Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

Summary

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

Abstract

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

Introduction

The retina is an integral part of the central nervous system (CNS) and has a limited capacity to spontaneously regenerate following a traumatic injury or diseases. Therefore, degenerative pathologies causing definitive retinal cell loss, such as age-related macular degeneration (AMD), retinitis pigmentosa (RP), glaucoma, and diabetic retinopathy, typically lead to irreversible blindness. Rescuing the degenerated retina is a major challenge for which stem cell-based therapies aiming to replace the damaged or lost cells are one of the most promising approaches^1,2,3. Pluripotent stem cells as human embryonic stem cells (ESCs) cells or human-induced pluripotent stem cells (iPSCs) have the capacity to be expanded indefinitely in culture, and they have the potential to produce any cell types. Advances in our understanding of retinal development and the improvement of in vitro protocols for human iPSC differentiation have resulted in the generation of retinal organoids^{7,8,9,10,11,12}. All of the major retinal cells, including retinal ganglion cells (RGCs), photoreceptors, and retinal pigmented epithelial (RPE) cells, have been successfully differentiated from human ESCs and iPSCs^4,5,6. Based on the SFEB (serum-free culture of embryoid body-like aggregates) method developed by Eiraku et al.¹³, self-formation of retinal organoids can be obtained from ESC- or iPSC-derived embryoid body-like aggregates in defined extracellular matrix components^7,10,14. But these protocols are intricate, requiring a large number of steps not always compatible with the large production of cells for therapeutic approaches or drug screening. Thus, the choice of the method to produce human retinal cells is critical and the method needs to be robust, scalable, and efficient.

Here, based on our previous publication¹⁵, we describe each step for a simple and efficient generation of retinal cells through retinal organoid self-formation from adherent human iPSCs cultivated in a feeder-free and xeno-free condition. Starting from routine cultures of adherent human iPSCs, this protocol requires only a simple successive medium changing to allow the generation of both iPS-derived RPE (hiRPE) cells and neuroretinal structures in 4 weeks. After a manual isolation, hiRPE can be expanded and the retinal structures can be cultured as floating organoids where the retinal progenitor cells are able to differentiate into all retinal cell types in a sequential order consistent with the in vivo human retinogenesis. Finally, for research advancement or clinical translation, we describe a cryopreservation method allowing the long-term storage of whole retinal organoids and hiRPE cells without affecting their phenotypic characteristics and functionality.

Protocol

The protocol described in this paper follows the guidelines of the Institut de la Vision's research ethics committee. The Institut de la Vision has been allowed the manipulation of human specimen according to the current French regulation. Specimen handling follows patient data protection in accordance with the Tenets of Helsinki, and national regulations after the ethical approval of the "Comité de Protection des Personnes (CPP) Ile-de-France V".

