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In vitro and animal models of SARS Cov-2
- 1. IN VITRO AND ANIMAL MODELS FOR SARS COV-2 Dr. Mohit Kher Senior Resident-I LHMC 1
- 2. INTRODUCTION In December 2019, pneumonia of an unknown etiology was confirmed in China. Chinese Center for Disease Control and Prevention (CCDC) identified a novel coronavirus infection as the cause of this pneumonia. WHO named the disease ‘2019-new coronavirus disease’ (COVID-19) and the International Committee on Taxonomy of Viruses named the virus ‘severe acute respiratory syndrome coronavirus 2’ (SARS CoV-2). On March 11 2020, COVID-19 outbreak was declared as a pandemic by WHO. COVID-19 has affected more than 80 million people around the world.
- 6. IN VITRO MODELS 2D- CELL LINES 3D- SPHEROIDS ORGANOIDS CONVENTIONAL 3D SCAFFOLDING 3D BIOPRINTED MODELS
- 7. CELL LINES Human airway epithelial cells Vero E6 Cells CACO-2 cells CALU-3 CELLS HEK293T cells Huh7 cells
- 8. Human airway epithelial cells Highly express the ACE2 and TMPRSS2. Cytopathic effects 96 hrs after the infection. Proliferating cells lineages - 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. Disadvantages - limited replication capacity, requiring constantly acquisition of new stocks and expensive.
- 9. VERO E6 CELLS Isolated from the kidney epithelial cells of an African green monkey. IFN deficiency allows SARS CoV-2 to replicate. Most widely used to replicate and isolate SARS-CoV-2, because these cells highly express ACE2. Expression level of TMPRSS2 is quite low. Chloroquine and hydroxychloroquine (HCQ) could inhibit SARS-CoV-2 in Vero E6 cells.
- 10. CALU-3 CELLS Isolated from non-small cell lung cancer. Great permissiveness and increased viral load when infected with SARS-CoV-2.
- 11. CACO-2 cells Isolated from human colon adenocarcinoma. SARS-CoV-2 could replicate in Caco-2 cells. Low levels of SARS-CoV-2 replication in culture.
- 12. HEK293T cells Isolated from human embryonic kidney (HEK) cells grown in tissue culture. Cells showed only modest viral replication.
- 13. SPHEROIDS Cellular aggregates, preserving cell–cell interactions and tissue-specific phenotype. Spheroids (lungs and brain in vitro models) composed of human bronchial tracheal cells/BEAS-2B cells and human neural progenitor cells. Both spheroids types showed cytopathic effects. Suggesting a similar behavior to the native system.
- 15. ORGANOIDS Self-organizing structures established from organ-specific cell types [iPSCs or multipotent adult tissue stem cells (ASCs)] Mimic multiple types of tissues, such as brain, lungs, intestine and liver Organoids simulated the native tissues morphologically and functionally
- 16. LUNG ORGANOIDS Fabricated using commercial human bronchial epithelial cells presented high expression of ACE2. Camostat, a therapeutic candidate against COVID-19 acting through transmembrane serine protease 2 (TMPRSS2) inhibition, was tested. Imatinib, mycophenolic acid, quinacrine dihydrochloride, and chloroquine were evaluate for their capacity to inhibit SARS-CoV-2 entry.
- 17. Vascular and kidney organoids Virus spreads from the lungs to the kidney. Evaluate the infectivity of SARS-CoV-2, as well as the inhibitory effect of human recombinant soluble ACE2 (hrsACE2). SARS-CoV-2 entry into the organoids was significantly reduced by hrsACE2.
- 18. Intestinal Organoids Fabricated from primary gut epithelial stem cells. Viruses were capable to infect enterocyte lineage cells in the organoids. Virus levels increasing about 1000-fold after 24 h of infection. Induction of type III IFNs and inflammatory mediators.
- 20. Liver ductal organoids Cholangiocytes, epithelial cells of the bile ducts that express both ACE2 and TMPRSS2. This model was permissive to SARS-CoV-2 infection and supported viral replication.
- 21. Brain Organoids Human iPSCs-derived brain organoids to study the neurodegenerative effects of SARS-CoV-2 in central nervous system. Virus preferably targeted human cortical neurons instead of neural stem cells. Embryonic brains are less vulnerable. Metabolic changes seen 24 h post-infection. Virus may not spread among neurons.
