| Usage |
This kit can generate more than 500 midbrain organoids in total.
1. Experimental Instruments and Materials:
Instruments: Biological safety cabinet, cell incubator, horizontal centrifuge, inverted microscope, low-temperature refrigerator
Materials: Centrifuge tubes (15 mL, 50 mL), pipettes (10 μL, 100 μL, 1000 μL, multi-channel 100 μL), sterile pipette tips (10 μL, 200 μL, 1000 μL), serological pipettes (10 mL, 50 mL), 37 μm reversible cell strainers, wide-bore 200 μL / 1 mL pipette tips, AggreWell™800 24-well plates, TC-treated non-low-adhesion 6-well plates, ultra-low attachment 6-well plates
2. Experimental Contents and Methods:
1. Preparation of hESCs / hiPSCs
Culture hESCs / hiPSCs in TC-treated non-low-adhesion 6-well plates with hESC/iPSC Culture Kit until cell confluency reaches 70–80%.
2. Midbrain Organoid Formation (Day 0–Day 6)
Preparation of Cell Seeding Medium
Cell Seeding Medium (2.5 mL): Warm reagents to room temperature before use; mix 2.5 mL Basal Medium 1 + 5 μL Supplement A, prepare freshly right before use.
Thaw supplements at 15–25 °C and mix thoroughly.
Note: Store unused supplements at -20 °C, do not exceed shelf life. Use immediately after thawing, avoid repeated freeze-thaw cycles.
Day 0
(1) Prepare AggreWell™800 24-well plates; adjust operation volumes proportionally if other culture vessels are used. Warm all plates, medium and reagents to 15–25 °C in advance.
a. Add 500 µL anti-adhesion rinse solution to each well.
b. Centrifuge the plate at 1300 × g for 5 min with plate balance matched.
c. Observe under microscope to confirm all air bubbles are removed from microwells; re-centrifuge at 1300 × g for another 5 min if bubbles remain trapped.
d. Aspirate and discard anti-adhesion rinse solution from wells.
(2) Add 1 mL Cell Seeding Medium to each AggreWell™800 well and set aside.
(3) Aspirate spent medium from hPSC 6-well plates, rinse cells gently with 1 mL DPBS (Ca2+/Mg2+ free) per well and discard wash buffer.
Note: Cells harvested from 4–6 wells provide sufficient quantity for subsequent steps.
(4) Add 1 mL Accutase per well and incubate at 37 °C for 3 min.
Note: Incubation time varies depending on cell line or alternative enzyme-free dissociation reagents.
(5) Gently triturate cells up and down 3–5 times with a 1 mL pipette, transfer single-cell suspension into sterile 15 mL conical tube.
(6) Rinse each well with 1–2 mL Basal Medium 2 and combine wash liquid with cell suspension.
(7) Count viable cells with trypan blue staining and automatic cell counter. Calculate required suspension volume to obtain total 4.5 × 106 cells (dilute later to final concentration of 3 × 106 cells/mL).
(8) Centrifuge cell suspension at 300 × g for 5 min.
(9) Carefully aspirate supernatant, resuspend cell pellet in 1 mL pre-prepared Cell Seeding Medium to reach final concentration of 3 × 106 cells/mL.
(10) Add 1 mL single-cell suspension (3 × 106 cells total) into each AggreWell™ well pre-filled with 1 mL Cell Seeding Medium. This yields 10,000 cells per microwell.
Note: Pipette up and down gently several times to evenly distribute cells across the well surface.
(11) Centrifuge AggreWell™ plate at 100 × g for 3 min with balanced plate to capture cells inside microwells.
(12) Check uniform cell distribution in microwells under microscope, incubate at 37 °C, 5% CO2.
Day 1–Day 5
Preparation of Midbrain Organoid Formation Medium
Midbrain Organoid Formation Medium (15 mL): Warm to room temperature before use, composed of pure 15 mL Basal Medium 1.
Note: Can be stored at 2–8 °C for maximum 3 weeks if unused, do not exceed basal medium expiry date. Use immediately after thawing, avoid repeated freeze-thaw cycles.
(13) Take the plate out of incubator carefully without disturbing aggregates inside microwells.
Note: Rough handling causes premature organoid fusion and reduces total yield.
(14) Gently remove two aliquots of 750 µL spent medium per well using 1 mL pipette and discard.
Note: Keep pipette tip at liquid surface only to avoid touching organoids.
(15) Slowly add two aliquots of 750 µL fresh Midbrain Organoid Formation Medium per well.
Note: Support pipette tip against well wall at liquid surface and release medium slowly; rapid flow will flush organoids out of microwells.
(16) Incubate at 37 °C, 5% CO2 for 24 h.
(17) Repeat steps 13–16 daily from Day 2 to Day 5.
(18) Proceed to expansion operation on Day 6.
3. Midbrain Organoid Expansion (Day 6–Day 25)
Warm all culture vessels, medium and reagents to 15–25 °C before operation.
If ultra-low attachment plates are unavailable, coat standard TC-treated plates with anti-adhesion rinse solution to prevent cell attachment: add 1 mL rinse solution per well, incubate briefly then fully aspirate and discard.
