Assembling Retinal Organoids with Microglia

This study was approved by the Institutional Ethics Committee of Beijing Tongren Hospital, Capital Medical University. HESCs cell line H9 was from the WiCell Research Institute. Pre-warm the cell culture medium at room temperature (RT) for 30 min before the experiment.

1. Generation of human microglia

1. Culture the hESCs in stem cell medium until the cell density reaches 80%-90%. Seed at least 1 x 10⁶ cells in each well.
2. Aspirate the stem cell medium and rinse the cells with 1x DPBS (1 mL/well of 6 well plate). The next step requires 2 x 10⁷-1 x 10⁸ cells. If not enough, combine cells of 2-5 wells to do the following steps.
3. Dissociate the stem cell colonies using the 0.5 mM EDTA solution for about 5 min in a 37 °C incubator (1 mL/well of 6 well plate).
4. Check colony morphology after 5 min. When the colony edges are slightly curled up with loose gaps, cells are ready to be differentiated and aspirate the EDTA. If not, incubate cells at 37 °C for another 1-2 min. Do not incubate for longer than 8 min.
5. Gently rinse the cells in each well using 6 mL of Medium A (Table 1) with 6 µL of 10 mM ROCK inhibitor Y-27632 solution. Gently tap the bottom of the dish to help rinse the cells. Set the day as day 0.
   NOTE: Do not pipette the cells. Cells will form embryoid bodies (EBs) the next day.
6. After 24 h, observe the cells under the microscope to confirm the EB formation. Collect the EBs into a 15 mL centrifugal tube with a 10 mL pipet and wait for the EBs to settle to the bottom in 5 min.
7. Carefully aspirate the supernatant and resuspend the EBs with 6 mL of fresh Medium A and transfer them into a new low-adhesion well of 6-well plate. Culture in a 37 °C incubator.
   NOTE: The purpose of the well transferring on day 1 is to reduce the influence of a large number of dead cells during the process of EB formation, which may affect the state of cell growth.
8. Refresh the fresh Medium A daily until day 4 (6 mL/well of 6-well plate).
9. On day 4, coat a 10 cm dish with 3 mL of 0.1% fish gelatin solution for at least 1 h in a 5% CO₂, 37 °C incubator.
   NOTE: If the coating time is less than 1 h, it is difficult to carry out the following steps.
10. Carefully collect the EBs into a 15 mL centrifugal tube with a 10 mL pipet and wait for the EBs to settle to the bottom in 5 min.
11. Remove the 0.1% gelatin solution and rinse the 10 cm dish once with 5-6 mL of 1x DPBS. Remove the DPBS.
12. Gently resuspend the EBs with 15 mL of Medium B (Table 1) in the 15 mL centrifugal tube and transfer them to the coated 10 cm dish. Culture in a 37 °C, 5% CO₂ incubator for 1 week.
    NOTE: In the first 7 days, do not move the dish.
13. Refresh with fresh Medium B once a week until day 49 (15 mL/10 cm dish), and examine the cells under the microscope once a week.
    NOTE: After day 49, several secretory cells can be found in the supernatant in the culture dish.
14. Centrifuge the supernatant cells at 200 x g for 5 min at RT.
15. Carefully aspirate the supernatant and resuspend the cells with 2 mL of fresh Medium B and transfer into a new low-adhesion well of 6-well plate. Incubate in a 5% CO₂, 37 °C incubator.
    NOTE: In the next 7 days, do not move the plate. The microglia could adhere to the bottom of the low-adhesion well.
16. On day 56, replace with Medium C (Table 1) (2 mL/well of 6-well plate). The microglia adhere to the bottom of the low-adhesion well and will look branched under the microscope.
    NOTE: By changing Medium C every 3 days (2 mL/well of 6-well plate), the microglia could be cultivated in Medium C for about 15 days.

2. Generation of human ROs and co-culture the ROs with microglia

1. Cultured the hESCs in stem cell medium until the cell density reaches 80%-90%. Seed at least 1 x 10⁶ cells in each well.
2. Add Dispase (1 mL/well of 6 well plate) to the culture cell. Incubate at 37 °C for 5 min. Aspirate the Dispase solution from the well.
3. Add Medium D (Table 1) (1 mL/well of 6 well plate) to the well. Cut the cell into small pieces with a 10 µL pipette.
4. Gently collect all the cell pieces and medium in a 1.5 mL microcentrifuge tube.
5. Centrifuge at 200 x g for 5 min and remove the supernatant.
6. Gently resuspend the cells in 200 µL of a cold matrix. Move the 1.5 microcentrifuge tube into an incubator for 20 min. Operate fast because the matrix will solidify at room temperature (RT). After 20 min in the incubator, the matrix will solidify.
7. Prepare a 10 cm dish with 15 mL Medium D. Resuspend the Matrix with Medium D and shake the Petri dish gently.
8. Then, put the dish in an incubator for 5 days. Do not move the plate. Set the day as day 0 (ROs). The cells will adhere in 3 days.
9. On day 5 (ROs) and day 10 (ROs), refresh the medium with Medium D.
10. On day 12 (ROs), remove the medium and add 3 mL of Dispase to each dish for 5 min.
11. Aspirate the Dispase and add 15 mL of Medium E (Table 1).
12. Harvest microglia: On day 56 (MGs), gently aspirate the Medium C, add Accutase (1 mL/well of a 6-well plate), and incubate at 37 °C for 3 min.
13. Collect the microglia cells into a 15 mL centrifugal tube with a 5 mL pipet. Centrifuge cells at 200 x g at RT for 5 min.
14. Gently aspirate the supernatant and suspend the microglia with 1 mL of Medium E. Add microglia to digested ROs on day 12.
    NOTE: On day 12 (ROs), the cells are adhesive. On day 13 (ROs), the cells are found suspending in Medium E. If the medium changes yellow from day 12 (ROs) to day 19 (ROs), refresh the medium with Medium E.
15. On day 19 (ROs), aggregate the organoids to the center of the dish by gently swirling the plate. Gently aspirate Medium E and refresh the medium with Medium F.
16. Transfer the organoids with Medium F to a new suspension dish.
17. Change the medium once a week and select organoids for experiments.