Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Summary

We describe a method to engineer a retinal tissue composed of retinal pigment epithelial cells derived from human pluripotent stem cells cultured on top of human amniotic membranes and its preparation for grafting in animal models.

Abstract

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms of retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Cell therapy is one of the most promising therapeutic strategies proposed to cure these diseases, with already encouraging preliminary results in humans. However, the method of preparation of the graft has a significant impact on its functional outcomes in vivo. Indeed, RPE cells grafted as a cell suspension are less functional than the same cells transplanted as a retinal tissue. Herein, we describe a simple and reproducible method to engineer RPE tissue and its preparation for an in vivo implantation. RPE cells derived from human pluripotent stem cells are seeded on a biological support, the human amniotic membrane (hAM). Compared to artificial scaffolds, this support has the advantage of having a basement membrane that is close to the Bruch's membrane where endogenous RPE cells are attached. However, its manipulation is not easy, and we developed several strategies for its proper culturing and preparation for grafting in vivo.

Introduction

RPE is crucial for the survival and homeostasis of the photoreceptors with which it is tightly associated¹. Several pathological conditions alter its functionality and/or survival, including RP and AMD.

RP is a group of inherited monogenic mutations that affect the functions of photoreceptors or RPE cells or both^2,3. It is estimated that mutations that affect specifically the RPE cells account for 5% of RP². AMD is another condition where the RPE layer is altered, leading ultimately to central vision loss. AMD is caused by the complex interactions of genetic and environmental factors and affects the elderly^4,5,6. According to projections, AMD will be a concern for 196 million patients worldwide by 2020⁷. For these disorders, no effective cure exists, and one of the strategies proposed is the transplantation of new RPE cells in order to compensate for dead/nonfunctional preexisting RPE cells⁸.

The mode of formulation of the final product to be grafted is essential to ensure the best functional outcomes. RPE cells injected as a cell suspension, despite being an easy and straightforward method of delivery, raise concerns regarding their survival, integration, and functionality^{9,10,11,12,13}. Scientists are now developing more complex formulations to deliver engineered retinal tissue^{9,13,14,15,16}. In this context, we developed an original method to generate in vitro RPE tissue that could be used for transplantation⁹.

RPE cell banks derived from human embryonic stem (ES) cells are used in this protocol. However, alternative RPE cell banks from different cell sources (human-induced pluripotent stem cells, primary RPE cells, etc.) and differentiated with a different method are also suitable for this protocol. It includes directed differentiation protocols using cytokines and/or small molecules^{17,18,19,20,21,22}.

To be transplanted, the engineered tissue should be prepared on a scaffold. In the past few years, different scaffolds were developed based on a polymer or on a matrix of biological origin^13,23,24. Here, the biological substrate used is the hAM, but other substrates, like denuded Bruch membranes, could be implemented. The method described herein has the advantage of using a biological scaffold that is more relevant to the RPE native environment.

Human ES cell-derived RPE cells are cultured for at least 4 weeks in order to be fully organized as a cobblestone monolayer. At that stage, the epithelium obtained is functional and polarized⁹. Finally, as this tissue wrinkles easily, it is embedded in a thin layer of a hydrogel carrier to give it more rigidity and elasticity and to protect it during the injection procedure. This product is then stored at 4 °C until grafting.

Protocol

All human materials used in this protocol were used in accordance with European Union regulations. The human ES cell line used in this study was derived from a unique embryo. The couple who had donated the embryo was fully informed and gave their consent for an anonymous donation. A clinical-grade human ES cell line was derived from this embryo, banked, qualified, and properly documented by Roslin Cells (UK). hAMs were procured under sterile conditions during a cesarean section in mothers who signed an informed consent for placenta donation according to hospital guidelines (APHP, Hôpital Saint Louis).

