Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

The induction process from hPSCs to retinal cells is complicated and time-consuming. This optimized protocol can generate retinal tissues with high reproducibility and no cost. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction from hPSCs.

With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate disease modeling and cell therapy for retinal degenerative diseases. Demonstrating the procedure, we are with Yuanyuan Guan, a PhD student, and Bingbing Xie, a technician from the laboratory.

Begin by preparing 50 mL of ECM solution by adding 1 mL of the third 50 times stock solution to 50 mL of DMEM. Then, add 1 mL of this prepared ECM solution to each well of a six-well plate and incubate it for one hour at 37 degrees Celsius and 5%carbon dioxide. Prepare hPSC maintenance medium according to the manufacturer's instruction and prewarm it to room temperature for 30 minutes.

Pour a cryogenic vial of hPSCs from a liquid nitrogen tank by incubating it in a water bath at 37 degrees Celsius for 30 seconds. Carefully disinfect it using a 75%alcohol spray and put it in a bio-safety cabinet. Transfer the cell suspension from the vial to a 15 millimeter tube.

Then, add 5 mL of pre-warmed maintenance medium drop-by-drop to the tube using a 5 mL pipette, while gently shaking the tube to blend the hPSCs. Centrifuge the tube at 170 times G for five minutes. Carefully remove most of the supernatant using a 1 mL pipette, leaving behind approximately 50 microliters of supernatant to avoid losing the cells.

Re-suspend the pellet with 1 mL of maintenance medium by pipetting up and down. Remove ECM from the pre-coated wells and add 1.5 milliliters of maintenance medium to each well. Then distribute 0.5 milliliters of cell suspension into each well.

Gently shake the plate to distribute the hPSCs uniformly. Put the plate in an incubator at 37 degrees Celsius and 5%carbon dioxide for at least 24 hours to promote cell adherence. Change medium every day and passage hPSCs at 80%confluence.

On Day-0, initiate the differentiation by removing the 80%confluence cells from one well of the six-well plate. Collect the cells with EDTA dissociation solution as described in the text manuscript. Remove the EDTA solution and add 1 mL of maintenance medium containing 10 micromolar fluvastatin to stop cell dissociation.

Collect the cells with a 1 mL pipette. Transfer the cell suspension to a 100 millimeter ultra-low attached Petri dish and add 9 mL of maintenance medium containing 10 micromolar fluvastatin to the dish. Gently shake the dish twice to distribute the cells uniformly.

Then, put it in the incubator at 37 degrees Celsius and 5%carbon dioxide. After the cells are cultured for at least 24 hours, observe them under the microscope. Add 9 mL of maintenance medium and 3 mL of NIM to a 15 mL tube.

Transfer the cell cultures to a 15 mL centrifuge tube and add 10 mL of the pre-warmed NIM mixture to the dish. Centrifuge the tube at 60 times G for 3 minutes to collect the aggregates. Then remove the supernatant using a 5 mL pipette, leaving behind approximately 500 microliters to avoid losing cells.

Add 2 mL of the mixture to the tube and transfer the suspension back to the same Petri dish. Gently shake the dish to uniformly distribute the cell aggregates. Then, put the dish back in the incubator.

On Day-5, remove ECM from the pre-coated dishes and add 10 mL pre-warmed NIM to each dish. Take out the dish containing Ebs and check the quality of EBs under the microscope, making sure that they are bright and round. Collect all EBs in a 15 mL tube and allow them to settle for 5 minutes.

Then, remove most of the supernatant, leaving behind about 2 mL of medium. After counting the EBs, seed them drop-by-drop at a density of approximately 2-3 EBs per square centimeter into coated dishes containing 10 mL of NIM. Gently shake the dishes to distribute the EBs uniformly and put them in the incubator for at least 24 hours.

Use a tungsten needle or a needle with a 1 mL syringe to mechanically detach the morphologically identifiable optic vesicles, along with the adjacent retinal pigment epithelium on days 28 to 35. Culture them in suspension. Put 50-60 optic vesicles into each 100 millimeter low attachment culture dish, containing 15 mL of RDM for retinal organoid formation.

Change the medium every 2 to 3 days until Day-42. To initiate the retinal differentiation, hPSCs were dissociated into small clumps and cultured in suspension, which formed EBs since Day-1. On Day-5 EBs were plated onto the ECM-coated culture dishes and the cells gradually migrated out of the EBs.

On Day-16, the induction medium was replaced by RDM, causing the neural retina domains to form and gradually protrude from the dish, as well as, cell forming optic vesicle-like structures surrounded by RPE cells. During Days-28 to 35, retinal organoids consisting of the neural retina, attached to an RPE sphere. At one side, cells formed.

As retinal differentiation and specification progressed, hPSCs produced subtypes of neural retina that gradually lined up in layers, mimicking the architectural features of a native human retina. Retinal ganglion cells were first generated from retinal progenitors and accumulated in the basal side of the neuro retina. With this protocol, retinal organoids developed into highly mature photoreceptors with both, rich rods and cones.

Photoreceptor cells were located in the apical side, while amacrine cells, horizontal cells, bipolar cells and muller glial cells were all located in the intermediate layer of the neural retina. The key points of this protocol are creating high quality of Ebs and seeding them at the right density.