in the ECM or not, providing mechanical strength, physical stability, and biological features that stimulate cell behavior, such as migration, proliferation, and differentiation [83] . In SARS-CoV-2 infection studies, the presence of an ECM may have important effects on cellular responses, as coronaviruses bind to ECM components, such as heparan sulfate, to assist its infection in the host [84] [85] [86] .

Aiming the fabrication of a more realistic model of lung tissue to study influenza A infections, Bhowmick et al. used airway epithelial cells mixed to a collagen-chitosan polymeric blend, simulating the alveolar barrier structure [10] . Cells cultured in the 3D matrix presented increased resemblance to the native tissue concerning cell morphology, specific markers expression, and differentiation, as compared to a 2D

culture. In addition, the 3D model showed immune responses that better resembled that of the native system when infected with H1N1 and H3N2 strains. Reproduced with permission from Elsevier [52] .

The 3D bioprinting is an emerging technology that has been widely employed in the tissue engineering field aiming to optimize the conventional 3D cell culture. Due to its capacity to deposit layer-by-layer cells and biomaterials in an organized and automatized manner, through a computer-aided process, the fabrication of complex architectures that mimic the structure of organs and tissues became possible [87, 88] .

Several bioprinting methods have been employed to construct mimetic tissues, each presenting advantages and limitations. For instance, extrusion-based bioprinting, inkjet/drop-on-demand, laser-assisted, stereolithography, and electronspinning-based are some methods used in biofabrication, being the extrusion-based bioprinting technique the most commonly used, due to its easy of handling and low cost [89, 90] .

Therefore, the construction of more realistic models in terms of cell heterogeneity, ECM presence, and the complex organization has greatly improved the physiological and pathological cell microenvironment, contributing significantly to cell response and prediction of results of in vitro studies [91] , including those involving viral infections [92] [93] [94] .

To study the mechanisms of SARS-CoV-2 infection in the lung, the alveolar tissue can be mimicked using a 3D bioprinted airway epithelium [95, 96] . Horvath et al.

reported the 3D bioprinting of an alveolar barrier by using a micro-extrusion bioprinter [95] . The biofabricated structure was morphologically similar to the native tissue, being highly organized in a thin layer. On the other hand, cells manually mixed to Matrigel TM J o u r n a l P r e -p r o o f formed multi-layered clusters with tick ECM between the epithelial and endothelial cells, which can affect the permeability of biomolecules. This work showed that 3D

bioprinting technology offers relevant advantages over conventional 3D scaffolding, with potential to be used for testing drug candidates that inhibit SARS-CoV-2 infection and replication. The effects of influenza A infection was modeled using an engineered respiratory tract [94] . For this, human alveolar epithelial (A549) cells were embedded in a polymeric blend composed of gelatin (to assure printability), alginate (for structural stability), and Matrigel TM (to assure biocompatibility), being extruded using microextrusion bioprinting technology ( Figure 5A ). Results showed that the virus was homogeneously distributed throughout the bioprinted construct, which reflects the natural condition of infection ( Figure 5B) 

As the COVID-19 outbreak continues to affect thousands of people around the world, it is urgent the development of strategies that lead to effective treatments and vaccines. Although 2D conventional cell culture has shown to be an important tool in virology studies, this model fails in replicating the cellular microenvironment in terms of architecture, composition, physiological function, and mechanical stimulus, which may lead to low prediction of results [7] . The establishment of 3D cell culture and the biofabrication of tissue-like structures can mimic the complex microenvironment found in the many organs affected by SARS-CoV-2 with higher accuracy, providing robust data to elucidate cellular and molecular mechanisms of virus infection, replication kinetics, and host-virus interaction.

In this work, we reviewed the strategies offered by the tissue engineering field to biofabricate useful in vitro 3D models to understand the effects of SARS-CoV-2 infection. Spheroids have been an important tool to study the effects of Zika virus in neurogenesis during brain development [15, 55] , and have shown cytopathic effects in lung and brain spheroids in response to SARS-CoV infection [60] . Given the numerous evidences of neurological disorders in COVID-19, neurospheres can elucidate the mechanisms of neural injury. Although spheroids lack in biological function and structure complexity, they can be fabricated by using simple methodologies. Organoids, in contrast, are more complex and organized in terms of structure and composition, resembling the natural environment of the target organ [80] . Due to their J o u r n a l P r e -p r o o f cytoarchitecture, organoids have been widely used to construct mimetic organs to study SARS-CoV-2 and its subsequent effects after infection. Although this model face some limitations [82, 97] , similarities to native tissues morphology, physiology, and functionality are leading to reliable results, which can accelerate the process of developing new drugs and consequent control of the disease.

Regarding scaffold-based strategies, these 3D models have been used to study the infectivity of different types of viruses, indicating they can greatly contribute to the understanding of the mechanisms of SARS-CoV-2 infection. By using 3D bioprinting technology, it is possible to fabricate complex architectures that resembles native tissues in a reproducible manner, providing molecular and cellular machinery to effectively replicate SARS-CoV-2, holding promises to future drug tests [95] .

Based on results of previous studies, in vitro 3D models are attractive alternatives to be used in virology, and this review hopes to shed light on different strategies to study the effects of SARS-CoV-2 in human organs. We believe that the 3D cell culture approach can greatly contribute to the development of faster diagnostics and therapeutics, due to its capacity to mimic with greater fidelity the in vivo system, providing increased prediction of results and faster translational applications to treat patients affected by SARS-CoV-2.

The authors declare no conflict of interest.

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