Differentiating Human Neural Stem Cells into Neural and Astrocyte Progenitors

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 5-milliliter portions into a clean 15-milliliter tube labeled "CM" for a conditioned medium. Take care not to aspirate any of the cells settled in the flask. Transfer 3 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 5 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 5-milliliter pipette to pipette up and down approximately 5 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 5 to 10 times. Then, incubate the sample in the 37 degrees 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 premixture onto a small weigh boat, and add 5 microliters of the cell suspension. Pipette up and down 3 to 5 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 5 x 10⁶ cells per flask.

If the volume is less than 5 milliliters, add additional conditioned medium up to 5 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 in 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 1 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 4 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 1 x 10⁵ cells per square centimeter.