This study investigates the effects of Zika virus on human cerebral organoids, which model early brain development. The research highlights how viral infection can disrupt neural progenitor cell function and organoid morphology.
Take a multiwell plate containing human cerebral organoids in a neural induction medium.
The organoids contain stem cells, radial glial cells, neural progenitor cells or NPCs, and neurons arranged in a radial pattern called a rosette, mimicking the developing brain.
Remove the medium and wash the organoids with a buffer.
Add Zika virus suspended in a buffer to the test wells and only buffer to the control wells.
Incubate, allowing the virus to bind to its receptors on the NPCs and become internalized.
Wash to remove non-internalized viruses, then add fresh neural induction medium and incubate.
The internalized viruses use the host cell machinery to form new viral particles.
Viral multiplication triggers a cellular stress response that induces apoptosis, reducing the number of NPCs available for organoid development.
Over time, the infected organoids exhibit reduced size and disrupted morphology, mirroring abnormal brain development in Zika virus-infected humans.
Bring Earle's 1X Balanced Salt Solution, 1X PBS, and Neural Induction medium to 37 degrees Celsius in a hot water bath. Next, dilute Zika virus of a known concentration in 1X Earle's solution to the target multiplicity of infection which is typically between 0.1 and 10. Using a P200 pipette, carefully remove all medium from each organoid well, then, quickly wash the organoids with 200 microliters of warm 1X PBS.
When removing the media from each well, it is critical that one does not touch the organoid or suck it into the pipette. This can cause irreparable damage to the organoid. Should this happen, it is best to discard the organoid.
Next, carefully remove all the remaining liquid from each organoid well using a single-channel P200 pipette. Then, quickly add 50 microliters of either 1x Earle's solution for a mock infection or Zika virus solution to each well. Ensure that each organoid is completely submerged. When finished, place the plate back into a 37 degrees Celsius incubator for two hours.
After a viral exposure is completed. Wash each well of the organoids with 200 microliters of PBS. Allow organoids to settle for 15 seconds, and then remove the PBS using a single-channel P200 pipette. Finally, add 200 microliters of fresh neural induction medium to each well, and place the plate back into the incubator.
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