Device and method for drying a film for artificial endothelial keratoplasty graft and methods of preparation thereof

Abstract

The present invention discloses a device and method for drying a support layer to be used for artificial endothelial keratoplasty graft. More specifically, the present invention provides an array of at least one case, having a base sized and shaped for enclosing at least one type of liquid solution, wherein said at least one case is rotatable at a predetermined angular velocity around a main longitudinal axis, being substantially perpendicular to the horizonal plane, such rotatable movement of said at least one case results in drying of said liquid solution to provide a film for the generation of a scaffold support layer for artificial endothelial keratoplasty graft.

Description

[0001] DEVICE AND METHOD FOR DRYING A FILM FOR ARTIFICIAL ENDOTHELIAL

[0002] KERATOPLASTY GRAFT AND METHODS OF PREPARATION THEREOF

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of priority of US provisional patent application no. 63 / 451,681, filed on March 13, 2023, the contents of which is all incorporated by reference herein in their entirety.

[0005] FIELD OF INVENTION

[0006] The present invention generally pertains to an artificial endothelial keratoplasty graft and to methods of its use and preparation.

[0007] BACKGROUND OF THE INVENTION

[0008] Comeal blindness is estimated to have effect on the order of 10 million people worldwide. It is further estimated that only about 1% of the people suffering from comeal blindness receive treatment.

[0009] The term comeal transplantation is used to describe several different medical procedures, in which different parts of the cornea is being implanted, including: (A) Penetrating Keratoplasty (PK), in which the entire thickness of the cornea is transplanted, (B) Descemet Stripping Endothelial Keratoplasty (DSEK), in which the endothelium membrane, Descemet membrane and part of the posterior comeal stroma are transplanted, and (C) Descemet Membrane Endothelial Keratoplasty (DMEK), in which the endothelium and the Descemet membrane are transplanted, without additional stroma layer.

[0010] Among the procedures mentioned above, DSEK is the most common one. In the US, 60% of the total comeal transplantations are DSEK. However, the DMEK procedure has major advantages over it, including lower rejection rate (1% for DMEK compared with 12% of DSEK), higher probability for visual acuity above 0.8 (79% in DMEK compared with 47% in DSEK), and shorter healing period (weeks in DMEK compared with months to year in DSEK). In the Precise Vision Endothelial Keratoplasty, PVEK, which mimics the DMEK procedure, according to the present invention, engineering a scaffold (that is a support layer for the artificial graft) is required. Such scaffold is mainly provided with collagen. During the preparation of the scaffold, namely, for comeal grafts / implants, a drying step is mandatory.

[0011] Therefore, it is a long felt need to provide a simple, a reliable and efficient tool and method for drying the scaffold whilst not damaging the healthy and functioning endothelial cells therewithin under repeatable conditions.

[0012] SUMMARY OF THE INVENTION

[0013] It is one object of the present invention to provide an array of at least one case, having a base sized and shaped for enclosing at least one type of liquid solution, wherein said at least one case is rotatable at a predetermined velocity around a main longitudinal axis, being substantially perpendicular to the horizonal plane, such rotatable movement of said at least one case results in drying of said liquid solution to provide a film for the generation of a scaffold support layer for artificial endothelial keratoplasty graft.

[0014] It is another object of the present invention to provide the array as defined above, wherein said at least one case comprising at least one cover for sealingly fitting said base.

[0015] It is another object of the present invention to provide the array as defined above, wherein said base is characterized by having a predetermined radius of curvature to shape said artificial endothelial keratoplasty graft accordingly.

[0016] It is another object of the present invention to provide the array as defined above, comprising a plurality of cases.

[0017] It is another object of the present invention to provide the array as defined above, wherein each case is aligned with a neighboring case.

[0018] It is another object of the present invention to provide the array as defined above, additionally comprising at least one alignment element in communication with at least two neighboring cases. It is another object of the present invention to provide the array as defined above, wherein said at least one alignment element is provided so as to facilitate substantially the same rotational velocity or angular velocity to all of said cases.

[0019] It is another object of the present invention to provide the array as defined above, wherein said at least one alignment element is at least one pin (also referred to as a common rotation axis) being in mechanical communication with said at least two neighboring cases.