1. Preparation of Culture Media and Reagents

1. Preparation of the RPE cell culture medium
   1. To prepare the RPE cell culture medium, add 4% serum substitute (20 mL for a final volume of 500 mL), 1,000x diluted 2-mercaptoethanol (500 µL for a final volume of 500 mL), and 1% Eagle′s minimum essential medium (MEM) non-essential amino acids solution (5 mL for a final volume of 500 mL) to Dulbecco's modified Eagle's medium (DMEM) high glucose (475 mL for a final volume of 500 mL).
2. Preparation of the transport/conservation medium
   1. To prepare the transport/conservation medium, add 1% of penicillin-streptomycin (5 mL for a final volume of 500 mL) to CO₂-independent medium (495 mL for a final volume of 500 mL) and keep it at 4 °C.
3. Resuspension of the thermolysin enzyme
   1. Preparation of a stock solution of thermolysin
      1. To prepare a stock solution of thermolysin, thaw the thermolysin powder, which was stored at -20 °C, at room temperature.
      2. As the enzymatic activity may be variable from batch to batch, calculate this activity based on the certificate of analysis provided with the thermolysin powder of the batch to be used. Divide the "Activity Neutral Protease calculated = ANPC" (indicated in the Certificate of Analysis) by 181 (which is tyrosine's molecular weight). The result obtained corresponds to the total enzymatic activity of the supplied thermolysin powder (U/vial).
      3. Prepare a stock solution at 200 U/mL by adding the volume of water corresponding to the total enzymatic activity (U/vial) divided by 200 (U/mL).
      4. Dissolve the enzyme by pipetting the water that was added up and down. Vortex the solution for 30 s and check whether the powder has completely been suspended. More pipetting may be required for a full dissolution.
      5. Aliquot the stock solution and store it at -20 °C.
   2. Preparation of a working solution of thermolysin
      NOTE: The thermolysin solution must be prepared on the day of the hAM treatment.
      1. Thaw the stock aliquots of thermolysin at 200 U/mL stored at -20 °C. Dilute the stock solution 200x in phosphate-buffered saline (PBS) in order to obtain a final enzymatic activity of 1 U/mL. Prepare 40 mL to treat 1–4 hAM patches.
      2. Filter the thermolysin solution through a 0.2 µm filter prior to use.
4. Resuspension of gelatin
   1. Preparation of 20% gelatin solution and block
      NOTE: The 20% gelatin solution and block may be prepared up to 1 week before use.
      1. Warm the CO₂-independent medium to 42 °C in a water bath for 30 min (up to 1 h). In a 50 mL tube, add 10 g of gelatin to 40 mL of warmed CO₂-independent medium and vortex the solution.
      2. Dissolve the gelatin for 30–60 min at 42 °C. Vortex each 10 min to homogenize the solution.
      3. Once the solution is homogeneous, add 4 mL of the 20% gelatin solution to four 6 cm culture dishes. Avoid bubbles. Add a plastic paraffin film to protect the dishes, and allow the solution to solidify at 4 °C.
   2. Preparation of 8% gelatin solution
      NOTE: The 8% gelatin solution may be prepared the day before use and stored at 4 °C.
      1. Warm the CO₂-independent medium to 42 °C for 30 min (up to 1 h). In a 50 mL tube, add 4 g of gelatin to 46 mL of warmed CO₂-independent medium and vortex the solution.
      2. Dissolve the gelatin for 30–60 min at 42 °C. Add a plastic paraffin film to protect the tube and keep it in a water bath at 37 °C if it is to be used the same day, or store it at 4 °C if it is to be used later.

2. Thermolysin Treatment of Human Amniotic Membranes

1. Human amniotic membrane wash
   1. Have hAMs supplied as small pieces (approximately 30 mm x 30 mm) fixed in a nylon scaffold, either already at 4 °C in PBS or frozen. If provided frozen, thaw the membranes at 37 °C in an incubator for 30 min.
   2. Wash the membranes:
      1. Place 1–4 membranes in a 250 mL bottle containing 80 mL of PBS. Use as many bottles as required.
      2. Shake each bottle in a plate shaker at a high speed for 5 min, then discard the PBS. Add 80 mL of PBS and repeat.
2. Thermolysin treatment
   1. Add 40 mL of the working solution of thermolysin (1 U/mL) per bottle containing the membranes (1–4 membranes per bottles; up to 3 bottles at a time).
   2. Shake the bottles for 5 min at 450 rpm and then vortex them for 30 s. Repeat 1x.
   3. Discard the thermolysin solution and add 80 mL of PBS.
   4. Shake the bottles for 5 min at 450 rpm. Discard the PBS and add 80 mL of PBS. Repeat 3x.

