Age-related Macular Degeneration is considered to be one of the main causes of central vision loss. The progressive degeneration and dysfunction of the RPE layer is a hallmark of the early form of AMD. In spite of advances in therapy for retinal diseases, the development of an effective treatment modality remains challenging.
One of the most promising treatment concepts is the replacement of the RPE by an in vitro cultured RPE cells of different origin. The RPE have usually been delivered into subretinal space in the form of a cell suspension, the self-supporting cell sheet, or a cell monolayer supported by an artificial carrier. The main advantage of the two late methods is that after the implantation, the cells are found in a differentiated monolayer state in the subretinal space.
Pre-clinical animal studies performed in vivo aim to establish a reproducible and safe surgical procedure on a large eye animal model in order to obtain data which could be useful and potentially applicable in humans. With this protocol, we aim to report the principles of the subretinal surgical technique used for in vivo subretinal implantation of a cell carrier in a large animal model that could be used for in vivo experimental studies with implantation of different types of cells and cell carriers. With this video, we would like to present a step-by-step protocol of the surgical procedure of subretinal implantation of the cell carrier in the eyes of Libechov minipig.
Insert the lid speculum. Open the conjunctiva on the nasal side two to three millimeters from the limbus in order to expose the sclera for sclerotomies. Insert the three 25 gauge trocars three millimeters from limbus in the area of the pars plana.
Keep the cornea wet, or coat it with methyl cellulose during the whole surgery to prevent osmotic corneal edema. Remove the middle portion of the vitreous body with the standard three-port pars plana vitrectomy approach. Carefully remove the vitreous behind the lens in the area of the future large sclerotomy.
Use the intravitreal triamcinolone acetonide injection to stain the posterior vitreous, which remains usually adherent to the retina in order to perform a controlled posterior vitreous detachment. After that, slowly perform a subretinal injection of BSS with a 41 gauge cannula more centrally, avoiding the formation of the bleb towards the periphery. Reduce the intraocular pressure setting of the irrigation system down to 15 millimeters of mercury during the subretinal injection in order to prevent transient retinal vascular occlusion.
Perform a linear large endodiathermy of the retina with the 27 gauge endodiathermy probe near the nasal bleb base. Afterwards, make a three millimeter large retinotomy using a 25 gauge MVR blade, or vertical scissors with elevated intraocular pressure setting of the irrigation system up to 60 millimeters of mercury for three to five minutes. Then make a three millimeter large sclerotomy three millimeters from the limbus using a 2.75 millimeter phaco knife.
Pay attention to the exodiathermy of possible bleeding of the scleral vessels and ciliary body inside the large sclerotomy. Remove the prolapsed vitreous body on the side of the large sclerotomy with the vitrector. Then gently insert the injector with a dominating hand into the vitreous cavity through the large sclerotomy.
Implant the cell carrier through the retinotomy into the subretinal space. Withdraw the injector from the eye, and close the large sclerotomy with an 8-0 Vicryl suture to avoid complications associated with intraocular hypotony. Perform a complete fluid-air exchange with the silicone-tipped cannula.
After that, inject the silicone oil into the vitreous cavity until the intraocular pressure will be normal. At the end of the surgery, remove the trocars, and close the three sclerotomies and the conjunctiva with 8-0 Vicryl sutures. For further details, please see the section Surgical Procedure.
Clean the periocular area. Use the forceps and scissors to remove the upper and lower eyelid. Remove the third eyelid.
Cut through the conjunctiva. Cut the eye muscles with scissors. Proceed the cutting circularly from the surface to the depth.
Cut the optic nerve. Remove the eye from the orbit. Clarity of the vitreous cavity filled with silicone oil allows one to visualize the condition of the retina with subretinal cell carrier at any time point of the post-operative period.
The red-free images show the reflectivity of the cultivated human RPE cells within the cell carrier frame doesn't differ from the reflectivity of the indigenous porcine RPE layer. In vivo OCT imaging reveals that the cell carrier implanted into the subretinal space doesn't significantly increase the entire retinal thickness. The Bruch's membrane appears to remain undamaged as well.
The frame of the cell carrier causes only minor shadowing of the OCT signal. Histological study of post-mortem cryo sections of the retina demonstrates the formation of the continuous and irregular pigmented cell layer in the area of the implanted primary human RPE, which proves the survival of the cultivated on the cell carrier cells. Immunohistochemical analysis of the tissue section shows the human RPE cells, which are present in the area of the implantation, and express the typical RPE marker similar to the indigenous minipig RPE cells.
In spite of this, the implanted RPE cells don't appear as a cell monolayer. Still, these cells are located within the defined subretinal space. A big eye animal model of minipigs, in this case, Libechov minipigs, we consider to be one of the most appropriate models for the future application of novel concepts for treatment of degenerative retinal diseases, due to their similarity to the human eye anatomy and physiology.
These similarities allow to develop the surgical techniques and instrumentation for subretinal implantation of cell carrier, and this technique could be transferred easily to the treatment of human eye disorders. It is important to assure that the surgeries on minipigs are performed with the same instrumentation, including implantation delivery tools, which would also be utilized in human surgeries, thus making the application of gained experience and know-how to humans without difficulties. The detailed description of the preoperative surgical and post-operative care procedures could be helpful for future studies by increasing efficient and standardized data generation.
