This method can help answer question regarding differentiation of pluripotent stem cell in the retinal field, such as the generation of different retinal cell types from human-induced pluripotent stem cell or iPS cells. The main advantage of this technique is that adherent human iPS cell can be directly used for the generation of retinal organoids by passing embryoid to formation only by successive change of culture media. Laboratory engineer Amelie Slembrouck-Brec will be demonstrating the method to generate and isolate retinal organoids and laboratory engineer Celine Nanteau will be demonstrating the strategy for retinal pigmented epithelial or RPE cell production.
Start the iPS cell differentiation following this schematic, which can be found in the text protocol when the colonies reach 60 to 70%confluence. Prepare four milliliters of basal iPS medium per six-centimeter dish and warm the medium to 37 degrees Celsius. Then, change the iPS cell medium to basal iPS medium.
Note this time as Day Zero. At Day Two, switch the cultures in the basal iPS medium to a basal iPS N-2 medium previously warmed at 37 degrees Celsius. Change the medium every two to three days.
Identify emergent cell-forming retinal organoids by neuroepithelium buds. At Day 28, prepare six-well plates containing four milliliters per well of Pro B-27 medium, initially supplemented with 10 nanograms per milliliter of FGF2. Recover the retinal structures from the six-centimeter dishes manually.
To do this, isolate the structures by making perpendicular striations with a needle around the neuroepithelial bud and detach the organoids by gently scratching them with the needle. Aspirate 10 to 15 organoids using a 1, 000-microliter pipette and transfer them in a single well of the six-well plate containing Pro B-27 medium. Keep the retinal organoids in floating culture conditions in Pro B-27 medium in a cell culture incubator at 37 degrees Celsius in 5%carbon dioxide.
Change half of the medium every two to three days. At Day 28, change the medium to a fresh Pro N-2 medium previously warmed at 37 degrees Celsius and continue to change the Pro N-2 medium every two to three days. At Day 42, prepare a 24-well plate previously coated with a matrix as described in the text protocol and containing one milliliter of Pro N-2 medium per well.
Place the plate for 15 minutes in the incubator at 37 degrees Celsius in 5%carbon dioxide before use. Manually recover the human iRPE patches from the six-centimeter dish. To isolate the patches, make a perpendicular striation with the needle around the pigmented epithelium and detach the sheet by gently scratching it with the needle.
Aspirate 10 pigmented patches using a 1, 000-microliter pipette and transfer them in a single well of the 24-well plate. Keep the human iPS-derived RPE patches in Pro N-2 medium in a cell culture incubator at 37 degrees Celsius and 5%carbon dioxide for 48 hours before changing the medium. When the cells are confluent, remove the medium using vacuum aspiration systems and wash each well once with one milliliter of PBS using a five-milliliter pipette.
After discarding the PBS using vacuum aspiration systems, add 200 microliters per well of 0.25%trypsin. Incubate the cells for a minimum of 15 minutes in an incubator at 37 degrees Celsius, then add 800 microliters of Pro B-27 medium to inactivate the trypsin. Now dissociate the sheet of human iRPE cells by pipetting it up and down.
Place the cell suspension in a 15-milliliter tube and centrifuge for five minutes at 110 times g. Remove the supernatant and gently resuspend the cell pellet in two milliliters of Pro N-2 medium prewarmed at 37 degrees Celsius, then count the cells with a cell counter. Now take 1.25 million cells and place them in a matrix-coated T25 square centimeter flask containing five milliliters of Pro N-2 medium that has been prewarmed to 37 degrees Celsius.
For whole retinal organoids, select five to 20 retinal organoids using a transfer pipette and place them in a cryogenic vial. Remove any excess medium with a 1, 000-microliter pipette without touching the organoids at the bottom of the tube and add 250 microliters of cold cryopreservation medium. Freeze the vials in an isopropranol-based freezing container at minus 80 degrees Celsius for a minimum of four hours.