1. Preparation of Culture Media and Dishes

1. Culture media
   1. Use iPSC medium, a chemically defined medium dedicated to pluripotent stem cell culture in feeder-free conditions¹⁶. Prepare 500 mL of medium according to the manufacturer's protocol.
   2. Use Basal iPSC (Bi) medium, the iPSC chemically defined medium without fibroblast growth factor 2 (FGF2) or transforming growth factor Beta (TGFß).
   3. Prepare 500 mL of Bi medium supplemented with 1% N2 supplement (BiN2 medium), 10 units/mL of penicillin, and 10 mg/mL of streptomycin.
   4. Prepare 500 mL of proneural N2-based medium (ProN2 medium) composed of DMEM:Nutrient Mixture F-12 (DMEM/F12, 1:1, L-Glutamine), 1% MEM nonessential amino acids, 1% N2 supplement, 10 units/mL of penicillin, and 10 mg/mL of streptomycin.
   5. Prepare proneural B27-based medium (ProB27 medium) composed of DMEM:Nutrient Mixture F-12 (DMEM/F12, 1:1, L-Glutamine), 1% MEM nonessential amino acids, 2% B27 supplement, 10 units/mL of penicillin and 10 mg/mL of streptomycin.
2. Culture vessel preparation
   1. For human iPSC culture
      1. Prepare 10 mL of a vitronectin solution containing 5 μg/mL of vitronectin in 1x PBS. In 10 mL of 1x PBS, add 100 µL of the thawed vitronectin stock solution (100x).
      2. Distribute 2 mL of the vitronectin solution per 6-cm culture dish corresponding to 0.5 µg/cm². Incubate it for 1 h at room temperature (RT). Remove the vitronectin solution by aspiration using a vacuum-aspiration system or a 5 mL pipette.
      3. Add 4 mL of iPSC medium per 6-cm dish.
      4. Incubate the dishes in a cell culture incubator at 37 °C and 5% CO₂ for a minimum of 30 min before use.
   2. For human iPSC-derived RPE (hiRPE) cell culture
   3. Use a pluripotent stem cell-qualified matrix or substrate to coat wells according to the manufacturer's protocol. Add sufficient matrix or substrate to cover the entire growth surface area. For example, put 300 µL of matrix per well in 24-well plate and 3 mL of matrix in a T-25 cm² flask.
   4. Incubate the coated culture vessels for a minimum of 1 h at 37 °C. Remove the matrix using vacuum-aspiration systems or a 5 mL pipette.
   5. Add 1 mL of ProN2 medium per well on 24-well plates and return the culture vessel to the incubator for a minimum of 30 min to warm and equilibrate the medium. For T-25 cm² flasks, see step 5.2.

2. Maintenance and Expansion of Human iPSCs

1. Maintenance of human iPSCs
   1. Prepare 3 mL of iPSC medium in a 15 mL tube and keep it at RT for a minimum of 15 min before use. Prepare a coated 6-cm dish as described in 1.2.1.
   2. Thaw a cryogenic sample of human iPSCs from a liquid nitrogen tank or a -150 °C freezer by incubation in a water bath at 37 °C for 30 s.
   3. Carefully disinfect the cryogenic vial using a disinfectant solution spray. Transfer the thawed human iPSCs from the cryotube to the 15 mL tube containing 3 mL of iPSC medium pre-warmed at RT.
   4. Centrifuge the tube at 110 x g for 3 min. Remove the supernatant by aspiration using a vacuum-aspiration system or a 5 mL pipette.
   5. Resuspend the cell pellet with 1 mL of iPSC medium from a vitronectin-coated 6-cm culture dish using a 2 mL pipette and transfer the cells back to the dish. Return the dish to the incubator at 37 °C and 5% CO₂ for a minimum of 24 h before changing the medium.
      NOTE: A ROCK inhibitor, such as Y-27632 at 10 µM, can be added to the iPSC medium in the 6-cm culture dishes to reduce apoptosis.
   6. Change the medium daily and pass the human iPSCs every week.
2. Passaging of human iPSCs
   1. Pass the human iPSC at a 70 - 80% confluence, classically after 7 days in culture.
   2. Prepare 6-cm dishes for the passaging as described in step 1.2.1. Remove the iPSC medium from the dishes with confluent iPSCs and add 2 mL of a dissociation solution for 6 min at RT.
      NOTE: The incubation time depends on the iPSC clones. Try a 6 min incubation as a start.
   3. Remove the dissociation solution using vacuum-aspiration systems or a 5 mL pipette and add 2 mL of iPSC medium pre-warmed at RT. Resuspend the iPSC colonies by pipetting them up and down 5 - 10x with a 1,000 µL pipette.
      CAUTION: Avoid single cell dissociation from excessive pipetting.
   4. Transfer 30 to 200 µL of resuspended cell clumps in a new 6-cm culture dish. Return the dishes to the cell culture incubator at 37 °C and 5% CO₂ for a minimum of 24 h before changing the medium.
      NOTE: The volume of resuspended cell clumps needed for the passaging depends on the iPSC clones.
   5. Change the medium daily and pass the human iPSCs every week.