- 22. EYE ORGANOIDS Organoid produced from human pluripotent stem cell enabled the researchers to observe SARS-CoV-2 RNA present in several eye regions. Due to the high expression of ACE2 and TMPRSS2, ocular surface ectoderm permissive to virus infection.
- 23. BENEFITS: Multiple cell types and model the physiological conditions of human organs. Ability to self-replicate. CHALLENGES: Lack of vascularization, neuronal circuit, immune system, reproducibility, and preclinical validation. Reproduce the pathology of COVID-19 in specific tissues
- 24. Conventional 3D scaffolding Advantages over spheroids and organoids, especially due to the presence of biomaterials (heparin sulfate) that simulate the ECM microenvironment. Provide mechanical strength, physical stability, and biological features. Presence of an ECM assist its infection in the host. Increased resemblance to the native tissue. Showed immune responses that better resembled to native system.
- 25. 3D BIOPRINTED MODELS Emerging technology Fabrication of complex architectures through a computer-aided process. Bioprinting methods - Extrusion-based bioprinting, inkjet/drop-on- demand, laser-assisted, stereolithography, and electronspinning- based. Showed in vivo-like immune response.
- 26. ANIMAL MODELS
- 27. MOUSE MODEL
- 28. BALB/c mice Moderate pneumonia and inflammation. Histopathological changes - inflammation in pulmonary alveoli, detection of viral antigen in the trachea, bronchiole, in type II pneumocytes. Monoclonal antibodies and vaccine efficacy were evaluated.
- 29. TRANSGENIC hACE2 mice Significant decrease in body weight and interstitial pneumonia. hACE2 mouse model using CRISPR/Cas9 knock compared it with the wild type C57BL/6 mice model. High viral titre in brain, lungs and trachea of hACE2 mice than wild type mice. K18-hACE2 transgenic mouse – Best mouse model
- 30. HAMSTER MODEL 3 0 Rapid breathing, weight loss, and alveolar damage with extensive apoptosis. Changes of inflammation, cellular viral N protein expression, and high viral load during the first week. Passively infused IgG antibodies protected golden Syrian hamsters against lung viral burden, as did passive transfer of convalescent serum to naïve animals. Hydroxychloroquine (with or without azithromycin) and favipiravir were evaluated.
- 31. LARGER ANIMALS
- 32. FERRET MODEL Ferrets were more susceptible to infection and transmission. Infected ferrets exhibited more replication of the virus in the upper respiratory tract. Nonlethal acute bronchiolitis in the lungs.
- 33. Primate cynomolgus macaques Primate cynomolgus macaques is currently closest to humans. Compare MERS-CoV, SARS-CoV, and SARS-CoV-2. Infect type I pneumocytes. Pulmonary edema and formation of hyaline membrane was reported.
- 34. RHESUS MACAQUES Three species of non-human primates were infected with SARS-CoV-2 (rhesus macaque, Macaca fascicularis, and common marmoset). Rhesus macaque model was more susceptible to SARS-CoV-2. High viral loads in the upper and lower respiratory tract, humoral and cellular immune responses, and pathologic evidence of viral pneumonia. Old rhesus macaques also observed to have diffuse severe interstitial pneumonia.
- 35. Adenovirus-vectored vaccine, DNA vaccine candidates expressing S protein, ChAdOx-1 vaccine, mRNA-1273 vaccine and remdesivir treatment evaluated. African green monkeys supported a high level of SARS-CoV-2 replication. Limitation - Reproduction rate in cynomolgus and rhesus monkeys is less and slower.
- 36. CONCLUSION
- 37. REFERENCES 1. World Health Organization . 2020. Coronavirus disease (COVID- 19) pandemic 2020. 2. Li W., Moore M.J., Vasilieva N., Sui J. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature. 2003;426 3. Duval K., Grover H., Han L.H., Mou Y., Pegoraro A.F., Fredberg J. Modeling physiological events in 2D vs. 3D cell culture. Physiology. 4. Zhou J., Li C., Liu X., Chiu M.C., Zhao X., Wang D. Infection of bat and human intestinal organoids by SARS-CoV-2. Nat Med. 2020 5. Yang W., Sirajuddin A., Zhang X., Liu G., Teng Z., Zhao S. The role of imaging in 2019 novel coronavirus pneumonia (COVID-19) Eur Radiol. 2020:1. 6. Devaux C.A., Rolain J.M., Raoult D. ACE2 receptor polymorphism: susceptibility to SARS-CoV-2, hypertension, multi-organ failure, and COVID-19 disease outcome. J Microbiol Immunol Infect. 2020
- 38. Thank you
- 39. Huh7 cells Isolated from hepatocyte-derived cellular carcinoma cells.