For human iRPE cells, dissociate the cells when they are confluent at Passage 1. Aspirate the medium from the T25 square centimeter flask using vacuum aspiration systems and wash it once with three milliliters of PBS using a five-milliliter pipette. After removing the PBS, add one milliliter of 0.25%trypsin, then incubate the cells for a minimum of 15 minutes in an incubator at 37 degrees Celsius.
Following incubation, add five milliliters of Pro B-27 medium to stop the trypsin activity. Dissociate the sheet of human iRPE cells by pipetting them up and down using a 10-milliliter pipette. Place the cell suspension in a 15-milliliter tube and centrifuge it for five minutes at 110 times g.
Aspirate the supernatant using vacuum aspiration and gently resuspend cells to obtain two million cells in 250 microliters of cryopreservation medium. Transfer 250 microliters of cell suspension per cryogenic vial, then freeze the vials in an isopropranol-based freezing container at minus 80 degrees Celsius for a minimum of four hours. From the liquid nitrogen tank or minus 150-degrees Celsius freezer, thaw a cryogenic vial containing retinal organoids in a water bath at 37 degrees Celsius for 30 seconds.
Disinfect the cryogenic vial carefully using a disinfectant solution spray. Open the vial and add one milliliter of prewarmed Pro B-27 medium. Now transfer the organoids to 1.5-milliliter tube with a transfer pipette.
Remove the medium gently by pipetting it without touching the retinal structures at the bottom of the tube. Then wash the organoids one more time with one milliliter of the prewarmed Pro B-27 medium. Aspirate the organoids with a transfer pipette and place them in one well of a six-well plate containing Pro B-27 medium prewarmed and equilibrated in an incubator at 37 degrees Celsius and 5%carbon dioxide.
To thaw the human iRPE cells, warm Pro N-2 medium at 37 degrees Celsius. From the liquid nitrogen tank or minus 150-degrees Celsius freezer, thaw a cryogenic sample of human iRPE Passage 1 cells by incubating them in a water bath at 37 degrees Celsius for 30 seconds. After disinfecting the cryogenic vial as before, open the tube and add one milliliter of prewarmed Pro N-2 medium, then transfer the cell suspension to a 15-milliliter tube containing two milliliters of prewarmed Pro N-2 medium.
Following centrifugation for five minutes at 110 times g, remove the supernatant and gently resuspend the cell pellet in two milliliters of Pro N-2 medium prewarmed at 37 degrees Celsius. Cell-forming neuroretinal structures can be isolated using a needle and transferred to culture plates to allow the maturation of the retinal organoids in floating culture conditions. Emerging retinal structures contain mainly retinal progenitor cells, which give rise to seven major classes of retinal cell types, as visualized by waves of early and late-born retinal cell generations during an in vitro maturation process.
Thus, the culture time defines the cell types present in the organoids. Depending on the human iPS cell clone, pigmented patches can be detected before or after the cell formation of retinal structures, generally one or two weeks after the use of Pro N-2 medium at Day 28. A representative brightfield image of human iRPE cells expanded from the isolated patches at Passage 0 is shown.
Longterm cultures result in a mature and functional epithelium. Shown here are mature human iRPE Passage 2 cells at Week 52, characterized by a classical cuboidal cobblestone morphology. While attempting this procedure, it's important to remember that the efficiency of the method represented here largely depends on the quality of the human iPS cell cultures.
Following this procedure, all retinal cell types can be generated from retinal progenitors present in organoid at early stage. They constitute powerful tools to treat and to model different retinal diseases. Patient-specific iPS cell could be used to better understand the etiology of complex or genetic diseases by the exploration of the molecular and cellular mechanism underlying these pathologies.
After its development, this technique paved the way for researchers in the field of retinal development to explore regenerative medicine in humans. Don't forget that working with human iPS cell requires important precautions such as specific cell culture confinement.