Preparation of Midbrain Organoid Expansion Medium
Midbrain Organoid Expansion Medium (250 mL): Warm to room temperature, mix 245 mL Basal Medium 3 + 5 mL Supplement B + 0.5 mL Supplement C + 0.5 mL Supplement D.
Note: Can be stored at 2–8 °C for maximum 3 weeks if unused, do not exceed expiry dates of basal medium and supplements. Use immediately after thawing, avoid repeated freeze-thaw cycles. Mild viscosity of Basal Medium 3 is normal; shake thoroughly and pipette slowly during transfer. Store undiluted supplements at -20 °C if not mixed, do not exceed shelf life.
(1) Add 2 mL Midbrain Organoid Expansion Medium into each ultra-low attachment 6-well plate well.
Note: Organoids harvested from one AggreWell™ well can be evenly split into one 6-well plate, with recommended density of 25–40 organoids per well. Controlling density effectively prevents premature fusion and yield loss.
(2) Place a 37 μm reversible cell strainer on top of a 50 mL waste conical tube (arrow mark of strainer facing upward). Use a new strainer and collection tube for each AggreWell™ well harvest.
(3) Aspirate top layer spent medium from AggreWell™ well, firmly flush microwell bottom with wide-bore 1 mL pipette tip to release organoid suspension.
(4) Collect suspension with wide-bore tip and pass through 37 μm strainer; organoids retain on strainer surface while single cells flow into waste tube.
(5) Flush the original AggreWell well with 1 mL DMEM/F-12 using wide-bore tip, immediately transfer flush liquid through the same strainer to recover residual organoids.
(6) Repeat step 5 once or twice until all organoids are fully recovered; verify complete harvest under microscope.
(7) Invert strainer onto a new 50 mL collection tube, elute trapped organoids with 2 mL Midbrain Organoid Expansion Medium.
Note: Medium viscosity requires sufficient pipette suction force to fully recover organoids from strainer mesh.
(8) Gently invert tube to homogenize organoid suspension, transfer into prepared ultra-low attachment 6-well plates with wide-bore pipette, maximum 40 organoids per well.
(9) Rock plate crosswise gently to evenly distribute organoids, observe under microscope to minimize contact between individual organoids.
(10) Place plate horizontally inside incubator without disturbance, culture at 37 °C, 5% CO2 for 48 h.
Day 8–Day 25
(11) Tilt plate gently and wait 15–30 s for organoids to settle at well bottom.
(12) Level plate again, aspirate spent medium from liquid surface with 1 mL pipette.
(13) Add 2 mL fresh Midbrain Organoid Expansion Medium per well, rock crosswise gently before returning to incubator to disperse organoids evenly. Ensure plate remains undisturbed at 37 °C, 5% CO2.
(14) Perform full medium exchange every other day from Day 8 to Day 25 following steps 11–13.
(15) Proceed to differentiation stage on Day 25.
4. Midbrain Organoid Differentiation (Day 25–Day 43)
Preparation of Midbrain Organoid Differentiation Medium
Midbrain Organoid Differentiation Medium (250 mL): Warm to room temperature, mix 245 mL Basal Medium 3 + 5 mL Supplement B + 0.25 mL Supplement E.
Note: Can be stored at 2–8 °C for maximum 3 weeks if unused, do not exceed expiry dates of basal medium and supplements. Use immediately after thawing, avoid repeated freeze-thaw cycles. Mild viscosity of Basal Medium 3 is normal; shake thoroughly before use. Store undiluted supplements at -20 °C if not mixed.
(1) Tilt plate gently and wait 15–30 s for organoids to settle at well bottom.
(2) Level plate, aspirate spent medium from liquid surface.
(3) Add 2 mL fresh Midbrain Organoid Differentiation Medium per well, incubate at 37 °C, 5% CO2.
(4) Refresh medium every other day from Day 27 to Day 43 following steps 1–3.
(5) Proceed to long-term maintenance culture starting Day 43.
5. Long-Term Midbrain Organoid Maintenance (Day 43 onwards)
Preparation of Midbrain Organoid Maintenance Medium
Midbrain Organoid Maintenance Medium (100 mL): Warm to room temperature, mix 98 mL Basal Medium 3 + 2 mL Supplement B.
Note: Can be stored at 2–8 °C for maximum 3 weeks if unused, do not exceed expiry dates of basal medium and supplements. Use immediately after thawing, avoid repeated freeze-thaw cycles. Mild viscosity of Basal Medium 3 is normal; shake thoroughly before use. Store undiluted supplements at -20 °C if not mixed.
(1) Tilt plate gently and wait 15–30 s for organoids to settle at well bottom.
(2) Level plate, aspirate spent medium from liquid surface.
(3) Add 2 mL fresh Midbrain Organoid Maintenance Medium per well.
(4) Refresh medium every 2–3 days.
Note: Increase medium volume to 3 mL per well after Day 50 when organoids expand to larger size to ensure full submersion.
(5) Immunofluorescence staining can be performed starting Day 50. Specific midbrain lineage markers can be detected in peripheral organoid regions, including midbrain progenitor markers FOXA2+/LMX1A+ and dopaminergic neuron markers TH+/GIRK2+. Organoids can be stably cultured over 50 days with maintenance medium.
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