Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

All experiments involving hiPSCs were carried out aseptically, in adherence to the standard laboratory practices, ethical and biosafety guidelines, and with the approvals of regulatory bodies such as the Institutional Ethics Committee (IEC), Institutional Committee for Stem Cell Research (IC-SCR), and Institutional Bio-Safety Committee (IBSC).

1. Preparation of iPSC culture and retinal differentiation medium and reagents

1. iPSC culture and maintenance medium
   1. Culture and maintain the normal hiPSC line²³ (hiPSC-F2-3F1) and a CRISPR edited, RB1^-/- hiPSC line (LVIP15-RB1-CS3, with biallelic, frameshift deletion of 10 bp within the exon 18 of the human RB1 gene) in Essential 8 medium on matrigel-coated (basement membrane matrix coated; see Table of Materials) culture plates under feeder-free culture conditions.
      NOTE: This protocol can be made totally xeno-free by replacing the basement membrane matrix with the recombinant vitronectin (VTN-N) coating. Prepare complete Essential 8 medium by adding 50x Essential 8 supplement (supplied with the Essential 8 medium kit; see Table of Materials) and 100 U/mL Penicillin-Streptomycin solutions. Alternately, the hiPSCs can also be cultured using the complete mTeSR1 medium.
2. Cell Dissociation Solution (1x CDS)
   1. For an enzyme-free dissociation and passaging of human iPSCs, prepare the CDS containing 0.5 mM EDTA, pH 8.0, and 30 mM NaCl in 1x Dulbecco's phosphate-buffered saline (DPBS) (see Table of Materials).
   2. To prepare 100 mL of 1x CDS, add 100 µL of 0.5M EDTA and 1 mL of 3 M NaCl stock solutions to 99 mL of 1x DPBS, mix well, and filter sterilize using a 0.22 µm filter.
3. Differentiation Induction Medium (DIM)
   1. Prepare DIM using DMEM-F12 Basal medium supplemented with 10% Knockout Serum Replacement (KOSR), 1x Non-Essential Amino Acid (NEAA), 2 mM GlutaMax, 100 U/mL Penicillin-Streptomycin, 200 µM L-Ascorbic acid, and 1% N2 supplement (see Table of Materials).
4. Retinal Differentiation Medium (RDM)
   1. Prepare RDM using DMEM-F12 Basal medium supplemented with 10% Knockout Serum (KOSR), 1x Non-Essential Amino Acid (NEAA), 2 mM GlutaMax, 100 U/mL Penicillin-Streptomycin, 200 µM L-Ascorbic acid, and 2% B27 supplement (with vitamin A) (see Table of Materials).
5. Extracellular matrix-coated cell culture surfaces
   1. Thaw the hESC-qualified basement membrane matrix overnight in an ice bucket, preferably inside a fridge at 4-8 °C. Dilute the thawed 100x matrix stock (5 mL) at a ratio of 1:5 by adding 20 mL of ice-cold DMEM-F12 basal medium and mix by gentle swirling to prepare a 20x stock solution.
      1. Prepare 0.5 mL aliquots on ice using pre-chilled pipette tips and sterile microcentrifuge tubes. Label the vials as 20x stocks and store them frozen in a -80 °C freezer for up to 6 months.
   2. For coating the cell culture surfaces (culture dishes or 6-well plates), thaw an aliquot of 20x matrix on ice and dilute it at a ratio of 1:20 using ice-cold DMEM-F12 basal medium to prepare 10 mL of 1x matrix coating solution, which is sufficient to coat a 100 mm dish or a 6-well plate (1.5 mL/well).
      NOTE: Prechill the pipettes/microtips by aspirating ice-cold and sterile DPBS before handling the matrix solution. Thawing and all handling of the matrix must be done on ice, using pre-chilled pipettes/microtips to avoid polymerization and gelling at room temperatures.
   3. Add 1.5 mL of 1x matrix coating solution to each well of a 6-well plate, gently swirl the plate to ensure even coating, and incubate the plates at 37 °C in a 5% CO₂ incubator. Leave the plates undisturbed for a minimum of 1 h to ensure uniform coating of the matrix on the culture surfaces.
   4. Prior to seeding the cells, aspirate out the coating solution using a 5 mL sterile pipette and discard the liquid waste. Add fresh culture medium immediately (2 mL/well of a 6-well plate) and seed the cells at a density of 150,000-200,000 cells/well. Do not let the plates dry during handling.
      NOTE: Alternately, recombinant vitronectin (VTN-N) coating at a final concentration of 0.5 µg/mL can be used for a xeno-free culture protocol.

