This method can be performed in any laboratory equipped for cell culture experiments. Using this method, we can further our understanding of renal development and disease pathways. This is a relatively simple and inexpensive method compared to others for generating kidney organoids.
The biggest challenge of this technique is maintaining high-quality human pluripotent stem cell culture. It is critical that the cells are maintained at less than 80%confluency and split regularly. Researchers new to human pluripotent stem cell culture should familiarize themselves with the stem cell lines before proceeding to differentiation.
Begin the kidney organoid assay by adding two milliliters of complete E5-ILP medium into each well of a six-well ultra-low attachment plate. Culture human pluripotent stem cells, or hPSCs, in 100-millimeter tissue culture-treated plates until they reach 60%confluency. Wash the hPSCs twice with approximately eight milliliters of DPBS.
Aspirate the DPBS, then add two milliliters of Dispase dropwise to cover the cells, and incubate them for six minutes at 37 degrees Celsius. Wash the cells three times with approximately 10 milliliters of DPBS. Aspirate the DPBS, then tilt the plate by 45 degrees, and scrape the cells down with a cell lifter.
Wash the colonies down from the top with six milliliters of complete E5-ILP medium using a 10-milliliter serological pipette. Then, holding the pipette vertically, break up large colonies by gently pipetting up and down, taking care not to touch the sides of the culture plate. Distribute the colony clusters evenly by adding one milliliter per well into the six-well plate, then place the plate on an orbital shaker inside a 37-degree Celsius incubator.
Alternatively, if an orbital shaker is not available, the cells can be statically cultured. On day two, after letting the embryoid bodies settle at the bottom of the plate, tilt the plate by 45 degrees, and aspirate the medium slowly from the top, leaving one milliliter per well. Add two milliliters of prepared complete medium per well, and return the plate back to the shaker.
On day three, after aspirating the medium as previously demonstrated, collect all the embryoid bodies carefully from each well using a 10-milliliter serological pipette, and transfer them to a 50-milliliter conical tube. To collect any remaining embryoid bodies, rinse each well with one milliliter of low-glucose DMEM, and add them to the same 50-milliliter conical tube. While waiting for the embryoid bodies to settle at the bottom of the tube, add two milliliters of Stage II medium to each well of the six-well plate.
Next, sieve out large embryoid bodies by placing a 200-micrometer cell strainer on top of a new 50-milliliter conical tube and pipetting all the embryoid bodies carefully over the cell strainer. Rinse the cell strainer with an additional five milliliters of DMEM to collect any embryoid bodies stuck in the strainer. At later stages, first use a 500-micrometer strainer to sieve out very large embryoid bodies, then pass the filtrate through a 200-micrometer strainer to sieve out very small embryoid bodies without tubules.
Collect the correct size embryoid bodies, between 200 to 500 micrometers, by inverting the 200-micrometer strainer into a new 50-milliliter tube and washing the strainer with DPBS. After straining, allow the embryoid bodies to settle to the bottom of the conical tube, then aspirate the supernatant, and wash with approximately 10 milliliters of DMEM. Then, resuspend the embryoid bodies in six milliliters of Stage II medium.
Transfer the embryoid bodies back into the six-well ultra-low attachment plate, distributing them evenly among the wells. Since most of the organoids collect at the top of the pipette, leave approximately half a milliliter at the end, then gently touch the tip to the media in each well, allowing organoids to flow into the well. Transfer the embryoid bodies into a 125-milliliter spinner flask with 45 milliliters of Stage II medium.
Set the magnetic stir speed to 120 rpm, and place the flask into the incubator. To feed the embryoid bodies or kidney organoids, let them settle briefly to the bottom of the spinner flask, then lift the lid from one side arm of the flask, and place the aspirating pipette inside with the tip touching the opposite inside wall. Slowly angle the pipette down, and aspirate approximately half of the medium.
Replenish with 20 milliliters of fresh Stage II medium by pipetting it through the same opening. Use long sterile forceps to carefully place one magnetic stir bar into each well of a six-well plate with embryoid bodies or kidney organoids. Place the plate onto the six-wheel magnetic stir plate, and set the speed to 120 rpm.
For the magnetic stir bars to snap into position and start spinning, first set the power level to 100. Once all bars start spinning, bring the power level down to 25. Maintain the kidney organoids by changing half the medium as previously demonstrated.
Immunofluorescence of paraffin sections of day 14 kidney organoids show presence of nephron segments such as renal tubules expressing hepatocyte nuclear factor-1 beta and Lotus tetragonolobus lectin, as well as podocyte clusters expressing V-maf musculoaponeurotic fibrosarcoma oncogene homolog B and nephrin. The modifications in this protocol can support expansion of endothelial cells, as confirmed by staining with platelet and endothelial cell adhesion molecule-1 at day 26 of culture. Human pluripotent stem cells need to be treated with Dispase for the appropriate amount of time, then thoroughly washed to remove all traces.
If Dispase is not completely removed, it can lead to cell death and clustering of embryoid bodies. This method has helped us examine the development of congenital kidney diseases, as well as various kidney injury pathways and potential for therapeutic interventions in vitro.