3. Generation of Retinal Organoids

1. Start the iPSC differentiation following the protocol schematized in Figure 1A when the colonies reach a 60 - 70% confluence (Figure 1B).
2. Prepare 4 mL of Bi medium per 6-cm dish. Warm the Bi medium to 37 °C. Change the iPSC medium to Bi medium. Note this time as day 0 (D0).
3. At D2, switch the cultures in the Bi medium to a BiN2 medium, previously warmed at 37 °C. Change the medium every 2 - 3 days.
4. Identify emergent self-forming retinal organoids by neuroepithelium buds, as shown in Figure 1C - 1E.
   NOTE: These early retinal organoids, corresponding in their developmental stage to the optic cup, can be manually isolated for downstream experimentation.

4. Maturation of Retinal Organoids

1. At D28, prepare 6-well plates containing 4 mL/well of ProB27 medium initially supplemented with 10 ng/mL of FGF2 (Figure 1A).
   NOTE: Add FGF2 immediately before the media is added to the plates.
   Caution: Do not filter FGF2.
2. Recover the retinal structures from the 6-cm dishes manually. To do this, isolate the structures making perpendicular striations with the needle around the neuroepithelial bud, as shown in Figure 1E, and detach the organoids by gently scratching them with the needle.
3. Aspirate 10 - 15 organoids using a 1,000 µL pipette and transfer them in a single well of the 6-well plate containing ProB27 medium.
4. Keep the retinal organoids in floating culture conditions in ProB27 medium in a cell culture incubator at 37 °C and 5% CO₂. Change half of the medium every 2 - 3 days.
   NOTE: Treat the organoids with FGF2 until D35.
5. At D35, remove half of the medium and add fresh prewarmed ProB27 medium at 37 °C without FGF2.
6. Change half of the medium every 2 - 3 days during the time required to obtain the desired retinal cell types according to the emergence of retinal cell types, as depicted in Figure 2.

5. Generation and Amplification of Human iPSC-derived RPE (hiRPE) Cells

1. Generation of human hiRPE cells
   1. Engage the iPSCs in differentiation when the colonies reach a 60 - 70% confluence, following the protocol schematized in Figure 3A.
   2. Prepare 4 mL per 6-cm dish of Bi medium and warm the Bi medium at 37 °C. Change the iPSC medium to Bi medium. Note this time as day 0 (D0).
   3. At D2, switch the cultures in the Bi medium to a BiN2 medium previously warmed at 37 °C. Change the medium every 2 - 3 days.
   4. At D28, change the medium to a fresh ProN2 medium previously warmed at 37 °C as depicted in Figure 3A.
      NOTE: This step can be done after the organoid picking described in step 4.
   5. Change the ProN2 medium every 2 - 3 days.
   6. At D42, identify emergent hiRPE cells by the observation of pigmented patches as shown in Figure 3B.
      NOTE: The pigmentation of hiRPE cells appears between D35 to D56 depending on the iPSC clones.
2. Amplification of hiRPE Cells
   1. At D42, prepare a 24-well plate previously coated with the matrix as described in step 1.2.2 and containing 1 mL of ProN2 medium per well. Place the plate for 15 min in the incubator at 37 °C and 5% CO₂ before use.
   2. Recover the hiRPE patches from the 6-cm dish manually. To isolate 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 µL pipette and transfer them in a single well of the 24-well plate.
   3. Keep the hiRPE patches in ProN2 medium in a cell culture incubator at 37 °C and 5% CO₂ for 48 h before changing the medium. Note this hiRPE cell passage as 0 (hiRPEp0). Change the medium every 2 - 3 days with ProN2 medium.
   4. Pass the hiRPEp0 cells when the cells are confluent.
      1. Remove the medium using vacuum-aspiration systems and wash each well 1x with 1 mL of PBS using a 5 mL pipette.
      2. Discard the PBS using vacuum-aspiration systems, add 200 µL per well of 0.25% trypsin, and incubate it for a minimum of 15 min in an incubator at 37 °C. Add 800 µL of ProB27 medium to inactivate the trypsin.
         NOTE: The use of ProN2 medium to stop the trypsin activity is not recommended.
      3. Dissociate the sheet of hiRPE cells by pipetting it up and down. Place the cell suspension in a 15 mL tube and centrifuge it for 5 min at 110 x g.
         NOTE: An additional wash can be done with ProN2 medium to remove the excess of ProB27 medium.
      4. Remove the supernatant and gently resuspend the cell pellet in 2 mL of ProN2 medium pre-warmed at 37 °C. Count the cells with a cell counter.
      5. Take 1.25 million cells and place them in a matrix-coated T-25 cm² flask (see step 2.2.2) containing 5 mL of ProN2 medium pre-warmed at 37 °C. Note this hiRPE cell passage as 1 (hiRPEp1).
   5. Keep the hiRPE cells in the ProN2 medium in a cell culture incubator at 37 °C and 5% CO₂ for 48 h before changing the medium.
   6. Change the media every 2 - 3 days. When the hiRPEp1 cells reach confluence, perform the next passage or cryopreservation.