2. Establishing human iPSC cultures

1. Thawing and revival of hiPSCs
   1. Coat one well of a 6-well plate with 1x membrane matrix solution. Incubate at 37 °C for 1 h to allow polymerization and uniform coating of the culture surface.
   2. After 1 h of incubation, remove the coating solution and add 1 mL of prewarmed complete Essential 8 medium containing 10 µM of Rho-kinase inhibitor, Y-27632 (1 µL/mL of 10 mM stock; see Table of Materials), before reviving the cells.
   3. Remove an hiPSC cryovial stock (1 x 10⁶ cells/vial) from the liquid nitrogen container. Quickly thaw the cryovial in a 37 °C water bath with gentle swirling.
      NOTE: Do not thaw the vial completely; note down the passage number, surface sterilize the vial, and wipe it dry using a lint-free swab containing 70% isopropyl alcohol.
   4. Using a sterile 1 mL pipette tip, aspirate the cryovial contents into a fresh 15 mL tube consisting of 2 mL of prewarmed complete Essential 8 medium without Y-27632. Centrifuge the tube at 1,000 x g for 4 min at room temperature. Discard the supernatant.
   5. Resuspend the cell pellet in 1.0 mL of complete Essential 8 medium containing 10 µM Y-27632.
   6. Add this cell suspension onto the matrix-coated surfaces without disturbing the matrix by dispensing it along the walls. Rock the plate gently crosswise to ensure the even distribution of cells.
   7. Incubate the plates at 37 °C in a 5% CO₂ incubator to allow the cells to adhere and start proliferating.
   8. After 12-24 h, replace the spent medium and maintain the cultures in prewarmed complete Essential 8 medium without Y-27632.
   9. Change the culture medium every 24 h and passage the cultures once they reach 70%-80% confluence.
      NOTE: A split ratio of 1:6 is routinely followed for hiPSC cultures and is passaged at regular intervals of 3-4 days.
2. Passaging and plating of hiPSCs to initiate retinal lineage differentiation
   1. Aspirate the spent medium from 70%-80% confluent human iPSC cultures in 6-well plates.
   2. Add 1 mL of CDS (step 1.2) to each well and incubate at 37 °C for 5-7 min until the cells round up. Carefully remove the CDS, taking care not to detach the cells, and add 2 mL of fresh Essential 8 medium and gently triturate using a pipette.
   3. Collect the cell suspension from one well of a 6-cell plate into a 15 mL centrifuge tube and spin the tube at 1,000 x g for 4 min at room temperature. Discard the supernatant.
   4. Resuspend the cell pellet in 1.2 mL of Essential 8 medium and dispense 200 µL of the cell 200μL of the cell suspension (1:6 split ratio) into each well of a matrix-coated 6-well plate containing 1.5 mL of iPSC culture medium containing 10 µM Y-27632, as described in step 1.5.
   5. After 12-24 h, replace the spent medium and maintain the cultures in prewarmed complete Essential 8 medium without Y-27632.
   6. Change the culture medium every 24 h till the cultures reach 70%-80% confluence.

3. Differentiation of hiPSCs into eye fields and retinal lineage

NOTE: A schematic outline of the differentiation process is shown in Figure 1.

