This article describes a method for differentiating stem cell-derived neuronal progenitor cells into cortical neurons and their subsequent transplantation into human cortical tissue. The process involves detaching the cells, resuspending them in a basement membrane matrix, and injecting them into a cortical slice for analysis.
Begin with an adherent culture of stem cell-derived neuronal progenitor cells primed for differentiation into cortical neurons.
Remove the medium, then add an enzyme to disrupt the extracellular matrix and detach the cells.
Use an inhibitor to neutralize the enzyme, then add a fresh medium.
Transfer the suspension into a tube, centrifuge, and discard the supernatant to isolate the cells.
Resuspend the cells in a basement membrane matrix to facilitate transplantation into brain tissue, then load them into a cold capillary tube.
Take a human cortical slice onto a transwell assembly containing a medium.
Remove part of the medium, then inject droplets of the cell suspension into the tissue.
Incubate to allow the matrix to solidify.
Add a medium to immerse the tissue, then incubate to enable progenitor cells to differentiate into neurons, extend their neurites, and form synapses with host cortical neurons.
The cortical slice is now ready for analysis.
Remove the media from the cell culture flask. On day seven of differentiation, detach cortically-primed GFP-lt-NES cells by adding 500 microliters of trypsin and incubate for 5 to 10 minutes at room temperature.
Next, add an equal volume of the trypsin inhibitor followed by five milliliters of DDM medium. Resuspend the cells, gently transfer the cell suspension into a 15-milliliter tube, and centrifuge for five minutes at 300 G. Meanwhile, transfer the plate containing the human tissue from the incubator to the hood, and remove two milliliters of media from the top of the insert. At the end of the centrifugation, remove the supernatant, resuspend the cells in a cold, pure, basement membrane matrix, and transfer the suspension to a smaller tube.
Collect the cell suspension into a cold glass capillary connected to a rubber teat for suction. Inject the cell suspension as tiny drops by stabbing the semi-dry tissue slice at various sites. Allow the gel to solidify at 37 degrees Celsius for 30 minutes.
Then, transfer the plate from the incubator back to the hood and carefully add two milliliters of human adult cortical medium to the top of the insert to completely submerge the tissue.
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