This is a model for retinal disease and emulates the in vivo retina. To some extent, it could replace animal experiments. The protocol is optimized for the quality and quantity of the photoreceptor precursors in the retinal organoids, and the photoreceptor is the key to several irreversible blindness conditions.
Begin by culturing the human embryotic stem cells, or hESCs, under feeder-free conditions. To do so, coat one well of a six-well plate with one milliliter of 100 micrograms per milliliter of reagent A, and incubate the plate at 37 degrees Celsius for at least half an hour. Thaw an aliquot of one million hESCs, and centrifuge it at 200 xg for five minutes.
After removing the supernatant, seed the cells into the previously coated plate, along with two milliliters of reagent B.Passage the cells after reaching approximately 80%confluency at around four days. After reaching confluency, wash the cells with pre-warmed DPBS, and transfer them into 500 microliters of freshly prepared reagent. Then, incubate the cells for 3.5 minutes at 37 degrees Celsius and 5%carbon dioxide.
Detach the cells by flicking the side and bottom of the plate for a few seconds, and add 500 microliters of prepared medium one. Harvest the detached cells into a new 1.5-milliliter tube, and pipette the cell suspension up and down. For cell counting, add 100 microliters of cell suspension to 900 microliters of DPBS.
Dilute the 900 microliters of the remaining cell suspension with medium one in the Petri dish to achieve nine times 10 to the fourth power cells per milliliter. Add 100 microliters of diluted cell suspension to each well of a non-adherent, V-bottom, 96-well plate. Lightly spin down the plate on a low-speed shaker for five minutes, and incubate at 37 degrees Celsius and 5%carbon dioxide until day two.
On day two, add 20 microliters of 1%reagent A to each well of the 96-well plate, and pipette twice in the center to scatter the dead cells. Clean the bottom of the plate before incubating it at 37 degrees Celsius and 5%carbon dioxide until day six. On day six, replace 58 microliters of the medium with 60 microliters of fresh medium one, and continue the incubation.
On day 12, harvest the cell pellets from the 96-well plate in a 15-milliliter conical tube, and allow the pellets to settle naturally for five minutes at room temperature. When pellets settle, remove the supernatant without disturbing the organoids, and transfer the cell aggregates to a 10-centimeter suspension dish containing 18 milliliters of medium two with reagent A.Incubate the dish at 37 degrees Celsius and 5%carbon dioxide until day 18. On day 18, confirm the generation of a semi-transparent optic vesicle in the dish, and cut the generated organoids using a microsurgical knife.
Aggregate all the cut pellets to the center of the dish. Then, harvest the cells into a 15-milliliter conical Falcon tube. When the cells settle, gently remove the supernatant, and resuspend the pellets in two 10-centimeter Petri dishes with 18 milliliters of medium, three per dish.
Gently transfer the dishes to the incubator to avoid cell aggregation. Continue culturing the cells, changing the medium weekly. Analyze the CRX expression after day 45 until day 120.
In this representative analysis, various stages of human retinal generation are displayed. On day six, organoids appeared as an aggregate of dense connections inside a bright rim with a diameter of around 600 micrometers. On day 12, the initial generation of the optic vesicle-like structures took place.
The organoids without proper vesicle-like architecture were discarded before culturing in a Petri dish on day 18. By day 30 of the culture, the vesicle-like architecture was more obvious, and inferior ROs could be easily distinguished and removed. Starting on day 45, the organoids expressed various markers of photoreceptor precursors, such as CRX, RCVRN, and OTX2.
By cleaving the superior retinal organoids, the differentiation efficiency is significantly improved, and more than 100 superior organoids from 96 plates could be harvested.