3. Fixation of Human Amniotic Membranes on a Culture Insert

1. Use long sterile forceps (that can reach the bottom of the bottle) to remove the hAMs one by one, and transfer each hAM to a 10 cm culture dish containing 10 mL of PBS.
2. In a new 10 cm culture dish containing 10 mL of PBS, place one of the membranes with the nylon facing down. Detach two of the four clips that are used to fix the membrane to nylon.
3. Insert the smaller ring of the culture insert between the nylon and the membrane.
4. Make sure that the membrane covers the smaller ring completely. Clip the second part of the culture insert on top of the membrane. The basement membrane of the hAM is facing up inside the culture insert. Detach the last two clips.
5. Cut, with sterile scissors, the excess of membrane outside the culture insert if necessary. Using forceps, transfer the membrane fixed in the culture insert to a 12-well plate, add PBS, and store the plate in an incubator at 37 °C and 5% CO₂.
6. Repeat these operations (from step 3.2 to 3.5) with the other membranes.

4. Thawing and Seeding of Retinal Pigment Epithelium Cells on Human Amniotic Membranes

1. Thawing of retinal pigment epithelium cells
   1. The RPE cell bank is frozen at 1 million cells per cryogenic vial in liquid nitrogen. Thaw as many cryogenic vials as required (350,000 cells are seeded per membrane).
   2. Prepare a 15 mL tube containing 3 mL of RPE medium per cryogenic vial. Once the vial is thawed, transfer the cells to each tube. Repeat this operation for the other vials.
   3. Homogenize (pipette up and down a few times to resuspend the cells in the RPE medium) and take 10 µL of the cell solution and transfer it to a 1.5 mL tube. Add 10 µL of trypan blue. Place 15 µL of this solution in a counting chamber, then count the number of viable cells (not colored in blue) under a microscope with a 4X objective. Repeat this operation for the other vials.
   4. In the meantime, centrifuge the 15 mL tubes at 110 x g for 5 min. Discard the supernatant and resuspend the cells at 700,000 cells/mL.
2. Seeding of retinal pigment epithelium cells into human amniotic membranes
   1. Remove the 12-well plate containing the membranes from the incubator.
   2. Aspirate the PBS. Add 500 µL of the cell suspension prepared in step 4.1.4 in the center of the culture insert. Add 1 mL of RPE medium outside the culture insert (inside the well). Repeat for the other membranes.
   3. Place the 12-well plate containing the membranes in the incubator.

5. Maintenance of Retinal Pigment Epithelium Cell Cultures on Human Amniotic Membranes

1. Renew the medium 2x per week, usually on Monday and Friday, until at least day 30 of the culture.
2. Aspirate the medium inside and outside the culture insert for each well and renew it with fresh medium (1 mL outside the culture insert and 500 µL inside). Place the plate in the incubator at 37 °C and 5% CO₂.

6. Preparation of the Retinal Pigment Epithelium Patch for Transplantation

NOTE: Starting at day 30 of the culture, the tissue is ready for transplantation.