1. Initiate the differentiation procedure once the hiPSC cultures reach 70%-80% confluence.
2. On day 0, change the hiPSC maintenance medium to DIM (step 1.3) containing 1 ng/mL bFGF and 1 ng/mL Noggin. Add 2.0 mL of medium per well of a 6-well plate and maintain the cells at 37 °C in a 5% CO₂ incubator.
   NOTE: Gradual withdrawal of bFGF induces the differentiation of PSCs, and the addition of increasing concentrations of Noggin (inhibitor of several BMPs) during the initial stages induces ectodermal lineage differentiation and neuralization^11,12 and blocks the mesoderm and endoderm commitment. Alternatively, Noggin can be replaced by LDN193189. Unlike the dual SMAD inhibition strategy reported earlier^20,21, this protocol does not require the addition of Activin or SB-431542.
3. On day 1, change the spent medium and add DIM containing 1 ng/mL bFGF and 10 ng/mL Noggin. Add 2.0 mL per well of a 6-well plate. Incubate and maintain the cells at 37 °C in a 5% CO₂ incubator.
4. On day 2-3, remove the spent medium and add DIM containing only 10 ng/mL Noggin. Add 2.0 mL per well of a 6-well plate and change the medium every 24 h. Incubate and maintain the cells at 37 °C in a 5% CO₂ incubator.
   NOTE: Always pre-warm the culture medium to 30-37 °C before use. Excess floaters and dead cells may be observed in early differentiation cultures. Under such conditions, wash the cultures once with 1x DPBS and add the retinal differentiation medium. Sterility tests on the spent medium can be done weekly or as required.
5. On day 4, remove the spent medium and add the RDM (step 1.4). Add 2.0 mL per well of a 6-well plate and continue to maintain the cultures in RDM at 37 °C in a 5% CO₂ incubator, with a fresh medium change every day.
   NOTE : Alternately, the PSCs can be grown as suspension cultures from day 1-3, in non-adherent and round-bottomed 96-well plates, at a cell density of 5 x 10³ cells/100 µL/well to form embryoid bodies (EBs) under identical culture conditions in DIM. Well-formed EBs on day 4 can be plated onto matrix-coated plates containing RDM and are allowed to adhere, proliferate, and differentiate as described below.
6. At around day 14-18, observe the cultures under a microscope at 10x magnification for the emergence of neural rosette-like domains consisting of early eye field progenitors (Figure 2B).
7. At around day 21-28 (3-4 weeks), observe cultures under a microscope at 4x and 10x magnification to observe the emergence of self-organized, distinct EFPs, with a central island of circular 3D neuro-retinal structures surrounded by contiguous outgrowths of neuro epithelium and ocular surface epithelium (Figure 2C,D).
   ​NOTE: About 20-30 EFPs can be observed per well of a 6-well plate of a normal hiPSC retinal differentiation culture. This number can vary with other disease-specific hiPSC lines, based on their genetic background and retinal lineage differentiation potential.