6. Cryopreservation of Retinal Organoids and hiRPE Cells

1. For whole retinal organoids
   1. Select 5 to 20 retinal organoids using a transfer pipet and place them in a cryogenic vial. Remove any excess medium with a 1,000 µL pipette without touching the organoids at the bottom of the tube and add 250 µL of cold cryopreservation medium (see Table of Materials).
   2. Freeze the vials in an isopropanol-based freezing container at -80 °C for a minimum of 4 h. Transfer the frozen vials to a -150 °C freezer or liquid nitrogen tank for long-term storage.
2. For hiRPE cells
   1. At passage 1, dissociate the hiRPE cells when the cells are confluent.
      NOTE: A cryopreservation of hiRPEp0 cells is not recommended.
   2. Aspirate the medium from the T-25 cm² flask using vacuum-aspiration systems and wash it 1x with 3 mL of PBS using a 5 mL pipette.
   3. Remove the PBS using vacuum-aspiration systems, add 1 mL of 0.25% trypsin, and incubate it for a minimum of 15 min in an incubator at 37 °C. Add 5 mL of ProB27 medium to stop the trypsin activity.
      NOTE: The use of ProN2 medium to inactivate trypsin is not recommended.
   4. Dissociate the sheet of hiRPE cells by pipetting it up and down using a 10 mL pipette. Count the cells with cell counter. Place the cell suspension in a 15 mL tube and centrifuge it for 5 min at 110 x g.
      NOTE: An additional wash can be performed with ProN2 medium to remove the excess of ProB27 medium.
   5. Aspirate the supernatant using vacuum-aspiration and gently resuspend cells to obtain 2 million cells in 250 µL of cryopreservation medium.
   6. Transfer 250 µL of cell suspension per cryogenic vial. Freeze the vials in an isopropanol-based freezing container at -80 °C for a minimum of 4 h.
   7. Transfer the frozen vials to a -150 °C freezer or a liquid nitrogen tank for long-term storage.

7. Thawing of Retinal Organoids and hiRPE Cells

1. Thawing of whole retinal organoids
   1. Warm ProB27 medium at 37 °C (2 mL will be required for each cryogenic vial).
   2. From the liquid nitrogen tank or -150 °C freezer, thaw a cryogenic vial containing retinal organoids in a water bath at 37 °C for 30 s. Disinfect the cryogenic vial carefully using a disinfectant solution spray.
   3. Open the vial and add 1 mL of pre-warmed ProB27 medium. Transfer the organoids to a 1.5 mL tube with a transfer pipette. 
   4. Remove the medium gently by pipetting it without touching the retinal structures at the bottom of the tube. Wash the organoids 1 more time with 1 mL of the pre-warmed ProB27 medium.
   5. Aspirate the organoids with a transfer pipet and place them in 1 well of a 6-well plate containing ProB27 medium pre-warmed and equilibrated in an incubator at 37 °C and 5% CO₂.
      NOTE: Place 10 - 15 retinal organoids per well of a 6-well plate.
   6. Change half of the medium every 2 - 3 days during the time required to obtain the desired retinal cell types according to their emergence, as described in Figure 2.
2. Thawing of hiRPE cells
   1. Warm ProN2 medium at 37 °C (8 mL will be required for each cryogenic vial).
   2. From the liquid nitrogen tank or -150 °C freezer, thaw a cryogenic sample of hiRPEp1 cells by an incubation into a water bath at 37 °C for 30 s. Disinfect the cryogenic vial carefully using disinfectant solution spray.
   3. Open the tube and add 1 mL of pre-warmed ProN2 medium and transfer the cell suspension to a 15 mL tube containing 2 mL of pre-warmed ProN2 medium. Centrifuge it for 5 min at 110 x g.
   4. Remove the supernatant and gently resuspend the cell pellet in 2 mL of ProN2 medium pre-warmed at 37 °C.
   5. Count the cells with a cell counter.
   6. Take 1.25 million cells and place them in the matrix-coated T-25 cm² flask (see step 2.2.2) containing 5 mL of ProN2 medium pre-warmed at 37 °C. At this stage, note the passage as 2 (hiRPEp2).
   7. Keep the hiRPEp2 cells in ProN2 medium in the cell culture incubator at 37 °C and 5% CO₂ for 48 h before changing the medium.
   8. Change the media every 2 - 3 days. When the hiRPE cells reach confluence, perform the next passage or cryopreservation.
      NOTE: After 2 weeks in culture, 8 - 10 million hiRPE cells can be collected for further experimentations.

