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1. Creating Cerebral Organoids from iPSC / hESC 2D Culture (Day 0)
Note: This protocol assumes that stem cell (SC) maintenance is conducted in xeno-free stem cell maintenance media (SCMM) with a vitronectin or Geltrex substrate. If using an alternative, high-protein stem cell culturing media, it is advisable to transition SC cultures to SCMM for at least two passages before initiating organoid formation. The protocol has not been tested with feeder-based SC culture methods.
- Using regular SC maintenance methods, bring cultures to 50%-70% confluence. This is typically 3-4 days after passaging, though this can be dependent on culture method and cell line.
Note: Approximately 2 wells from a 6-well plate are needed to produce a full 96-well plate of organoids.
- Inspect cultures via brightfield microscopy at 10X-20X magnification to ensure healthy colony morphology, with no detectable differentiation.
- Bring SCMM to 37 °C in a hot water bath and thaw a 50 µL vial of 50 mM Y-27632 (Rock inhibitor) and 2 mL of enzymatic detachment reagent to room temperature.
- Prepare an ultra-low attachment (ULA) U-bottom 96 well plate, a multichannel P200 pipette, and a 25 mL reagent reservoir.
- Aliquot 45 mL of SCMM in a 50-mL conical tube and add 45 µL of 50 mM Y-27632. Mix thoroughly by triturating.
- Vacuum aspirate the two wells containing SCs, and quickly add 1 mL of enzyme-free detachment reagent to each well using a P1000 pipette. Incubate the plate in a 37 °C incubator for 4 min.
- Vacuum aspirate the treated wells and add 1 mL of enzymatic detachment reagent to each well using a P1000 pipette.
- Incubate the plate in a 37 °C incubator for an additional 5 min.
- Remove the plate and examine the treated wells. Gently tap on the plate to break up aggregated cells. If large cell clusters are still visible, incubate the plate for 2 additional min at 37 °C. Repeat this step until clusters are no longer visible by the naked eye.
- Once the cells are properly dissociated, add 1 mL of SCMM to each well to deactivate the dissociation enzymes. Pool the 4 mL of cell suspension in a 50-mL conical tube and take a small aliquot for cell counting.
- Return any remaining, untreated wells in the maintenance plate to the 37 °C incubator.
- While counting, centrifuge the cell suspension at 300 x g for 5 min.
- Determine the total number of cells and calculate the amount of SCMM + Y-27632 solution that will be needed to achieve a 60,000 cells/mL suspension.
- Carefully vacuum aspirate the supernatant and add the calculated volume of SCMM + Y-27632 solution. Mix 5-10 times with a 5-mL pipet, ensuring a uniform cell suspension.
- Immediately transfer the cell suspension into a reagent reservoir, and pipette 150 µL into each well of the ULA U-bottom 96-well plate using a multichannel P200 pipette. This produces 96 individual organoids, each initially consisting of 9,000 cells.
- Centrifuge the plate at 150 x g for 1 min.
- Place the plate into a 37 °C incubator, and do not disturb the plate for 48 h.
2. Cerebral Organoid Maintenance (Day 2)
- Prepare 17 mL of SCMM with 17 µL of 50 mM Y-27643 in a 50-mL conical tube. Warm the SCMM + Y-27632 solution to 37 °C in a hot water bath.
- Take the organoid plate from the incubator and using a multichannel P200 pipette set to 75 µL, slowly draw up medium from the edge of the well on the first row. Once the medium has been drawn, quickly expel the medium back into the well to lift up loose cells and organoids. Repeat this for each row.
Note: This technique, hereafter referred to as a "dispersion," lifts cellular debris and the organoid into suspension. The organoid rapidly sinks to the bottom of the well, while the smaller debris remains in suspension, allowing it to be removed with a media change. This helps to prevent the organoid from resting in cellular debris during culture.
- After all wells have been dispersed, wait for 15 s to ensure that the organoids have sunk to the bottom of each well.
- Slowly and carefully aspirate 75 µL of medium from each well using the multichannel P200 pipette and dispense into a waste reservoir.
