Generation of Human Brain Organoids for Mitochondrial Disease Modeling

This protocol aims to use brain organoids to study mitochondrial diseases. It was developed for generating reproducible brain organoids in the assessment of their mitochondrial properties. This method does not require expensive bioreactors and time-consuming embedding procedures.

Therefore, it is easy to implement and upscale. This protocol makes it possible to generate reproducible brain organoids and to test their mitochondrial properties. This can lead to identification of interventional targets for untreatable mitochondria diseases.

To begin, culture human iPSCs under feeder-free conditions in iPSC medium on coated six-well plates and keep them in a humidified tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide. Passage the iPSCs at 80%confluency using enzyme-free detachment medium in ratios ranging from one by four to one by 12. To increase cell survival, add 10 micromolar rho-associated protein kinase or ROCK inhibitor after each splitting.

Dissociate the 80%iPSCs at day zero. Prepare cortical differentiation medium one or CDM1 and pre-warm it at room temperature before adding it to the cells. Wash the wells containing the iPSCs with PBS to remove dead cells and debris.

Add 500 microliters of pre-warmed reagent A to each well and incubate for five minutes at 37 degrees Celsius. Check under the microscope to ensure cell detachment. Add one milliliter of iPSC medium to dilute reagent A to neutralize its activity.

Use a 1, 000 microliter pipette to dissociate the cells by piping up and down and transfer the cell suspension to a 15 milliliter centrifuge tube. Gently centrifuge the iPSCs at 125 times G for five minutes at room temperature, then carefully aspirate the supernatant to avoid disturbing the cell pellet. Resuspend the pellet with one milliliter of CDM1 to obtain a single cell suspension and count the cell number.

Prepare the seeding medium with 9, 000 iPSCs per 100 microliters in CDM1 supplemented with 20 micromolar ROCK inhibitor, three micromolar WNT catenin inhibitor or IWR-1, and five micromolar SB-431542. Add 100 microliters of seeding medium per well to a 96-well V bottom plate. Keep the plate in a humidified tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide.

To generate neurospheres, on day one, observe that round cell aggregates with defined smooth borders are forming. Note the dead cells around the aggregates. Continue to culture in the incubator at 37 degrees Celsius and 5%carbon dioxide.

On day three, agitate the plate by tapping on the sides three times to detach dead cells, then add 100 microliters of CDM1 supplemented with 20 micromolar ROCK inhibitor, three micromolar IWR-1, and five micromolar SB-431542 to each well. Keep the plate to the incubator at 37 degrees Celsius and 5%carbon dioxide. On day six, carefully remove 80 microliters of the supernatant medium from each well.

Avoid touching the bottom of the well. Add 100 microliters of CDM1 supplemented with three micromolar IWR-1 and five micromolar SB-431542 to each well and incubate the plate. Repeat the previous step after every three days until day 18.

On day 18, to transfer neurospheres, prepare CDM2 and add 10 milliliters to a 100 millimeter ultra low attachment cell culture plate. Use a 200 microliter pipette with the tip cut off to transfer the round neurospheres from the 96-well plate to the 100 millimeter ultra low attachment cell culture plate. Remove five milliliters of medium from the plate containing the neurospheres and add five milliliters of fresh CDM2.

Place the plate on an orbital shaker at 70 rotations per minute inside a humidified tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide. Every three days, carefully aspirate the supernatant medium and replace it with fresh CDM2. Leave a small amount of the medium to prevent neurospheres from drying out.

On day 35, prepare CDM3. Aspirate the medium from the plate and add 10 milliliters of cold CDM3. After changing the medium, place the plate back on an orbital shaker at 70 rotations per minute inside a humidified tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide.

Change the medium every three to five days depending on the rate of growth as indicated by the color of the medium. On day 70, prepare CDM4. Use CDM4 medium until the desired age of organoids is reached.

During this period, keep the plate on an orbital shaker set at 70 rotations per minute inside a humidified tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide. Change the medium every three to five days depending on the growth rate. Prepare 4%PFA solution and place it under a safety hood.

Collect brain organoids and gently transfer them with a blunt tipped three milliliter plastic Pasteur pipette to a six-well plate filled with PFA. Keep the organoids in the PFA solution for one hour at room temperature. Carefully remove the PFA with a three milliliter plastic Pasteur pipette and wash the fixed organoids three times using PBS.

Store the fixed organoids at four degrees Celsius in PBS until further use. The organoids generated using this protocol contain mature neurons that can be visualized using protein markers specific for axons and dendrites. Mature organoids contain not only neuronal cells, but also glial cells.

Using sliced brain organoids analysis, it is possible to monitor SMI 312 positive axons and MAP2 positive dendrites or the S100 beta glial cells. Further, confocal images help to investigate the detailed distribution and organization of SOX2 positive neural progenitors with respect to beta-3 tubulin positive neurons. Brain organoids were stained for mitochondria-specific markers, such as translocase of outer membrane or TOM20.

Bioenergetic profiling of brain organoids was performed by measuring both mitochondrial metabolisms using the oxygen consumption rate, or OCR, and the glycolytic metabolism using the extracellular acidification rate, or ECAR. Oligomycin causes a drop in the OCR profile and therefore identifies the OCR needed for ATP production. Upon oligomycin treatment, there may also be a compensatory increase in ECAR suggesting that the cells can upregulate glycolysis to prevent the metabolic stress.

When transferring the neurospheres from the 96-well plate to a culture plate or during the fixing procedure, it is crucial to handle the organoids gently to avoid damaging them. Following the generation of brain organoids, multi-electrode array or calcium imaging would be ideal methods to test the functionality of the organoids.