Results

The first step for human iPSC differentiation cultivated in feeder-free conditions¹⁶ is to shut down self-renewal machinery using Bi medium to encourage a spontaneous differentiation (Figure 1A). Then, at D2, the Bi medium is complemented with an N2 supplement to guide differentiating iPSCs cells towards the neural and retinal lineages. This process leads to the appearance of neuroretinal buds at around D28 (Figure...

Discussion

This protocol describes how to produce RPE cells and retinal organoids, containing retinal RGCs and photoreceptors, from human pluripotent stem cells in xeno-free and feeder-free conditions. Compatible with the Good Manufacturing Practice (GMP) process, the method cultivated presented here allows a large production of iPSC-derived retinal cells as RPE cells, RGCs, and photoreceptors for the development of stem cell-based therapies and drug discovery approaches for the future treatment of retinal degenerative diseases. Th...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Vitronectin (VTN-N) Recombinant Human Protein, Truncated	ThermoFisher Scientific	A14700	Coating
CTS Vitronectin (VTN-N) Recombinant Human Protein, Truncated	ThermoFisher Scientific	A27940	Coating
Essential 8 Medium	ThermoFisher Scientific	A1517001	medium
Essential 6 Medium	ThermoFisher Scientific	A1516401	medium
CTS (Cell Therapy Systems) N-2 Supplement	ThermoFisher Scientific	A1370701	supplement CTS
N-2 Supplement (100X)	ThermoFisher Scientific	17502048	supplement
B-27 Supplement (50X), serum free	ThermoFisher Scientific	17504044	supplement
CTS B-27 Supplement, XenoFree	ThermoFisher Scientific	A1486701	supplement CTS
DMEM/F-12	ThermoFisher Scientific	11320074	medium
MEM Non-Essential Amino Acids Solution (100X)	ThermoFisher Scientific	11140035	supplement
Penicillin-Streptomycin (10,000 U/mL)	ThermoFisher Scientific	15140122	antibiotic
CellStart CTS	ThermoFisher Scientific	A1014201	Matrix CTS
Geltrex hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix	ThermoFisher Scientific	A1569601	Matrix
Gentle Cell Dissociation Reagent	Stemcell Technologies	7174	dissociation solution
Cryostem Freezing Media	clinisciences	05-710-1D	Cryopreservation medium
Fibroblast growth factor 2 (FGF2)	Preprotech	100-18B	FGF2
Fibroblast growth factor 2 (FGF2) animal free	Preprotech	AF-100-18B	FGF2 Xeno free
AGANI needle 23G	Terumo	AN*2332R1	Needle
Flask 25 cm² Tissue Culture Treated	Falcon	353109	T-25 cm²
24 well plate Tissue Culture Treated	Costar	3526	24-well plate
6 well plate Tissue Culture Treated	Costar	3516	6-well plate