Published: September 6th, 2018
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.
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.
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 both2,3. 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 ....
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 f.......
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 .......
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.......
The authors would like to thank Jérôme Larghero and Valérie Vanneaux (Hôpital Saint Louis, Paris, France) for their input during the setting-up of the method described here.
This work was supported by grants from the ANR [GPiPS: ANR-2010-RFCS005; SightREPAIR: ANR-16-CE17-008-02], the Fondation pour la Recherche Médicale [Bio-engineering program - DBS20140930777] and from LABEX REVIVE [ANR-10-LABX-73] to Olivier Goureau and Christelle Monville. It was supported by NeurATRIS, a translational research infrastructure (Investissements d'Avenir) for biotherapies in Neurosciences [ANR-11-INBS-0011] and INGESTEM, the na....
|Sterile biosafety cabinet
|Liquid waste disposal system for aspiration
|CO2-controlled +37 °C cell incubator
|Thermo Electron Corporation
|BVC 21 NT
|200 µL pipette: P200
|1 mL pipette: P1000
|+4 °C refrigerator
|Grant subaqua pro
|Horizontal Rocking Shaker
|IKA MTS 214D
|LAB DANCER S40
|plastic paraffin film
|0.200 µm single use syringe filter
|Syringe without needle 50 mL
|15 mL sterile Falcon tubes
|50 mL sterile Falcon tubes
|60 mm cell culture disches: B6
|12 well cell culture plate
|6-well culture plates
|Ted Pella, Inc
|PBS 1X (500 mL)
|DMEM, high glucose, GlutaMAX
|KSR CTS (KnockOut SR XenoFree CTS)
|b-mercaptoethanol (50 mM)
|human amniotic membrane
|Tissue bank St Louis hospital (Paris, France)
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