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Fury vs. College Football: Select Games. According to their approach, RPE were isolated from 6- to 8-day old black eyed rats and grafted into the subretinal space by using a lesion paradigm which penetrates through the sclera and choroid.
However, while this technique is marginally appropriate for immature RPE cells, with mature cells it leads to activation and transformation of these cells which damages eye and retinal tissue. Lopez et al. In this procedure, RPE cells were obtained from normal, congenic, pigmented rat eyes by trypsin digestion. These freshly harvested, dissociated RPE cells were injected into the subretinal area of the eyes of dystrophic RCS rats via an incision through the sclera, choroid and neural retina.
Comparable to the Li and Turner approach discussed above, this procedure destroys the organized native structure of the transplanted RPE cells, which take the form of a confluent monolayer in a healthy eye. Moreover, the procedure is of questionable value for the transplantation of mature RPE cells.
When mature RPE cells are transplanted in dissociated form, experimental results indicate that they are likely Mammals - Lucifer Wong - Mammals (Vinyl) become activated, migrate into the subretinal space, and as noted by Lane, C.
This activation of the transplanted, mature RPE cells can result in such pathologies as retinal pucker, massive subretinal fibrosis, retinal rosette formation, retinal detachment, and proliferative vitreoretinopathy.
The difficulties discussed above associated with the transplantation of mature RPE cells is problematic for human transplantation since available supplies of immature human RPE donor tissue are extremely limited. Moreover, the inability to use mature cells effectively prevents transplantation using autologous RPE tissue, which otherwise would be desirable to avoid the complications involving potential immunological responses faced by non-autologous transplants. Since the victims of AMD are predominantly older adults, in most cases utilizing autologous tissues for transplants would necessarily entail the use of mature human RPE cells.
It is believed by the present inventor that it is necessary to maintain adult human RPE cells substantially as a monolayer to achieve their successful transplantation and to avoid the problems associated with activation of Mammals - Lucifer Wong - Mammals (Vinyl) cells.
Although not wishing to be limited to a particular theory, it is thought that the cell-to-cell contact inhibition provided by an intact monolayer, supplemented by adherence to a substrate, mitigates against RPE cell activation. Moreover, a monolayer structure for the RPE provides a proper foundation for the maintenance of the photoreceptor cells in an organized outer nuclear layer structure and for proper growth and arrangement of inner and outer segments, believed by this inventor to be advantagous to restore a reasonable degree of vision.
The requirement that the photoreceptors be maintained in an organized structure is based on the well known optical characteristics of photoreceptors outer segments act as light guides and clinical evidence showing that folds or similar, even minor, disruptions in the retinal geometry can severely degrade visual acuity. Additionally, in cases of AMD where subretinal neovascular membranes have appeared, prior to RPE transplantation, such membranes will need to be removed to prevent subretinal edemas and hemorrhaging of these membranes.
In practice, removal of the neovascular membrane results in removal of the native RPE and Bruch's membrane as well. A critical impediment to the transplantation of RPE cells as a monolayer is the fragility of the intercellular structure of RPE relative to the rigors of manipulation during transplantation to the subretinal area. Moreover, providing satisfactory support to the RPE cells during this process is complicated by the fact that the support must either be removed subsequent to transplantation, to avoid compromising metabolic exchange between the choroid and the overlying retina, or be compatible with such ongoing physiological activity.
Thus, a method is needed wherein an implant comprising a monolayer of RPE cells is prepared and transplanted in which the component supporting the monolayer of RPE cells, upon transplantation to the subretinal area and exposure to a set of predetermined conditions, does not impede normal eye tissue function. Further, a Bruch's-like membrane for attachment of the transplanted RPE cells will be needed in those cases where neovascularization has occurred and the native Bruch's membrane is removed.
Among the objects of the present invention, therefore, may be noted the provision of a method for preparation of a population of RPE cells as a monolayer for use in the reconstruction of a dystrophic RPE; the provision of such a method which allows for use of mature, and in particular, autologous, mature RPE cells as donor tissue in the reconstruction of a dystrophic RPE; the provision of an implant for use in the reconstruction of a dystrophic retina; the provision of such an implant which does not interfere with normal eye tissue function after transplantation and maintains photoreceptors and their inner and outer segments by allowing for maintenance of the native organization of the photoreceptor; the provision of such an implant which provides a membrane for attachment of the population of RPE cells especially when the native Bruch's membrane has been removed; and the provision of a method for transplantation of such implants to the subretinal area of an eye.
