Generating Induced Pluripotent Stem Cell-Derived Cerebral Organoids
Transcrição
Aspirate the medium with a pipette, and wash the cells once with DPBS. Then, add 500 microliters of cell detachment solution, or 0.5 millimolar EDTA, to dissociate the colonies, and incubate at 37 degrees Celsius for 3 to 5 minutes. Next, add 1 milliliter of fresh E8 media to each well, and pipette gently to detach all cells.
Transfer the entire cell suspension into a 15-millimeter centrifuge tube, and add another 1 milliliter of fresh E8 media. Then, spin down the cells at 290 g for 3 minutes at room temperature.
After discarding the supernatant, resuspend the cell pellet in 1 milliliter of E6 media, supplemented with 50 micromolar ROCK inhibitor, and count the cells using a hemocytometer.
Prepare a cell suspension of the desired seating density in E6 media, supplemented with the ROCK inhibitor. Then, add 150 microliters of the cell suspension into each well of a 96-well ultra-low attachment plate.
Next, centrifuge the plate at 290 g for 3 minutes at room temperature to force-aggregate the cells. Then, incubate the plate at 37 degrees Celsius in a humidified atmosphere with 5% carbon dioxide for embryoid body formation.
After 24 hours of incubation, transfer the plate to the hood, and carefully aspirate 120 microliters of the media using a pipette, taking care not to aspirate the embryoid body by lowering the pipette tip too far into the well.
Next, add 150 microliters of fresh E6 media, supplemented with 2 micromolar XAV939 and the SMAD inhibitors to each well. Transfer the plate back to the incubator, and replace the medium daily with freshly prepared E6, supplemented with the inhibitors.
At day seven of incubation, check whether all the embryoid bodies have reached a diameter of 550 to 600 micrometers and display a smooth, clear edge. At this stage, they are ready to be embedded in an extracellular matrix.
To embed the organoids, prepare dimpled embedding sheets by placing a 4-inch long thermoplastic sealing film sheet on an empty P200 box. Then use a 15-milliliter conical tube, or a 500-microliter microcentrifuge tube, and gently press down the film sheet into the holes to make 12, or the desired number of dimples.
Next, spray 70% ethanol on the film sheet, and let it dry inside the hood with the UV light on for at least 30 minutes. Meanwhile, thaw a sufficient quantity of basement membrane matrix on ice. Then, using a wide bore P200 tip, transfer one embryoid body from the 96-well plate to each dimple. Remove as much media as possible with a normal pipette tip, but do not let the embryoid body dry.
Next, with a regular pre-chilled P200 tip, add approximately 30 microliters of undiluted membrane matrix to each organoid, ensuring that the embryoid body is as close as possible to the center of the droplet.
Once all embryoid bodies are embedded in the matrix, place the film sheet in a sterile Petri dish. Then incubate the Petri dish at 37 degrees Celsius for 10 minutes in a humidified atmosphere with 5% carbon dioxide to allow the membrane matrix to solidify.
For organoid differentiation, add 5 milliliters of the prepared differentiation media with B27 without vitamin A to one well of an ultra-low attachment 6-well plate. Pre-warm the plate to 37 degrees Celsius in an incubator. Once the membrane matrix has solidified, transfer the embedded embryoid bodies to the 6-well plate by pushing out the dimples from the back of the film sheet. If necessary, use 1 milliliter of media from the well, and pipette it onto the sheet to detach the droplets from the film sheet.