Editor's Notes
- The novel coronavirus disease (COVID-19) emerged at the end of 2019 in the city of Wuhan, China, and by december 2020. It has affected more than 80 million people around the world, according to the World Health Organization (WHO), and the Johns Hopkins Coronavirus Resource Center. 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. Likely origin is bat but it is not confirmed.
- Coronoviruses are enveloped, positive sense, single-stranded RNA viruses of ~30 kb. They are largely divided into four genera; α, β, γ, and δ based on their genomic structure. α and β coronaviruses infect only mammals. SARS-CoV-2 are classified to β coronaviruses. Coronaviruses consist of four structural proteins; Spike (S), membrane (M), envelop (E) and nucleocapsid (N). Spike comprises two functional subunits; S1 subunit is responsible for binding to the host cell receptor and S2 subunit is for the fusion of the viral and cellular membranes.
- ACE2 is the main receptor for SARS-CoV-2. As ACE2 is widely distributed in mammalian cells, the virus infects the respiratory tract and rapidly spread throughout the human body infecting other organs, such as the heart, brain, liver, and intestine. Spread faster because of binding to ACE2 receptor which is present everywhere. 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 TMPRSS2-expressing cells. The life cycle of the virus with the host consists of the following 5 steps: attachment, penetration, biosynthesis, maturation and release. Spike is composed of a transmembrane trimetric glycoprotein protruding from the viral surface, which determines the diversity of coronaviruses and host tropism. Following the binding of SARS-CoV-2 to the host protein, the spike protein undergoes protease cleavage.
- Cellular and animal models of SARS-CoV-2 pathogenesis. Cell-based models for SARS-CoV-2 include Vero E6 cells, human ciliated airway epithelial (HAE) cells, human cells from infected patients, human primary upper and lower airway cells, and organoid culture models. Animal models for SARS-CoV-2 include golden Syrian hamsters, ferrets, mice and non-human primates (NHPs). Transgenic hACE2 mice, ACE2 adenovirus- and adeno-associated virus (AAV)-transduced mice and genetically diverse wild-type mice infected with mouse-adapted SARS-CoV-2 constitute a ‘second generation’ of models, which have been optimized based on insight from earlier research.
- In vitro models are crucial to validate therapeutics before being used in clinical trials. 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. These 3-D 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. 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. 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. These limitations exemplify the necessity to develop 3D in vitro modeling. A 3D cell culture approach represents a more realistic environment for cells, contributing to the cell adhesion, maintenance of cytoarchitecture, perception of the mechanical stimulus, and cell signaling, which in turn, regulate functional responses that differ from traditional 2D cultures.
- An in vitro cell model for SARS-CoV-2 research is essential for understanding the viral life cycle, for amplifying and isolating the virus for further research, and for preclinical evaluation of therapeutic molecules. Different cell lines accelerate SARS-CoV-2 studies and discoveries. Several infinitely proliferating cell lines such as Caco-2 [6], Calu-3 [7], HEK293T [8], and Huh7. These cell lines do not accurately mimic human physiological conditions and generate low titer of infectious SARS-CoV-2.
- These cells are expensive and do not proliferate indefinitely. Many studies have used the most diverse types of proliferating cells lineages, usually derived from tissue-specific carcinomas. E.g 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.
- Vero cells have given high titre of viral particles. For efficient SARS-CoV-2 research, these cells can easily replicate and isolate the virus. These cells were isolated from the kidney epithelial cells of an African green monkey in 1963 and have been shown to not produce interferon (IFN) when infected with Newcastle disease virus, rubella virus, and other viruses. A homozygous ~9 Mbp deletion on chromosome 12 causes the loss of the type I interferon (IFN-I) gene cluster and of cyclin-dependent kinase inhibitor genes. The IFN deficiency allows SARSCoV-2 to replicate in Vero cells. Among the several Vero cell clones, Vero E6 is the most widely used to replicate and isolate SARS-CoV-2, because these cells highly express ACE2 on the apical membrane domain. However, the expression level of TMPRSS2, the receptor that the virus uses to prime the S protein (spike protein of SARS-CoV-2) [4], is quite low in this clone. To enhance the replication and isolation efficiencies of SARS-CoV-2 in Vero E6 cells, Matsuyama et al. have used TMPRSS2-overexpressing Vero E6 cells. They reported that the viral RNA copies in the culture supernatants of these cells were N100 times higher than those of Vero E6 cells, suggesting that it would be possible to isolate higher titre virus using TMPRSS2-overexpressing Vero E6 cells. In February/March 2020, it was reported that the antimalarial compounds chloroquine and hydroxychloroquine (HCQ) could inhibit SARS-CoV-2 in Vero E6 cells.
