Generation of human midbrain organoids from induced pluripotent stem cells

The iPSC cells should not have been passaged more than 10 times.

The cells need to be a minimum of passage 2 after thawing for generation of midbrain organoids.

Do not work with colonies that present with differentiated areas (Figure 1a). We recommend removing the differentiated areas during daily maintenance and to never exceed 5% of differentiated areas prior to organoid generation for optimal organoid formation.

Sterile technique must be used at all times when working with cells or in preparing reagents and materials.

Human iPSCs lines are to be handled within a Class II biosafety laminar flow hood to protect the worker from possible biohazardous agents.

Start by thawing an aliquot of 150-200 µl Matrigel® hESC-qualified Matrix at 4°C, 15–20 minutes prior to use (depends on the dilution factor of each lot per manufacturer instructions).

Prepare DMEM/F-12 solution by removing 5 mL of DMEM/F-12 from a 500 mL bottle and adding 5 mL of anti-anti. Place the bottle at 4°C until cold.

Once the Matrigel® is thawed and DMEM/F-12 is cold, prepare a coating solution diluted as per manufacturer instructions and mix well.

Immediately use the diluted Matrigel® solution to evenly coat the tissue culture dishes (2 ml/ 60 mm dish and 5 ml/ 100mm dish) by swirling the tissue culture dish in each direction, multiple times. Incubate at 37°C for a minimum of one hour before use.

Meanwhile, warm up mTeSR^TM 1 and DMEM/F-12 medium at room temperature (RT) for a minimum of one hour. Do not use a water bath during this process because the media components would be degraded.

Once the tissue culture dish is coated and mTeSR^TM 1 and DMEM/F-12 medium are warmed, get the frozen cryovial of iPSCs from the liquid nitrogen storage container, and quickly thaw the vial by warming in a 37°C water bath. Continuously shake the cryovial until only a small frozen pellet remains.

Using a 5 mL glass pipette, transfer the contents of the cryovial to a 5 mL solution of DMEM-F12 (with anti-anti), in dropwise manner and gently pipette up and down 1–2 times.

Centrifuge the cells at 1200 rpm for 3 minutes at RT. After centrifugation, aspirate the medium, leaving the cell pellet intact. Using a 5 mL glass pipette, gently resuspend (1–2 times) the cell pellet in 3 mL of mTeSR^TM 1 medium containing Y27632 (1:1000 dilution).

Take the coated tissue culture dish from 37°C and aspirate out the coating medium. Transfer the cells in mTeSR^TM 1 medium to the coated dish and place in a 37°C, 5% CO₂ incubator. Do not disturb the dish for 24h, to allow the cells to attach.

Change the medium every day with mTeSR^TM 1 without Y27632, until the cell confluency reaches 70% and cells are close in contact.

Daily, visually identify regions of differentiation under the microscope and remove them by aspiration under the sterile hood.

When the cells reach 60-70% confluency, they are ready to be split.

Aspirate out the medium from the dish and wash the cells with 3 mL DMEM/F-12 (with anti-anti).

Add gentle cell dissociation medium (5 ml for 100 mm dish and 1 mL for 60 mm dish) at RT until the cells at the edge of the colony begin to detach from each other. (Note: Do not leave cells for longer as the cell viability will be affected)

Aspirate out the gentle cell dissociation medium from the dish and wash the dish with DMEM/F-12.

Add 5mL DMEM/F-12 to the dish and gently detach the colonies by scrapping with a cell scrapper. Using a glass pipette, transfer the detached cell aggregates in a Falcon tube.

Centrifuge the Falcon tube with cells at 1200 rpm for 3 minutes.

After the centrifugation is done, check the pellet and aspirate out the supernatant (do not let the pellet dry so leave 100 μL-200 μl DMEM after aspiration).

Using a glass pipette, resuspend the pellet gently to break the cell aggregates in mTeSR^TM 1 medium with 10μM Y27632.

For seeding cells, add 10% of the original cell suspension to a new coated dish when passaging between similar size dishes. For a small to large dish passage-take 20% of the original cell suspension and from a large to small dish, take 5%.

Incubate the dishes at 37°C until the colonies reach 70% confluency.

Start with one 10 cm dish of iPSCs at 70% confluency, cultured in mTeSR^TM 1 to obtain 96 midbrain organoids from a 96-well ultra-low attachment plate.

