To begin, wash the cultured iPSCs with Dulbecco's PBS or DPBS. And add one milliliter of proteolytic and collagenolytic mixture. Incubate the dish at 37 degrees Celsius for two minutes to detach the cells.
Next, add 1.5 milliliters of prewarmed iPSC culture medium to stop the reaction. Dissociate the cells from the cell culture dish by pipetting up and down seven to 10 times to obtain a single-cell suspension. Transfer the cell suspension to a 15-milliliter conical centrifuge tube.
Pellet the cells at 200xg for five minutes at room temperature. And aspirate the supernatant. Resuspend the pellet in two milliliters of iPSC culture medium supplemented with 50 micromolar Y27632.
Now, use 10 microliters of the cell suspension to count the cells using a Neubauer chamber. Adjust the concentration of the cell suspension to 9, 000 cells per 150 microliters using iPSC culture medium supplemented with 50 micromolar Y27632. To generate embryoid bodies or EBs, shake the cell suspension tube to prevent cell sedimentation before seeding 150 microliters of the cell suspension into each well of an ultra-low attachment 96-well plate.
Culture the EBs in a humified atmosphere for 48 hours. At the end of the incubation, remove 100 microliters of medium per well. And add 150 microliters of prewarmed fresh medium without Y27632.
Create a four by four dimple grid on the parafilm by placing the parafilm grid on the 0.2 milliliter tube rack with the paper-enveloped side facing up. And gently press a gloved finger against each rack hole to embed the EBs in the basement membrane matrix. Then remove the paper and cut the dimple grid out of the parafilm square using scissors to adjust its size to fit into a 60-milliliter cell culture dish.
Place the dimpled parafilm back on the 0.2 milliliter tube rack to provide a basis for the basement membrane matrix droplet generation. Carefully transfer the EBs one after another from the well of the culture dish to the parafilm dimples using a pipette with a cut 200 microliter pipette tip. After moving 16 EBs to the grid, take a new 200 microliters pipette tip and remove the remaining medium from the dimples.
Now, pipette one drop of basement membrane matrix onto each dimple containing one EB.Take a 10-microliter pipette tip and quickly move the EBs into the center of each droplet without disturbing the droplet borders. Place the dimpled parafilm with the basement membrane matrix drops in a 60-millimeter cell culture dish. And incubate for 15 to 30 minutes at 37 degrees Celsius to allow the matrix to polymerize.
Further, to detach the matrix-embedded EBs from the parafilm, add five milliliters of differentiation medium without vitamin A to the dish. And turn the parafilm square upside down using forceps so that the side with the EBs is facing the bottom of the dish. Carefully shake the dish to detach the basement membrane matrix drops containing the EBs from the parafilm.
If some of them are still attached, take an edge of the parafilm square using forceps and rapidly roll it up toward the center of the dish multiple times. Then culture the cerebral organoids on an orbital shaker at 55 rpm in a humidified atmosphere with 5%carbon dioxide and 95%air at 37 degrees Celsius. Keep the organoids in DM without vitamin A with medium changes every other day.
A brightfield image of a 32-day post-seeding human cerebral organoid with ventricle-like structures on the periphery and a compact, healthy morphology is shown.