Briefly, therefore, the present invention is directed to a method for the preparation of a population of RPE cells for transplantation to the subretinal area of a host eye. The method comprises providing donor tissue comprising RPE cells, harvesting from that tissue RPE cells, and apposing the harvested RPE cells as a monolayer to a non-toxic, flexible support that, upon transplantation to the subretinal area and exposure to a set of predetermined conditions, will not impede normal eye tissue function of the host eye and the transplanted population.
The present invention is further directed to an implant for transplantation to the subretinal area of a host eye. The implant comprises a laminate of a monolayer of retinal pigment epithelium cells and a non-toxic, flexible support that, upon transplantation to the subretinal area and exposure to a set of predetermined conditions, will not impede normal eye tissue function.
The present invention is also directed to a method for transplanting to the subretinal area of a host's eye an implant comprising a monolayer of RPE cells.
The method comprises providing an implant comprising a laminate of a monolayer of retinal pigment epithelium cells and a non-toxic, flexible support that, upon transplantation to the subretinal area and exposure to a set of predetermined conditions, will not impede normal eye tissue function, making an incision through the host's eye, at least partially detaching the retina to permit access to the subretinal area, and positioning the implant in the accessed subretinal area.
As used herein, the term "donor" shall mean the same or different organism relative to the host and the term "donor tissue" shall mean tissue harvested from or cultured from tissue harvested from the same or different organism relative to the host. Autologous tissue shall mean tissue harvested from or cultured from tissue harvested from the host organism. It is believed that in age-related macular degeneration AMDcompromise or degeneration of the RPE cells which underlie and form a close structural and metabolic support for the photoreceptors, leads to the loss or destruction of viable photoreceptors.
It has been discovered, however, that transplantation of a population of RPE cells as a monolayer, i. Moreover, as illustrated in Example 2 below, it is believed that it is essential to transplant mature RPE cells Mammals - Lucifer Wong - Mammals (Vinyl) a monolayer in order to prevent activation of such cells.
Activation leads RPE cells to transdifferentiate into wandering macrophages, fibroblast-like cells and other cell types, which cause a number of dystrophic effects in the eye and retina. In FIG. Referring now to FIG. The flexible composition serves as a stabilizing support for the RPE cells during transplantation.
The implant constitutes a laminate of a monolayer of RPE cells and a support composition approximately to microns thick, and preferably microns thick. The surface of the implant has a surface area greater than about 1 square millimeter, preferably greater than 2 square millimeters, and most preferably greater than 4 square millimeters, or as large as may be practically handled.
Thus constructed, the implant may subtend a considerable extent of the sub-retinal surface. In selecting donor tissue for the harvesting of RPE cells, it is noted that previously, transplantation of RPE cells was effectively limited to immature cells. Mature RPE cells transplanted according to prior art methods have been shown to undergo activation resulting in retinal pucker, sub-retinal fibrosis and proliferative vitreoretinopathy.
However, by utilizing the procedures for implanting a monolayer of RPE cells according to this invention, transplanted mature RPE cells have successfully maintained essentially normal structure and function.
Thus, the subject invention makes mature RPE cells available for transplantation, significantly increasing the supply of usable donor tissue beyond the narrowly limited supply of immature human donor tissue. It is also noted that the RPE forms part of the blood-retinal barrier and is thus exposed to lymphocytic attack. Accordingly, use of autologous RPE cells, now possible even for mature RPE cells, is preferable, since it will avoid immunological complications in clinical applications.
Donor tissue may be provided, for non-autologous, human RPE tissue, from eye banks, which make the RPE tissue available in connection with conducting corneal transplants. Harvesting donated tissue comprising non-autologous RPE cells can be accomplished by any suitable method. Specifically, to harvest non-autologous RPE cells, a donor eye is pinned by the optic nerve stump into an eye cage and placed in an eye jar immediately after corneal removal.
After loose connective tissue and muscle have been carefully trimmed from the eye, it is placed upright on a sterile plate and the anterior segment with the adherent vitreous is lifted out of the eye cup. The neural retina is separated at the optic disc and removed.
