Researchers from the Federal University of São Paulo (Unifesp) have developed – using mouse cells – a bio-printed three-dimensional model of the brain to study the neurological action of the SARS-CoV-2 virus. Additionally, the group managed to create an adapted version of the pathogen capable of infecting rodent nerve cells. The band’s expectation is that these two feats – describe in the magazine Advanced Biology – help make research into the effects of COVID-19 on the central nervous system cheaper and faster.
“Our proposal was to create three-dimensional bioprinted models that could be used to study mechanisms of viral invasion, drug action, and other topics. As the SARS-CoV-2 which infects humans does not infect mice, the option until now was to use genetically modified animals, which express the human receptor. [a proteína ACE-2, à qual o vírus se liga para invadir as células]. But we wanted an adapted version of the virus specific to the neural cells of these animals”, he specifies. Marimelia Porcionattoprofessor at the Escola Paulista de Medicina (EPM-Unifesp) and project coordinator, who is finance by FAPESP (Fundação de Amparo in Pesquisa do Estado de São Paulo).
The so-called bioink, created by the group, is a mixture of natural compounds with neural cells that powers a 3D printer. The model had been developed in previous work and allows cells to survive the bioprinting process, migrate through space and interact with each other as if they were in nerve tissue. According to the researchers, the protocol was developed with mouse cells, but uses biocompatible materials that could in the future be adapted to human cells (Read more on: agencia.fapesp.br/37767/).
“Now, in addition to astrocytes, we have added neurons to the model. However, since they are more sensitive, these cells were added later, as if seeded on the bioprinted material. So the neurons not only fitted into the model, but also interacted with the astrocytes,” he explains. Bruna Alice Gomes de Melofirst author of the work, produced during his post doctoral at EPM-Unifesp.
The researchers explain that more neural cell types could be added in the future, increasing the complexity of the model and bringing it closer to neural tissue.
Currently, the other three-dimensional models are organoids and spheroids, clusters of cells that self-organize in laboratory cultures. “Our model has greater reproducibility than organoids and spheroids. In addition, it can be produced on a larger scale,” informs Porcionatto.
Spheroids, structures formed from neural stem cells and also known as neurospheres, played a key role in the development of mouse-adapted SARS-CoV-2. For this, they were incubated for seven days with particles of SARS-CoV-2 which infects humans.
The few virus particles that survived and replicated were then isolated and placed in a new culture of neurospheres. The process was repeated four times, when enough virus was obtained to perform the tests.
Genetic sequencing of the resulting virus showed that it lost mutations compared to that used in the first incubation step. The adapted coronavirus turned out to be even closer to the original strain from Wuhan, which started the pandemic.
Vero cell lines, normally used as a model for human infection, were challenged with both normal and adapted virus, showing a weak ability of the latter to infect cells other than mice.
An interesting result obtained in the 3D bio-printed model was that the adapted virus had a 30 times higher replication capacity in astrocytes than in neurons, showing the relevance of these cells in the infection of the central nervous system by SARS- CoV-2.
“The pathogen could simply be present in the cell, but not replicate to the point of being a problem for the organism. The result shows that there are viral particles that reproduce and place themselves outside the cell, which can infect others,” reports Melo.
In addition, during infection, the researchers observed an increase in the expression of chemokines, molecules that attract immune system cells. Thanks to their presence, there was a decrease in so-called inflammatory cytokines.
“This means that our model replicates what would happen in the organism, being a better option for cells in two-dimensional plates, organoids and spheroids. Additionally, the bioink we developed can aggregate other cells that appear to be involved in SARS-CoV-2 infection in the brain, such as microglia and endothelial cells present in the blood-brain barrier, that lines the inside of the brain blood. ships,” says Porcionatto.
With the model, the researchers hope to reduce not only the costs of such research, but also the use of animals in the laboratory. The work was also supported by FAPESP through three other projects (18/12605-8, 19/01255-9 and 21/03684-4).
The article 3D bioprinted neural-like tissue as a platform to study mouse-adapted SARS-CoV-2 neurotropism can be read at: https://onlinelibrary.wiley.com/doi/10.1002/adbi.202200002.