Dissociate cells using Accutase by removing medium from cells, wash cells once with DMEM-F12 +Anti-Anti and add 5 ml Accutase at RT. Incubate cells for 3 minutes at 37°C, then stop reaction with DMEM-F12+Anti-Anti. Transfer the medium with the cells into a 15 mL Falcon tube and centrifuge for 3 min, at 1200 rpm in a regular Falcon centrifuge. Remove the supernatant.

Using a 1mL tip combined with a 200 µl tip resuspend cells in 5 ml of neuronal induction medium (Table 3) by pipetting gently up and down 3 times. Count cells. Note: The 200 µl tip is at the end of the 1 ml tip.

Plate 10,000 cells/well in an ultra-low attachment 96 well U-bottomed plate with a multichannel pipette and add neuronal induction medium (Table 3) to a total of 200 µl/well with a multichannel pipette.

Centrifuge the plate for 10 min at 1200 rpm at 37°C.

Incubate cells for 48 hrs at 37°C in a regular cell incubator.

Change medium to neuronal induction medium WITHOUT ROCK inhibitor using a multichannel pipette.

Incubate cells for 48 hrs at 37°C in a regular cell incubator.

Change medium to midbrain patterning medium (Table 4) using a multichannel pipette.

Incubate cells for 48 hrs at 37°C in regular cell incubator.

Transfer aliquots of Matrigel® reduced growth factors to ice and let it reach 4°C.

Remove the medium from each well with a multichannel pipette and add 30 µl of reduced growth factor Matrigel® with the manual repeater-pipette and sterile distritips.

Incubate the plate for 30 min at 37°C in a regular cell incubator.

Add 200 µl of tissue growth induction medium (Table 5) and incubate for 24 hours at 37°C in a regular cell incubator.

Autoclave a box of 1mL cut tips if not already done.

Add 3 mL of final differentiation medium per well of an ULTRA LOW ATTACHMENT 6 well plate.

Transfer the midbrain organoids with a cut 1000 µl pipette tip into the 6 well plate (5 organoids/well).

Put the 6 well plates on the orbital shaker (set at 70 rpm) in the 37°C regular cell incubator and change the medium every 2–3 days using final differentiation medium (Table 6). When the midbrain organoids reach 1 mm in diameter, increase the final differentiation media volume to 5 mL to provide enough nutrients (Figure 1f).

Remove medium from plates and fix organoids with submersion in fresh 4% formaldehyde solution for 1h at RT or O/N at 4°C in fume hood.

HAZARD WARNING: Use care handling formaldehyde solutions. Follow instructions according to the product MSDS.

Wash organoids with PBS 3 times for 5 min to remove formaldehyde.

Incubate organoids in 20% sucrose solution at 4°C until the organoids sink. This is usually achieved O/N or after 3 days.

NOTE: Do not extend the incubation for longer than 3 days, as this impacts the quality of sectioning. In rare occasions, the tissue does not sink after a long time because it includes low-density components such as Matrigel®. However, this does not impact subsequent procedures.

Transfer organoids from the sucrose solution to a cryomold using a pipette with a cut tip. We typically embed up to 9 organoids per block. If embedding different types of organoids (different ages, cell lines, etc.) in the same block, care must be taken not to mix the organoids.

After all organoids are placed in the mold, remove all the sucrose solution with a paper tissue, taking care that the organoids do not stick to the paper.

Slowly pour optimal cutting temperature (OCT) mounting medium directly on top of the organoids to ensure they stay on the bottom of the cryomold. If embedding different types of organoids, be careful to maintain their organization.

Use a needle to place organoids in the desired positions, while taking care not to move organoids upwards. Space the organoids about 1 mm from each other.

Freeze organoids by placing the mold in a -80°C freezer or in the gaseous phase of a liquid N₂ container. When moving the mold, take care not to tilt excessively, which may displace the organoids.

Once completely frozen, blocks may be stored long term inside a closed container to prevent drying in a -80°C freezer.

Set cryostat temperature and place all blocks to be cut in the same session inside the cryostat chamber to equilibrate the temperature.

NOTE: The relationship between the cryostat temperature and the actual temperature at the block surface after mounting varies according to the cryostat model. Using a thermal probe, we found that a surface temperature of -9°C allows easy production of high-quality samples. However, the precise setting necessary to achieve this temperature must be determined for each machine.