- 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. Compared with mock control, SARS-CoV-2 showed an over 500-fold increase in luciferase activities in Calu3 cells. Notably, SARS-CoV-2 grew in Calu3 cells, but not in A549 cells, although both cell lines derive from lung adenocarcinoma. SARS-CoV-2 replication was greatest in Calu3 and Caco2 cell lines.
- Data not available.
- 1. 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. However, both cell lines present low levels of SARS-CoV-2 replication in culture.
- Spheroids are cellular aggregates that self-assemble when cultured on non-adhesive surfaces, preserving cell–cell interactions and tissue-specific phenotype. SARS-CoV infection in lung and brain in vitro models was evaluated using spheroids composed of human bronchial tracheal cells/BEAS-2B cells and human neural progenitor cells. Both spheroids types showed cytopathic effects in response to SARS-CoV infection, suggesting a similar behavior to the native system, being promising for estimating the pathophysiology of the lung, brain, and other tissues infected with SARS-CoV-2.
- Fabricated by culturing cells in non-adherent cell culture flasks and wells, non-adherent spheroid molds, bioreactors or by the hanging drop method. Fabrication of spheroids using a rotating wall vessel bioreactor. Scheme illustrating the spheroids assembly and formation in the rotating wall vessel bioreactor. Image of the bioreactor system. Scanning electron microscopy of human neural progenitor spheroids used in SARS-CoV infection studies.
- Organoids retain the property of multicellular diversity and cytoarchitecture. Results showed that the organoids simulated the native tissues morphologically and functionally, indicating they are suitable to study infectivity.
- Successfully infected by SARS-CoV-2. 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.
- Because SARS-CoV-2 was identified in the urine of patients, it can be assumed that the virus spreads from the lungs to the kidney through the vasculature. While membrane-bound ACE2 may mediate cell entry of SARS-CoV-2, a genetically modified soluble form of ACE2, called hrsACE2, may decrease cell entry of SARS-CoV-2 competing for membrane-bound ACE2. APN001 is a hrsACE2 designed by Apeiron Biologics to imitate the human enzyme ACE2. Results showed that both vascular and kidney organoids were infected with SARS-CoV-2, and the supernatant was also capable to infect monolayers of Vero E6 cells. Besides, SARS-CoV-2 entry into the organoids was significantly reduced by hrsACE2 in the early stages of infection.
- ACE2 is also highly expressed in multiple digestive tract organs, which can lead to infection by SARS-CoV-2 and consequently gastrointestinal symptoms, being observed in many patients. Lamers et al. fabricated human small intestinal organoids from primary gut epithelial stem cells and evaluated SARS-CoV-2 infection capacity. Results showed that both viruses were capable to infect enterocyte lineage cells in the organoids. Replication was observed in human duodenum, ileum and colon organoids, with virus levels increasing about 1000-fold after 24 h of infection. Human epithelial organoids were also capable of replicating SARS-CoV-2 and inducing type III IFNs and inflammatory mediators, which are upregulated upon viral infections in the human intestine.
- SARS-CoV-2 infecting intestinal organoids. 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. Immunofluorescent images of dividing cells (KI67-positive) and post-mitotic enterocytes identified by Apolipoprotein A1 (APOA1) (pointed arrows). ACE2 staining in intestinal organoids in expansion (EXP) and differentiated (DIF). Reproduced with permission from AAAS [42].
- 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. Interestingly, virus infection impaired the bile acid transporting functions of cholangiocytes [20]. This effect might be the reason for the bile acid accumulation and consequent liver damage in patients with COVID-19.
- Organoids of specific brain regions, such as cerebral cortex, hippocampus, hypothalamus, and midbrain were exposed to SARS-CoV-2 for 8 h. Results showed an increased number of neurons infected in all organoids and a stabilization of the number of infected cells, indicating that the virus may not spread among neurons. Virus also preferably targeted human cortical neurons instead of neural stem cells within the brain organoids, demonstrating that developing embryonic brains are less vulnerable.