The RPE cells are released from Bruch's membrane by gently pipetting the culture medium in the shell. For implants containing autologous tissue, RPE cells may be harvested by performing a biopsy following the procedures disclosed by Lane, C.
The implant also comprises a support for the RPE cells so that the monolayer of RPE cells is less likely to be damaged and is more easily manipulated during the transplantation process. The support consists of a substrate, an overlayer or both, comprising a sheet or sheets of a non-toxic, flexible composition selected to provide mechanical strength and stability to the easily damaged monolayer of RPE cells. Because the support composition will be inserted into the eye as a laminate with the monolayer of RPE cells, the support is also comprised so that, upon transplantation to the subretinal area and exposure to a set of predetermined conditions, described herein, the support composition will not impede normal eye tissue function of the host eye or the population of transplanted RPE cells.
If the harvested RPE cells are to be cultured before transplantation, the composition providing support to the monolayer of RPE cells during transplantation may optionally be capable of serving as the attachment substrate for the RPE cells during culturing. A variety of compositions may be used as the support for the population of RPE cells, depending upon the specific set of conditions to which the composition will be exposed after transplantation. In short, the composition is selected either because any portion remaining within the subretinal area for more than approximately one week after transplantation is compatible with normal eye tissue function upon exposure to bodily fluids within the sub-retinal area, or for its susceptibility to elimination upon exposure to prescribed conditions.
An additional factor to consider in determining the make-up of compositions for support of the monolayer of RPE cells is whether Bruch's membrane has been or will be removed from the host eye prior to transplantation of the monolayer of RPE cells.
Bruch's membrane serves to anchor the RPE cells in a healthy eye. Removal of Bruch's membrane may occur in cases of AMD where a subretinal neovascular membrane has formed and Bruch's membrane is removed as a consequence of the removal of the neovascular membrane.
In cases where Bruch's membrane is removed, the support composition will comprise a layer of collagen less than microns in thickness, and preferably between 1 and 10 microns in thickness. The basal surfaces of the RPE cells attach readily to the collagen layer, which serves to anchor the RPE cells to the choroid in place of the removed Bruch's membrane. The layer of collagen also serves to inhibit the occurrence or reoccurrence of subretinal neovascularization through and around the transplanted RPE.
Such a collagen Mammals - Lucifer Wong - Mammals (Vinyl) is retained indefinitely in the sub-retinal area. However, it is known that Bruch's membrane is essentially comprised of collagen, and such microthin layers of collagen are permeable enough to avoid impeding normal eye tissue functions such as the metabolic exchange between the choroid and the retina.
However, collagen compositions of such a minimal thickness are not strong enough to prevent buckling or distortion of the monolayer of RPE cells during transplantation. Thus, such microthin collagen materials need to be used in combination with additional supporting materials which will be eliminated after transplantation, if they are to form part of the support for the transplanted RPE cells. The transplanted RPE cells may then anchor themselves directly to the Bruch's membrane.
Either to supplement a layer of collagen, as discussed above, or in cases where inclusion of a collagen anchor Mammals - Lucifer Wong - Mammals (Vinyl) not required, a support composition may be selected which is dissipated, for example, by exposure to a sufficient amount of heat, selected enzymes, or bodily fluids. Gelatin is an example of a preferred support material which is flexible, lacks toxicity to neural tissue and has the ability to dissolve at body temperature.
Another alternative is to use biodegradable polymers such as ethylene-vinyl acetate copolymer Elvax 40W, DuPont Chemical Co. Polysciences, Inc. See, e. Data Sheet Advantageously, the gelatin or other support composition may additionally serve as a carrier for any of a number of trophic factors such as pharmacologic agents including immunosuppressants such as cyclosporin A, anti-inflammation agents such as dexamethasone, anti-angiogenic factors, anti-glial agents and anti-mitotic factors.
Upon dissolution of the support composition, the factor or agent becomes available to impart the desired effect upon the surrounding tissue. The dosage can be determined by established experimental techniques. With appropriate enzymatic digestion, using techniques disclosed by Pfeffer, B. Such a freshly harvested intact monolayer of RPE cells may be immediately apposed as an intact monolayer to a support, such as a thin collagen sheet, supplemented with an overlayer or substrate of gelatin, allowing several hours for adhesion prior to transplantation.
