Begin by removing stage II medium from the iPSC cell suspension on the seventh culture day. Now wash the cells with two milliliters of DPBS. Pipette one milliliter of cell detachment solution to each well.
Then place the plate in an incubator at 37 degrees for five minutes. After incubation, add one milliliter of stage II medium to the cells and mix thoroughly until the cells have detached from the surface. Collect the cell suspension in a 15 milliliter tube, then centrifuge it at 400g for three minutes at room temperature.
After centrifugation, discard the supernatant and resuspend the cell pellet in two milliliters of stage III medium. Mix the solution gently. Now seed the suspension into a six-well, low-adhesion plate at a one-to-three ratio.
Place the plate on an orbital shaker in an incubator set at 37 degrees Celsius with 5%carbon dioxide. To remove the medium in the suspension culture, first cut off the tip of a one milliliter pipette using aseptic scissors. Then gently agitate the six-well plate to centralize the organoids.
Next, aspirate the organoids with the prepped pipettes and place them in a 15 milliliter tube, letting them stand for five minutes. Aspirate the supernatant, ensuring that the organoids remain at the tube's base. Then pipette stage III medium into the plate.
Pipette the mixture to resuspend the organoids. Replate the organoids back into a six-well, low-adhesion plate. Continue shaking the low-adhesion plate at 60 rotations per minute in the incubator.
On the 12th day, replace the medium with stage IV medium. The kidney organoids display tubular like structures and are easily observed in bright field images after 18 days of aggregation. CD31 endothelial cells were induced.
Dextran was taken into the proximal tubules of the kidney organoids.
