Modeling Mitochondrial Disease Using Brain Organoids: A Focus on Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episodes

The scope of our research is to understand mitochondrial disease meters, and find drugs to treat it. The challenge is to retain the capacity for high throughput assays, even though the technique contains organoid formation. To begin, remove the medium from induced pluripotent stem cell culture plates when they are approximately 70 to 80%confluent.

Wash the cells with PBS. Add 350 microliters of enzyme solution to the cells and incubate. Add 800 microliters of stem fit medium to stop the reaction.

After transferring the cell suspension to a fresh tube, centrifuge it at 190G for five minutes at 22 degrees Celsius. Next, re-suspend the pellet in one milliliter of differentiation medium. After counting the cells, seed 3000 cells in 100 microliters into each well of a U-shaped 96 well low attachment plate.

Incubate for six days at 37 degrees Celsius, with 5%carbon dioxide. After incubation, use a wide bore pipette to transfer the organoids from the 96 well plate into a 10 centimeter dish. Pipette 50 microliters of supplemented DMEM/F12 medium to each well of a new 96 well plate.

Collect the organoids into two milliliter tubes. Remove the medium carefully without aspirating the organoids, and add 1.5 milliliters of supplemented DMEM/F12 medium. After pipetting out the last wash, add 500 microliters of fresh medium.

Then transfer the organoids to a 10 centimeter dish. Add five milliliters of medium to facilitate picking each organoid. Transfer all organoids into each well of a 96 well plate using a micro pipette set to 50 microliters, and incubate for three days.

At day nine, collect the organoids into two milliliter tubes and wash three times with DMEM/F12. Then re suspend the organoids in differentiation medium. Transfer approximately 10 organoids per well into a six well plate and adjust the final volume of medium in each well to two milliliters.

Culture the plate statically for six days at 37 degrees Celsius with 5%carbon dioxide. On the 15th day, collect the organoids into 15 milliliter tubes. After letting the tubes to settle, add fresh differentiation medium supplemented with B27 and vitamin A after pipetting out the supernatant.

Transfer approximately 10 organoids per well into a six well plate. Culture the organoid statically for 15 days at 37 degrees Celsius with 5%carbon dioxide. Coat the culture plates with the dilute poly-L-Ornithine solution.

Incubate the plates overnight at 37 degrees Celsius. The next day, wash the wells three times for five minutes each with PBS. Now add diluted laminin solution to the wells.

Incubate the plates for three hours at 37 degrees Celsius, or overnight. Centrifuge the organoid suspension at 200G for five minutes. Remove the supernatant and add one milliliter of enzyme solution.

Mix well and incubate at 37 degrees Celsius for five minutes before centrifuging. After removing the supernatant and adding one milliliter of dispersion solution, carefully layer the isolation solution beneath the suspension. Then centrifuge the suspension at 200G for five minutes.

Now, pipette one milliliter of neuronal medium into the tube. Mix the solution and pass the suspension through a 70 micrometer strainer into a 50 milliliter tube. After counting the cells, seed the cell suspension at a density of 100, 000 cells per square centimeter on poly-L-Ornithine and Laminin coated plates.

Replace the medium every four to seven days until day 60. To fix the organoids for immunofluorescence, first harvest the organoids into micro tubes. Rinse the organoids with PBS, then fix the organoids in 4%Paraformaldehyde for 30 minutes at room temperature.

Post fixation, wash the fixed organoids three times with PBS. Store the organoids at four degrees Celsius until used. 30 days after seeding for 2D neuronal culture, remove the culture medium from the plates.

Then wash the cells with PBS. Fix the cells with 4%Paraformaldehyde for 15 minutes at room temperature. Pipette out the Paraformaldehyde, then wash the cells with PBS three times.

Store the fixed cells at four degrees Celsius until used. To collect the organoids for RTQPCR, transfer the organoids into micro tubes. Pipette PBS into the tubes to wash the organoids.

Now, add 350 microliters of lysis buffer to the organoids, then snap freeze them in liquid nitrogen. Store the tubes at minus 80 degrees Celsius until used. Brain organoids containing FOXG1 positive neurons were successfully induced from the healthy control IPSC line 414C2 on day 30, and 2D cultured neural networks were obtained on day 60.

Heteroplasmy levels of the M.3243A to G variant were confirmed by QPCR. Line two to eight had low heteroplasmy, and lines two to six had high heteroplasmy. Brain organoids derived from the low heteroplasmy line two to eight exhibited sizes and morphologies similar to those from healthy controls.

Organoids generated from the high heteroplasmy line two to six displayed reduced size of 0.3 square millimeters, and irregular morphology with a circularity value of 0.82. Most neurons induced from the 414C2 line and the two to eight line were FOXG1 positive on days 30 and 60. FOXG1 positive neurons were rarely detected in organoids or 2D cultured neurons derived from the two to six line.

Our results suggests that patient derived IPSC based organoid models represent a useful platform for studying mirrors, mechanisms, and for drug screening. Our conventional methods with a short culture time and cost effectiveness offer practical tools for investigating the pathophysiology of MELIS. We would like to search for potential drugs for MELIS by using compounds with patient delivered organized in the future.