- Eyes are also a potential entry route so ocular cells can be used for tropism of SARS COV-2. 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.
- Organoids are composed of multiple cell types and model the physiological conditions of human organs. Because organoids have the ability to self-replicate, they are also suitable models for large-scale screening in drug discovery and disease research. However, while organoids can reproduce the pathology of COVID-19 in specific tissues on which they are modeled, they cannot reproduce the systemic symptoms associated with whole body responses to the viral infection.
- Providing mechanical strength, physical stability, and biological features that stimulate cell behavior, such as migration, proliferation, and differentiation. Increased resemblance to the native tissue concerning cell morphology, specific markers expression, and differentiation.
- Widely employed to optimize the conventional 3D cell culture. Due to its capacity to deposit layer-by-layer cells and biomaterials in an organized manner, through a computer-aided process, the fabrication of complex architectures that mimic the structure of organs and tissues became possible. Extrusion-based bioprinting technique the most commonly used, due to its easy handling and low cost. Bioprinted cells also showed in vivo-like immune response, observed by the inflammatory cytokine interleukin 29 release. The effects of influenza A infection and adenovirus infection was modeled. There is still a void in the literature regarding 3D bioprinting to study viral infections.
- While cell lines and organoids are faster systems to study the virus and its interactions inside host cells, these can only reproduce the symptoms of COVID-19 in a specific cell type or organ, respectively. Several different animals are being used to study the disease and to test candidate therapeutic compounds.
- Interestingly, SARS-CoV-2 use all ACE2 proteins, except for mouse ACE2. Therefore, transgenic mice were developed that express human ACE2.
- SARS-CoV-2 significantly affected all groups of mice irrespective of their ages. A study reported higher viral load in lungs along with significant histopathological changes like denatured trachea, inflammation in pulmonary alveoli, detection of viral antigen in the trachea, bronchiole, in type II pneumocytes. Significant elevation of inflammatory chemokine and cytokines in sera and lungs.
- Sun SH et al. developed the hACE2 mouse model using CRISPR/Cas9 knock in technology to study the SARS-CoV-2 infection and compared it with the wild type C57BL/6 mice model. Human ACE2 transgenic (hACE2) mice allow for SARS-CoV-2 replication in the lungs, develop signs of disease and can die from lethal encephalitis. Disadvantage - Lethality caused by neuroinvasion, limited availability and relatively slow propagation speed of mice. (hACE2) expression is driven into the mouse epithelial cells under the control of the human cytokeratin 18 (K18) promoter
- Molecular docking studies were performed, with the finding that the Syrian hamster might be suitable because of significant binding of the novel coronavirus spike with the ACE2 receptor. Limited or no efficacy has been demonstrated for the repurposed drugs hydroxychloroquine (with or without azithromycin) and favipiravir—although high doses of favipiravir did reduce infectious virus titres in the lungs of infected hamsters
- Small animals like mice and Syrian hamster are advantageous to study SARS-CoV-2, as they reproduce faster; however, to faithfully reproduce COVID-19 pathology in humans, larger animal models are preferred.
- Kim et al. reported nonlethal acute bronchiolitis in the lungs of a ferret model. Another study showed that SARS-CoV-2 can replicate in ferrets and cats, but not in pigs, chickens, and ducks. Ferrets were more susceptible to infection and transmission. Virus can spread from infected ferret to naive ferrets by direct or indirect physical contact. Also showed viral shedding
- SARS-CoV-2 infect type I pneumocytes. Damage of type I pneumocytes led to pulmonary edema and the formation of hyaline membranes.
- A comparative study was performed in three species of non-human primates (rhesus macaque, Macaca fascicularis, and common marmoset) and infected with SARS Cov-2. The study found that the rhesus macaque model was more susceptible to SARS-CoV-2, as compared to others.
- NHP models are the gold standard in pre-clinical animal testing prior to the initiation of Phase I human clinical trials. chimpanzee adenovirus-based (ChAdOx-1) vaccine
- COVID-19 has spread rapidly all over the world in the past 5 months. Even now, the number of infected people and deaths continues to rise. At this time, there are no therapeutic prevention or intervention methods available. The only ways to control the pandemic and reduce associated loss of lives has been to change peoples’ behavior, like quarantine and social distancing. While multiple clinical trials are currently underway, in parallel, preclinical research on in vitro and model organisms is also needed, both to understand the virus and to test therapeutic agents for safety and efficacy.
- 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. Advantages of 3D over 2D: 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.