Our research presents a comprehensive and efficient method for producing kidney organoids from induced pluripotent stem cells, using suspension cultural conditions. We emphasize the importance of initial cell density and WNT agonist concentration during the inducing phase, which will educate new organoid researchers. Currently, it is difficult to obtain a single high-density cell line for iPSCs.
The batch impact was noticeable, particularly in various iPSC lines. Each batch's kidney organoids may have a varied form and size. Several attempts are necessary to get the best initial cell density and concentration of CHIR for one iPSC line.
We have established that the initial differentiation of the intermediary method is important for successful kidney organoid formation. Our protocol describes, in detail, how to choose the optimal cell densities and CHIR concentrations during the process because different iPSC lines showed variances in cell proliferation and differentiation ability. Researchers can quickly generate the specific kidney organoids from their iPSCs.
According to our protocol, by adjusting the cell density and the concentration of CHIR, it's undoubtedly beneficial for all our researchers to focus on their kidney disease. Our lab future research aims to optimize the method of the protocol in disease models, regenerate new medicine and the drug screening. In recent five years, our lab will focus on the epigenetics and the cell topic drug development of various hereditary kidney disease, based on patient development, iPSC, and the kidney organized disease models.
Begin by washing the induced pluripotent stem cells or iPSCs on the membrane matrix-coated 6-well plate with two milliliters DPBS. Aspirate the DPBS using a pipette. Next, add one milliliter of the commercially available cell detachment solution to detach the iPSCs.
Then, place the cell into an incubator at 37 degrees for five minutes. Now, pipette one milliliter of mTeSR onto the iPSCs, ensuring that the cells have separated from the plastic surface. Centrifuge these cells at 400 g for three minutes at room temperature.
Resuspend the cell pellet. Then, count the cell numbers using a hemocytometer. Next, seed a range of cell densities on a 6-well plate that has been coated with a membrane matrix.
Culture these with mTeSR supplemented with 10-micromolar Rho kinase inhibitor. Culture the plates overnight in a carbon dioxide incubator set at 37 degrees Celsius. The next day, aspirate the mTeSR and wash the cells with two milliliters of DPBS.
Next, add two milliliters of stage I medium to the cells. Then, place the cells in a 37 degrees Celsius incubator for four days. On day four, remove the stage I medium and wash the cells with two milliliters of DPBS.
Then, add two milliliters of stage II medium to the cells before incubating the cells as before. From day zero to day four, iPSCs rapidly expanded and took on rhomboid or triangular shapes. The confluence reached 90 to 100%and accumulated evenly until day seven.
Upon suspension culture, the aggregates spontaneously form nephron structures after dissociating on day seven. 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 400 g 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 6-well, low-adhesion plate at a 1:3 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 6-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 6-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.
