To begin, obtain human microglia from the human embryonic stem cells and harvest them on day 56. For retinal organoid derivation, culture the human embryonic stem cells in a stem cell medium until the cell density reaches 80 to 90%Add one milliliter dispase per well to the culture cells and incubate at 37 degrees Celsius for five minutes. After removing dispase, add one milliliter of medium D to each well.
Cut the cells into small pieces. With a 10 microliter pipette, gently collect all the cell pieces and medium into a 1.5 milliliter microcentrifuge tube. Centrifuge the tube at 200 x g for five minutes.
After removing the supernatant, gently resuspend the cells in 200 microliters of a cold matrix. Move the 1.5 milliliter microcentrifuge tube into an incubator for 20 minutes. After 20 minutes in the incubator, the matrix will solidify.
Prepare a 10 centimeter dish with 15 milliliters of medium D.Resuspend the matrix with medium D and shake the Petri dish gently. Place the dish in an incubator for five days. On day 12, replace the medium with three milliliters of dispase.
After five minutes, aspirate the dispase and add 15 milliliters of medium E.Introduce the harvested microglia to digested retinal organoids on day 12. On day 19, gently swirl the plate to aggregate the organoids to the center of the dish and replace the medium E with medium F.Finally, transfer the organoids with medium F to a new suspension dish. By day 30, retinal organoids co-cultured with microglia showed significant GFP autofluorescence, indicating the presence of microglia.
The co-cultured retinal organoids displayed clear immunofluorescence signals for the photoreceptor marker CRX and the microglial marker IBA1.