Prior to, or shortly after, removal of the block from mold, cut one corner of the block to keep track of the block orientation in the subsequent steps.

Trim the block edges using a razor blade, maintaining a margin of 1-2 mm of OCT around the area containing organoids.

WARNING: Use care when handling the razor blade inside of the cryostat. Avoid manually cutting blocks that are not equilibrated with the cryostat temperature, as these become harder and need more strength to be cut, which may lead to injuries.

Pour an amount of OCT on the sample holder (chuck) sufficient to cover the entire bottom surface of the block.

Press the block on top of the OCT layer on the sample holder using a heat extractor to orient the block as horizontally as possible. Wait until OCT freezes completely.

Place the mounted block in the microtome head and cut sections in the desired thickness. We routinely produce sections with a thickness ranging between 10 and 20 µm.

If necessary, flatten sections using a pair of brushes and pick sections using a slide kept at room temperature (direct mount method).

Let slides air dry for 30–60 min and proceed with histological staining. The slides may also be kept in boxes at -80°C for long term storage.

Observations: Cryosections on 30-day aged midbrain organoids reveal the presence of rosettes in the tissue (Figure 3a). The progenitors are localized in the center of rosettes while the more mature neurons localized on the external layer. The differentiated cells are positive for neuronal markers such as MAP2 and TUJ1. TH is more specific for dopaminergic neurons (Figure 3bc).

Figure 1a iPSC colonies.jpg (Quality of iPSCs suitable for midbrain organoids formation.)

Figure 1c EB with smooth edge 48 hours after formation.jpg (EB with smooth edge 48 hours after formation in neuronal induction media.)

Figure 1d Extrusion of buds on EB after midbrain patterning.jpg (Extrusion of buds on EB after midbrain patterning.)

Figure 1e Typical EB 24 hours after embedding in matrigel.jpg (Typical EB 24 hours after embedding in Matrigel®.)

Figure 1f Day 15 after transferring the tissue into final differentiation media.jpg (Typical hMo at day 15 after transferring the tissue into final differentiation media.)

Figure 1f Day 1 after transferring the tissue into final differentiation media.jpg (Typical hMo at day 1 after transferring the tissue into final differentiation media.)

Figure 1f Day 5 after transferring the tissue into final differentiation media.jpg (Typical hMo at day 5 after transferring the tissue into final differentiation media.)

Figure 2 Day 1 in final differentiation media.jpg (At day 1 in final differentiation media, hMOs are approximately 600μm.)

Figure 2 Day 50 in final differentiation media.jpg (At day 50 in final differentiation media, hMOs are approximately 4mm.)

Figure 3a Cryosection of hMO.tif (Cryosection of hMO at day 30. The immunofluorescence staining reveals dopaminergic neurons and multiple ventricule-like structures or “rosettes”.)

Figure 3b map2.tif (MAP2 staining reveals the presence of neurons on the outside layer of rosettes.)

Figure 3b nuclei.tif (Nuclei staining of the section.)

Figure 3b th.tif (TH staining reveals the presence of dopaminergic neurons on the outside layer of rosettes.)

Figure 3b tuj1.tif (TUJ1 staining reveals the presence of neurons on the outside layer of rosettes.)

Figure 3c map2.tif (MAP2 staining reveals the presence of neurons.)

Figure 3c nuclei.tif (Nuclei staining of the section.)

Figure 3c th.tif (TH staining reveals the presence of dopaminergic neurons.)

Figure 3c tuj1.tif (TUJ1 staining reveals the presence of neurons.)

Figure 4a 45 days hMO with dopamine treatment.JPG (Under dopamine treatment, brown/black areas appeared in hMos.)

Figure 4a 45 days hMO without dopamine treatment.JPG (hMos, not treated with dopamine, are used as controls.)

Figure 4b colorimetric extraction on 45 days hMO with dopamine treatment.jpeg (Colorimetric selection from GIMP software on hMos treated with dopamine.)

Figure 4b colorimetric extraction on 45 days hMO without dopamine treatment.jpeg (Colorimetric selection from GIMP software on control hMos not treated with dopamine.)

Figure 4b fontana masson on 45 days hMO with dopamine treatment.TIF (Fontana Masson staining on hMo treated with dopamine.)

Figure 4b fontana masson on 45 days hMO without dopamine treatment.TIF (Fontana Masson staining on hMo not treated with dopamine.)