4. Harvesting of retinal organoids

1. Flame pulling of glass Pasteur pipettes for manual picking of retinal cups.
   NOTE: Use autoclaved and sterile glass Pasteur pipettes for eye field picking.
   1. Switch on a Bunsen burner. Take a sterile Pasteur pipette and hold the base in one hand and the capillary tip in the other. Flame sterilize and heat the region near the middle of the capillary tip, with rotational movements until the glass becomes pliable. Then move away from the flame and pull outward swiftly to create a fine capillary tip with a closed lumen.
   2. Hold the fine tip horizontally in front of the flame and quickly pass it through the flame in an outward motion to create a smooth hook or an L-shaped capillary tip.
   3. Use the smooth outer curvature zone of the capillary hook as a fine scoop to gently lift and detach the intact neuro-retinal cups from the EFP clusters.
      NOTE: The smooth angle of the glass capillary hook neither causes damage to the cells nor creates scratches on culture surfaces. This simple tool can also be effectively used for individual hiPSC colony splitting in grid patterns and for passaging them as small cell clusters during clonal expansion.
2. Culture and maintenance of retinal cups to generate 3D retinal organoids.
   1. At day 25-30, add 4.0 mL of prewarmed retinal differentiation medium in a low-attachment 60 mm dish before preparing for the harvest of neuro-retinal cups.
   2. Work under a stereo microscope at 0.63x-4.5x magnification to observe and manually pick well-formed neuro-retinal cups from individual EFPs.
      NOTE: The appearance of pigmented RPE cell outgrowths helps in the easy identification of EFPs and the centrally placed neuro-retinal cups. The retinal cups appear as doughnut-shaped, circular, and translucent 3D structures, with clear radial striations, formed by the linearly arranged and self-assembled retinal stem cells, as opposed to the tightly packed and spherical or irregular clusters formed by the CNS neurons or the neural crest cells²³.
   3. Use the flame-pulled Pasture pipette hooks to gently nudge and scoop out the central neuro-retinal island within individual EFPs.
   4. Set a 1 mL micropipette to aspirate 100 µL and use 1 mL microtips with wide bore openings to aspirate and transfer the floating retinal cups into the fresh, low-adherent culture dishes prepared in step 4.2.1.
      NOTE: Using tips with a smaller bore size and higher suction pressure may cause shearing and affect the integrity of retinal cups. The approximate dimensions of the retinal cups are about 1-2 mm in diameter.
   5. Maintain the retinal cups in RDM as non-adherent suspension cultures and incubate them at 37 °C in a 5% CO₂ incubator.
   6. Day 30-45: Change the medium daily by gently tilting the dish and allowing the retinal cups to settle for about 30 s. Follow a partial feeding method, removing half volumes of spent medium and replacing with equal volumes of fresh medium.
      NOTE: Avoid repeated aspiration and transfers to prevent any damage to the retinal cups. After 1-2 weeks of suspension culture, the retinal cups slightly grow in size (1-3 mm) and develop into self-organized 3D retinal organoids (OC-1M) comprising of linearly arranged, early neuro-retinal progenitors expressing PAX6 and CHX10 (Figure 3Bi).
   7. Day 45-60: Culture the retinal organoids for a further 4 weeks in RDM containing 100 µM Taurine (see Table of Materials) to support better survival and lineage differentiation of neuro-retinal progenitors and the development of mature retinal cell type (OC-2M).
      NOTE: About 70%-80% of the retinal or optic cups picked from EFPs remain intact, retain their lamination, and develop into mature retinal organoids after 4 weeks of suspension culture (OC-2M).
   8. Check that the mature retinal organoids at 4 weeks of suspension culture show the emergence of RCVRN+ and CRX+ photoreceptor precursors and mature cells with a rudimentary inner segment-like extension in the outermost cell layers, thus recapitulating the normal retinal development and maturation process in vitro (Figure 3Bii).
      NOTE: After removing neuro-retinal cups, the differentiation cultures can be continuously maintained in RDM. The proximal epithelial outgrowths surrounding the neuro-retina contain retinal pigmented epithelial (RPE) cell precursors, which further expand and mature to form pigmented RPE monolayers with typical cobblestone morphology (Figure 4). The distal outgrowths predominantly consist of ocular surface epithelium that contributes to the lens and cornealepithelium. Desired cell types can be harvested from different zones of EFP outgrowths and enriched further to establish pure cultures.