Note: Most users occasionally aspirate an organoid during regular feeds. This can occur roughly 1% of the time. Please plan accordingly to ensure that enough organoids survive to experimental time points.
- Transfer SCMM + Y-27632 solution into a reagent reservoir and dispense 150 µL into each organoid well using the multichannel P200 pipette.
- Place the plate back into a 37 °C incubator.
3. Cerebral Organoid Maintenance (Day 3)
- Warm 17 mL of SCMM to 37 °C, this time without Y-27643.
- With the multichannel P200 pipette set to 100 µL, disperse all wells of the organoid plate (see step 2.2). Wait 15 s to ensure that organoids have sunk to the bottom of their wells.
- Prepare a waste reservoir and transfer warmed SCMM into a source reservoir.
- Carefully aspirate 125 µL of medium from each well and replace with 150 µL of fresh SCMM using the multichannel P200 pipette.
- Place the plate back into a 37 °C incubator.
4. Cerebral Organoid Maintenance (Day 4+)
Note: Conduct this regular maintenance every other day until infection.
- Before beginning the feed on day 4, prepare Neural Induction (NI) medium.
- Thaw a 250 µL aliquot of 2 mg/mL heparin, along with one 5 mL vial of 100X N-2 Supplement. Add the 250 µL of heparin, 5 mL of 100X N-2, and 5 mL of 100X Minimum Essential Medium-Non-Essential Amino Acids (MEM-NEAA) to 500 mL of Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) + GlutaMAX. Sterile filter the mixed solution in a 500 mL filter.
Note: Keep NI at 4 °C when not in use; NI lasts up to two weeks before fresh medium should be prepared.
- Warm 17 mL of NI medium to 37 °C.
- With the multichannel P200 pipette set to 100 µL, disperse all wells of the organoid plate (see step 2.2). Wait 15 s to ensure that organoids have sunk to the bottom of their wells.
- Prepare a waste reservoir and transfer warmed NI into a source reservoir.
- Carefully aspirate 125 µL of medium from each well and replace with 125 µL of fresh NI using the multichannel P200 pipette.
- Place the plate back into a 37 °C incubator.
5. Organoid Infection with zika virus (ZIKV) (~Day 24)
NOTE: After approximately 3 weeks of differentiation, there will be large neuroepithelial structures and rosettes visible within the organoid that will be highly susceptible to ZIKV infection.
- Bring Earle's 1X Balanced Salt Solution (EBSS), 1X phosphate-buffered saline (PBS), and NI to 37 °C in a hot water bath.
- Dilute ZIKV of known concentration in 1X EBSS to the target infection multiplicity of infection (MOI).
Note: Titrations of different MOIs are recommended to achieve a viral dose response. This may vary by virus strain; for ATCC VR-1838 and ATCC VR-1843, typical MOI values range between 0.1 and 10).
- Carefully remove all medium from organoid well using a single channel P200 pipette. Quickly add 200 µL of 1X PBS to each well using a P200 multichannel pipette to wash the organoids.
- Carefully remove all medium from organoid well using a single channel P200 pipette. Quickly add 50 µL of 1X EBSS-only (mock infection) or ZIKV virus solution to well and ensure that the organoid is completely submerged. Repeat for each organoid that is to be infected for the study.
- Place the plate back into a 37 °C incubator for 2 h.
Note: Incubating mock-infected organoids for this duration should not significantly impact their growth as compared to undisturbed organoids.
- Approximately 30 min before viral incubation is completed, aliquot 25 mL of NI into a 50-mL conical tube and bring to 37 °C in a hot water bath.
- After viral exposure is completed, wash the organoids with 200 µL of 1X PBS for each well, allow organoids to settle for 15 s, and then remove 1X PBS using a single channel P200 pipette.
- Add 200 µL of fresh NI to each well, and place back into the incubator.
- Optional: For a day 0 time point, organoids may be imaged one hour after infection.
- Continue to culture by replacing 125 µL NI every other day using the dispersion method (see step 4). Be sure to properly dispose of spent media and tips, as this contains infectious ZIKV particles.