1. Warm the 8% gelatin solution in a water bath at 37 °C for 30 min. Fill the internal chamber of the vibratome with cold CO₂-independent medium (at 4 °C).
2. Place the 20% gelatin block into the vibratome.
   1. Take the 6 cm culture dish containing the block of 20% gelatin from the fridge. Using a scalpel, cut out a 5 cm x 5 cm block. Paste the top side of the block to the support with cyanoacrylate glue or its equivalent.
   2. Place a new blade in the vibratome.
   3. Adjust the level of the block until it is at the same level of the razor blade.
   4. Fill the bath around the support with fresh CO₂-independent medium at 4 °C. Cut the block with the vibratome using a medium velocity until the block is cut uniformly, leading to a smooth surface. Set the position 0.
   5. Aspirate the medium around the block of gelatin until it is completely dry, and then take the 12-well culture plate containing the tissues from the incubator at 37 °C.
   6. Using forceps, carefully open the culture insert on top of the gelatin block. Cut, with the scissors, all parts of the membranes that do not contain cells (outside the ring where cells were not cultured). Aspirate the entire residual medium.
   7. Add 1 mL of the liquid 8% gelatin solution at 37 °C in order to cover the membranes. Carefully remove the excess. Wait 5–8 min to allow the gelatin to solidify.
   8. Add fresh CO₂-independent medium (at 4 °C) to the bath until the membrane is covered.
   9. Cut, with the vibratome at a position of -100 µm, with a medium velocity, remaining aware of how the block behaves. At the end of the section, be careful to maintain the orientation of the tissue.
   10. With a scalpel, cut a corner to identify the orientation of the section.
   11. Collect the membrane embedded in gelatin with a spatula and place it in a 6-well plate filled with the conservation medium at 4 °C, until grafting it into the recipient eye.
   12. At the time of the transplantation, adjust the size of the implant to the size of the recipient eye under a surgical microscope (1–3 for rats, 10–15 mm² for non-human primates).

Results

hAMs contain an epithelial layer that should be removed before the seeding of RPE cells. An enzymatic treatment of the membrane is performed with the thermolysin under shaking. In order not to not lose the polarity of the membrane (the epithelium is on one side), it is fixed on a support which composition could be different depending on the provider (Figure 1A). Check the adhesion of the membrane to its support at this step and add clips if necessary. At the ...

Discussion

We described a method for the culture of RPE cells on a biological scaffold and its preparation for implantation in animal models. One of the critical steps of the protocol is the maintenance of the orientation of the hAM all along the procedure until its inclusion into gelatin. Indeed, the native epithelium of the membrane is removed and its basement membrane becomes exposed⁹. The RPE cells have to be seeded on top of this basement membrane. Upon preparation for gelatin embedding, it is crucial t...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sterile biosafety cabinet	TechGen International	Not applicable
Liquid waste disposal system for aspiration	Vacuubrand	BVC 21
CO2-controlled +37 °C cell incubator	Thermo Electron Corporation	BVC 21 NT
200 µL pipette: P200	Gilson	F144565
1 mL pipette: P1000	Gilson	F144566
Pipet aid	Drummond	75001
+4 °C refrigerator	Liebherr	Not applicable
Vibratome	Leica	VT1000S
Fine scissors	WPI	501758
Forceps (x2)	WPI	555227F
Water bath	Grant subaqua pro	SUB6
Precision balance	Sartorius	CP225D
Centrifuge	Eppendorff	5804
Microscope	Olympus	SC30
Horizontal Rocking Shaker	IKA-WERKE	IKA MTS 214D
Vortex	VWR	LAB DANCER S40
Disposable Scalpel	WPI	500351
plastic paraffin film	VWR	PM992
0.200 µm single use syringe filter	SARTORIUS	16532
Syringe without needle 50 mL	Dutscher	50012
Bottles 250 mL	Dutscher	28024
15 mL sterile Falcon tubes	Dutscher	352097
50 mL sterile Falcon tubes	Dutscher	352098
culture insert	Scaffdex	C00001N
60 mm cell culture disches: B6	Dutscher	353004
12 well cell culture plate	Corning	3512
6-well culture plates	Corning	3506
Razor blades	Ted Pella, Inc	121-9
Cyanoacrylate glue	Castorama	3178040670105
PBS 1x (500 mL)	Sigma	D8537
Thermolysine	Roche	5339880001
DMEM, high glucose, GlutaMAX	Invitrogen	61965-026
KSR CTS (KnockOut SR XenoFree CTS)	Invitrogen	12618-013
MEM-NEAA (100x)	Invitrogen	11140-035
b-mercaptoethanol (50 mM)	Invitrogen	31350-010
Penicillin/Streptomycin	Invitrogen	15140122
CO2-independent medium	GIBCO	18045-054
Gelatin	MERCK	104078
human amniotic membrane	Tissue bank St Louis hospital (Paris, France)	Not applicable