The overall goal of this in vitro procedure is to observe the effect of Zika virus infection on survival in differentiation of human fetal neural stem cells. This method can help answer key questions in the neural stem cell and infectious disease fields such as how viral infection influences innate immune responses and how these changes contribute to the survival and differentiation of neural stem cells. The main advantage of this technique is that the cells used are human and not genetically modified which enhances the translational application of the results.

The implications of this technique extend toward therapy for the neurological deficits associated with the Zika virus because it provides a means to evaluate the mechanism of infection and screen therapeutics. After preparing culture medium and plating human neural stem cells according to the text protocol, retrieve from the incubator the flask of cells to be passaged and bring it into the biosafety cabinet, or BSC. Gently tilt the flask allowing the cells to sink to the bottom of the pooled medium.

Transfer 10 milliliters of medium into two five-milliliter portions into a clean 15-milliliter tube labeled CM for conditioned medium. Take care not to aspirate any of the cells settled in the flask. Transfer three milliliters of medium from the CM tube to a 15-milliliter tube labeled trypsin inhibitor solution, then set the CM tube aside.

Next, remove the five milliliters of medium and cells remaining in the T75 flask and place it in a clean 15-milliliter tube labeled cells. Then, using a serological pipette and 3.5-milliliter volumes of the CM, rinse the T75 flask twice transferring the CM rinse into the cells tube. This will remove all neurospheres from the T75 flask.

Centrifuge the cells tube at 100 times G in room temperature for five minutes. While the cells are spinning, obtain the previously prepared DPBS glucose solution from the 37-degrees-Celsius water bath and add the appropriate amount of trypsin and DNase. After the cells tube has stopped spinning, remove all the conditioned medium supernatant and transfer it to the CM tube.

Add trypsin solution to the cell pellet in the 15-milliliter tube and use a five-milliliter pipette to pipette up and down approximately five to 10 times. Close the cap of the tube and incubate it in a 37-degrees Celsius water bath for five minutes. To the trypsin inhibitor tube, add the appropriate volume of trypsin inhibitor adding the same volume of solution as was used for the trypsin solution.

Then, place the trypsin inhibitor solution in the 37-degrees-Celsius water bath until needed. Following the incubation, pipette the cells up and down five to 10 times. Then, incubate the sample in the 37-degree-Celsius water bath for an additional 10 to 15 minutes.

Following the cell incubation, pipette the cells up and down until the spheres are completely dissociated. Then, immediately add the trypsin inhibitor solution and pipette the cell suspension up and down 10 times. To count the number of cells in the suspension, pipette 15 microliters of Trypan Blue pre-mixture onto a small weigh boat and add five microliters of the cell suspension.

Pipette up and down three to five times to mix. Then, transfer 10 microliters of the Trypan Blue cell suspension onto either side of a hemocytometer covered with a glass coverslip. Observe the cells under a light microscope and count the total number of cells in each of the four corner grids.

Then, add these numbers together. After calculating the number of cells according to the text protocol, add a volume of cell suspension to each T75 flask to obtain five times 10 to the 6th cells per flask. If the volume is less than five milliliters, add additional conditioned medium up to five milliliters.

Then, add 10 milliliters of new DFHGFPS medium containing growth factors to the flask. Ensure the flask is labeled with the type of cells, the dates, the number of cells in the flask and the passage number. If desired, incubate the cells at this point.

To plate adherent HNSCs, the day before passaging, clean the BSC as explained in the text protocol and use forceps to place single sterile 12-millimeter glass coverslips into the wells of a 24-well plate. Coat the wells and coverslips with 250 microliters per well of 0.01%Poly-D-Lysine, or PDL, in sterile water and incubate the plates at 37 degrees Celsius for one hour. Remove the PDL from the wells and then coat them with one microgram per square centimeter of laminin DPBS.

Incubate the plate at 37 degrees Celsius overnight. If not needed the next day, wrap Parafilm around the edges of the plate to seal it and store it at four degrees Celsius. Remove any excess laminin solution from the wells through aspiration and use 0.5 milliliters of DPBS to rinse the wells once.

Then, seed the cells into each well at a density of 0.6 to one times 10 to the 5th cells per square centimeter. After passaging the cells according to the text protocol, transfer one milliliter of cell suspension into a 1.5-milliliter microcentrifuge tube and centrifuge the sample at 200 times G at room temperature for five minutes. Then, remove the supernatant by aspiration.

Re-suspend the pellet in ZIKV stock at a multiplicity of infection, or MOI, between one and 10. The volume of ZIKV solution should not exceed 0.5 milliliters. For mock treatment, re-suspend cells in the same volume and type of medium used in ZIKV stock.

Incubate the samples at 37 degrees Celsius for one hour. After, invert the tube two to three times and centrifuge the mixture at 216 times G at room temperature for five minutes to obtain a cell pellet. Remove the supernatant by aspiration, then re-suspend the cells with DPBS to rinse them.

Next, centrifuge the mixture at 216 times G for five minutes, and remove the supernatant by aspiration. Re-suspend the cells with 12 milliliters of the appropriate medium. Then, load 500 microliters of the infected cells into a 24-well plate.

As seen here, healthy spheres should be approximately one to two millimeters in diameter, nine to 10 days following a passage from non-adherent neurospheres. Healthy spheres will also appear translucent and display pseudocilia around the edges of the sphere. Following the appropriate priming steps in addition of differentiation medium, the cells will spread out across the surface of the culture vessel and grow as an interconnected monolayer of cells as shown in this figure.

This immunohistochemistry experiment identified nestin, an intermediate filament commonly expressed in NSCs that can be used to verify an HNSC culture. Class III beta-tubulin is labeled here to verify the presence of neurons in glial fibrillary acidic protein, or GFAP, reveals the presence of astrocytes following differentiation. Immunofluorescent staining with specific ZIKV antibodies showed that the K048 line of NSCs was infected by both African and Asian strains of ZIKV.

The virus tends to stay in the peripheral region of an infected cell one day after a one-hour infection, but fills the whole cell at three to seven days. Once mastered, the passaging technique can be done in one hour if it is performed properly. Medium changes can be performed in 20 minutes.

While attempting this procedure, it is important to remember to make sure neurospheres do not become too large before passaging. Following this procedure, other methods, like various drug treatments, can be performed in order to answer additional questions like the efficacy of pharmacotherapies on prevention or reversal of Zika-mediated deficits. After its development, this technique paved the way for researchers in the fields like infectious disease, addiction and trauma to explore how viruses, drugs of abuse and traumatic physical injury impact human neural stem cells.

After watching this video, you should have a good understanding of how to passage, infect and plate primary human fetal brain neural stem cell for your own investigations, specifically how the cells should be handled to yield the most successful culture. Don't forget that working with Zika virus can be extremely hazardous and precautions such as double gloves should always be taken while performing this procedure.