This procedure may be useful for transplantation of RPE cells obtained in a biopsy, provided sufficient RPE tissue is obtained from the biopsy as an intact monolayer. In most cases, however, it is preferable to culture the harvested RPE cells on an attachment substrate so that a monolayer of RPE cells may be prepared. Culturing the RPE cells before harvesting is also preferred both to allow for the production of larger populations of RPE cells from a small amount of harvested tissue and to allow for a period of observation to ensure that the RPE cells to be transplanted are healthy and functional.
For proper culturing, harvested RPE cells contained are apposed to a substrate to which they will attach and grow, and which is capable of being maintained in culture conditions appropriate for efficient growth of RPE cells.
Apposition of RPE cells may be accomplished by pipetting a solution containing a population of RPE cells onto the substrate. Intact sheets of harvested RPE cells in a physiological solution may also be physically released via a wide-bore pipette onto the substrate and manipulated with a fine camel-hair brush if necessary. Attachment substrates which are suitable for growth of RPE cell cultures include plastic culture ware or glass culture chamber slides coated with collagen for cell attachmant.
Culturing harvested RPE cells can be accomplished by any suitable method for obtaining a confluent monolayer of RPE cells. Appropriate culturing techniques are described in Pfeffer, B. An implant laminate 1 is formed as depicted in FIGS. Thus, a monolayer of RPE cells 5 is first cultured on a suitable substrate for growth. RPE cells will grow on plastic culture ware A. However, to aid in removal of the monolayer of RPE cells 5 from the culture ware A, the culture ware A may be coated with a substrate 9 for the RPE cells, such as agarose or fibrin.
See FIG. The substrate 9 is in turn coated with a thin microns layer of collagen 7, to which the monolayer of RPE cells 5 readily attach, and which will serve as an anchor for the RPE cells in the post-transplantation period. If agarose is used as the substrate 9, the agarose is activated so that the collagen 7 will adhere to it by treating the surface of the agarose with an activator such as cyanogen bromide, as disclosed by Axen, R. The implant laminate 1 comprising a monolayer of RPE cells 5, attached to the collagen anchor 7 and an overlayer of gelatin 3, may then be physically removed from the culture ware A by slicing between the culture ware A surface and the collagen 7 with a razor C, as depicted in FIG.
The implant laminate 1 may also be removed from the culture ware and substrate 9 enzymatically, as depicted in FIG. The underlying substrate 9 may be removed from the implant laminate 1 by application of an enzyme specific to the material comprising the substrate 9. If the substrate is agarose, agarase, an enzyme specific to agarose, may be used to dissipate the agarose.
To aid in dissipation of the substrate 9, the culture ware A may comprise a cell culture insert membrane, containing an inner membrane A-1 with a porous bottom, and an outer, solid membrane A The porous e. These pores facilitate penetration of the solution B containing the enzyme into the substrate 9 so that it may be broken apart, and the implant laminate 1 removed from the culture ware A. As depicted in FIG. The implant 1a is transplanted to the subretinal area at the posterior pole 76 of the host eye after detachment of the retina, as portrayed in FIG.
The gelatin overlayer dissolves within hours after insertion of the implant 1a into the host eye. After transplantation, the retina reattaches, and the monolayer of RPE cells, anchored to the layer of collagen, is sandwiched between the choroid and the retina. To transplant the implant, the host eye is prepared so as to reduce bleeding and surgical trauma. The preferred surgical approach in the human, FIG.
The instrument 20 is advanced through the sclera and choroid 78 and to the ora serrata 74 as illustrated in FIG. The instrument detaches the retina as it is advanced under the retina and into the sub-retinal space to the posterior pole 76 of the eye.
Preferably, an instrument comprising an elongate tube having a flat, wide cross-section may be used so that the implant may be drawn into the elongate tube for protection as it is transported through an appropriate sized incision in the sclera or choroid. Advantageously, the fluid may additionally contain anti-oxidants, anti-inflammation agents, anesthetics or agents that slow the metabolic demand of the host retina. If the instrument does not include a lumen, the retina is detached by subretinal irrigation or by the walls of the surgical instrument as it is advanced under the retina and into the subretinal space to the posterior pole 76 of the eye.
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