A Technique to Generate Brain Organoids from Human Embryonic Stem Cells

Dilute the protease stock solution to a working concentration by adding 1 milliliter of the stock solution to 5 milliliters of DMEM or F12 medium for each 6-well plate of hESCs. Aspirate and remove the cell culture media then cover the hESCs with the protease solution. Place the plates in the incubator for 10 to 15 minutes, or until the edges of the colonies round-up and begin to separate from the matrix, but before they round up completely.

Tilt the plate, aspirate the protease solution, and gently wash the cells with 2 milliliters of DMEM or F12 for each well three times. Make sure the colonies stay attached to the matrix.

So it's critical that the pieces of SL is the appropriate size. So make sure that they're in dispase for the appropriate amount of time. If they're in there for too short of a time, it can be very difficult to flush them off and break them apart. And if they're in there for too long, they can actually just come off during the wash steps.

Add back about 1 to 1.5 milliliters of fresh mTESR media to each well, and flush the cells of the plate into a 50-milliliter conical tube using gentle pipetting, aspirate and dispense hESCs within the plate until they reach approximately 1/30 of their original size. Now, the colony clusters resemble 250 to 350-micrometer-sized pieces.

Transfer the cells into a single ultra-low attachment T75 flask, containing milliliters of mTESR media without bFGF. The next day, tilt the flask, such that the live cells pool in the corner. If there are a large number of cells that have adhered to the bottom of the flask, use a 10-milliliter pipette to transfer the cells to a new flask.

Expect to have a high population of dead cells for the first few days. Once the cells settle, aspirate off the media and dead cells, leaving about 10 milliliters of media containing the live cells, and add 20 milliliters of low bFGF media, supplemented with 30 nanograms per milliliter of bFGF. After two days, check the cells. Most of the cells should look healthy and bright.

However, if more than a third of the cells appear dark, tilt the flask, and replace 20 milliliters of media with 20 milliliters of low bFGF media supplemented with 20 nanograms per milliliter of bFGF. On day 3, remove half of the media, and replace with 15 milliliters of HSC media, supplemented with 10 nanograms per milliliter of bFGF.

On day 5, replace half of the media with 50 milliliters of neural induction media. After that, every other day, replace half of the medium with neural induction media. After three weeks in culture, add 100x penicillin-streptomycin to the media at a final concentration of 1x if desired. Refresh half the media every other day.