5. Characterization of retinal organoids

1. Morphological and molecular characterization
   1. Observe the adherent EFPs and floating retinal organoids under a phase-contrast microscope at 4x and 10x magnification and document their morphology and dimensions.
   2. Collect about 20 retinal organoids at different stages of maturation (steps 4.2.3-4.2.6) into 1.5 mL microcentrifuge tubes using wide bore 1,000 µL tips. Let the organoids settle at the bottom and aspirate out the medium. Wash the cups with 1x PBS.
   3. Remove the excess PBS and add 1.0 mL of TRIzol reagent (see Table of Materials). Incubate at room temperature for 5 min. Homogenize the tissue using a tube pestle and triturate using a 1.0 mL pipette.
   4. Prepare total RNA by following the standard reagent method of RNA isolation and purification, as per the manufacturer's instructions (see Table of Materials).
   5. Check the RNA quality on the agarose gel and quantify it using the NanoDrop Spectrophotometer (see Table of Materials).
   6. Convert 1-2 µg of total RNA into cDNA using the reverse transcriptase enzyme as per the manufacturer's instructions (see Table of Materials). See Table 1 for a list of gene-specific primers.
   7. Briefly, prepare the RNA-primer master mix in 10 µL of total volume as mentioned in Table 2 and incubate the tube at 65 °C for 5 min in a thermal cycler. Transfer the tube from the thermal cycler to ice for 2 min.
   8. Meanwhile, prepare the master mix 2 with the reagents mentioned in Table 3. Add this master mix to the RNA-primer mix tube prepared in step 5.1.7. Mix gently.
   9. Incubate the sample at 50 °C for 50 min, then 85 °C for 5 min, and then hold at 4 °C to stop the reaction.
   10. Use the synthesized cDNA as a template in PCR reactions to check for the expression of neuro-retinal progenitor and mature retinal cell markers.
   11. Normalize the total cDNA templates of each sample, namely F2-UD (hiPSC-F2-3F1; undifferentiated cells), OC-1M (1-week-old optic cup in suspension culture), and OC-2M (4-week-old optic cup in suspension culture) by semi-quantitative PCR using the housekeeping genes such as hE1fα or GAPDH.
   12. Prepare the master mix for semi-quantitative PCR, as mentioned in Table 4, and place the test sample tubes for amplification in a thermal cycler. The PCR amplification conditions are mentioned in Table 5.
2. Histology and immunohistochemistry
   1. Collect the optic cups in a 2.0 mL microcentrifuge tube and aspirate the excess medium (steps 4.2.3-4.2.6). Rinse the organoids with 1x PBS and then wash and suspend them in 500 µL of 4% Paraformaldehyde to fix them overnight at room temperature.
   2. The next day, wash the cups in deionized water. Allow the cups to stand in 95% alcohol (three changes, 15-20 min each), followed by 100% alcohol (three changes, 15-20 min each), a 1:1 mix of absolute alcohol and xylene for 15 min, xylene (two changes, 15 min each), and paraffin (three changes, 15 min each). Embed this tissue to prepare a paraffin block following standard procedures. Allow it to cool and solidify.
   3. Prepare thin sections (~4-5 µm thickness) using a microtome and place them on silane-coated microscopic slides (see Table of Materials) following standard histology procedures.
   4. For deparaffinization, heat the slides at 70 °C on a heating block for 15-20 min. Once the wax melts, wash the slides with xylene (three changes, 3-4 min each), which will remove the paraffin completely.
      NOTE: Incomplete removal of paraffin results in poor/patchy staining of sections with a huge amount of background noise.
   5. Re-hydrate the slides using different percentages of ethanol (100%, 90%, and 80%) for 3 min each. Rinse the slides with distilled water and proceed with antigen retrieval.
   6. Before starting the antigen retrieval, pre-heat the citrate buffer (pH 6.0) in a Coplin jar (see Table of Materials) using a microwave oven till it reaches 95-100 °C.
   7. Immerse the slides in pre-heated citrate buffer and heat the jar for 15 min in a microwave oven. Remove the Coplin jar from the oven and allow it to cool at room temperature.
   8. Block the sections for endogenous peroxidase by adding a 1:1 mixture of methanol and hydrogen peroxide for 5 min, followed by washing the sections thrice with 1x PBS.
   9. Permeabilize the sections using 0.5% Triton-X 100 for 15 min. Wash the slides thrice with 1x PBS.
   10. Block the nonspecific binding of primary antibody by incubating the sections with 10% Fetal Bovine Serum (FBS) in 1x PBS for 1 h.
   11. Incubate the sections with primary antibody for 1 h at room temperature or overnight at 4 °C. Wash the slides thrice with 1x PBS for 3-5 min each to remove the unbound antibody.
   12. Add suitable fluorescent dye-conjugated secondary antibody and incubate for 45 min. Wash the slides thrice with 1x PBS for 3-5 min each.
       NOTE: The antibodies and their respective dilutions are mentioned in the Table of Materials. The retinal organoid sections were immunolabelled using PAX6 and CHX10 to detect early neuro-retinal precursor cells, and using Recoverin and CRX to detect committed retinal and photoreceptor precursor cells. Similarly, anti-PAX6 and anti-MITF were used to detect RPE precursors, and anti-CRALBP and anti-RPE65 were used to detect pigmented mature RPE cells by immunocytochemistry of 2D monolayer cultures.
   13. Counterstain the sections with DAPI or PI and mount them on a glass slide using the antifade mounting medium (see Table of Materials).
   14. Image and document the immunolabelled sections of retinal organoids and RPE cultures using a fluorescence or confocal laser scanning microscope to examine different cell layers expressing different retinal markers.