[0020] It is another object of the present invention to provide the array as defined above, wherein said predetermined velocity of each of said at least one case is independently controlled.

[0021] It is another object of the present invention to provide the array as defined above, wherein said predetermined velocity of each of said at least one case is changeable as a function of time.

[0022] It is another object of the present invention to provide the array as defined above, wherein said case is hingly connected to the array.

[0023] It is another object of the present invention to provide the array as defined above, wherein said liquid solution comprising hydrogel.

[0024] It is another object of the present invention to provide the array as defined above, wherein said hydrogel is collagen solution comprising at least one selected from a group consisting of collagen or collagen methacrylate and any combination thereof.

[0025] It is another object of the present invention to provide the array as defined above, wherein liquid solution is gelatin or gelatin methacrylate.

[0026] It is another object of the present invention to provide the array as defined above, wherein the source of said collagen or collagen methacrylate is human recombinant collagen, human skin source and any combination thereof.

[0027] It is another object of the present invention to provide the array as defined above, wherein the liquid solution is based on gelatin or gelatin methacrylate.

[0028] It is another object of the present invention to provide the array as defined above, wherein the total thickness of the graft is between 3 and 50 microns.

[0029] It is another object of the present invention to provide the array as defined above, wherein the total thickness of the graft is below 25 microns, therefore mimicking native DMEK graft. It is another object of the present invention to provide the array as defined above, wherein the mechanical properties of said support layer allows positioning and flattening of the graft inside the anterior chamber during implantation.

[0030] It is another object of the present invention to provide the array as defined above, wherein said cells remodeled the said support layers to form comeal endothelium extra-cellular matrix, by a long maturation period or by additional nutrients to the cells.

[0031] It is another object of the present invention to provide the array as defined above, additionally comprising introduction of EDC and NHS molecules to crosslink said film to thereby result in said scaffold support layer for artificial endothelial keratoplasty graft.

[0032] It is another object of the present invention to provide the array as defined above, wherein said introduction of EDC and NHS is obtained within said at least one case or outside thereof.

[0033] It is another object of the present invention to provide a matrix of x arrays, each comprising at least one case, each case having a base sized and shaped for enclosing at least one type of liquid solution, wherein said at least one case is rotatable at a predetermined velocity around a main longitudinal axis, being substantially perpendicular to the horizonal plane, such rotatable movement of said at least one case results in drying of said liquid solution to provide a film for the generation of a scaffold support layer for artificial endothelial keratoplasty graft; where x is an integer being equals to or greater than 0.

[0034] It is another object of the present invention to provide the matrix as defined above, wherein at least one of said cases comprising at least one cover for sealingly fitting said base.

[0035] It is another object of the present invention to provide the matrix as defined above, wherein at least one of said bases is characterized by having a predetermined radius of curvature to shape said artificial endothelial keratoplasty graft accordingly.

[0036] It is another object of the present invention to provide the matrix as defined above, wherein at least one of said arrays comprising a plurality of cases.

[0037] It is another object of the present invention to provide the matrix as defined above, wherein each case is aligned with a neighboring case.

[0038] It is another object of the present invention to provide the matrix as defined above, additionally comprising at least one alignment element in communication with at least two neighboring cases. It is another object of the present invention to provide the matrix as defined above, wherein said at least one alignment element is provided so as to facilitate substantially the same rotational velocity or angular velocity to all of said cases.

[0039] It is another object of the present invention to provide the matrix as defined above, wherein said at least one alignment element is at least one pin (also referred to as a common rotation axis).

[0040] It is another object of the present invention to provide the matrix as defined above, wherein said predetermined velocity of each of said at least one case is independently controlled.

[0041] It is another object of the present invention to provide the matrix as defined above, wherein said predetermined velocity of each of said at least one case is changeable as a function of time.

[0042] It is another object of the present invention to provide the matrix as defined above, wherein said liquid solution is collagen solution comprising at least one selected from a group consisting of collagen or collagen methacrylate and any combination thereof.

[0043] It is another object of the present invention to provide the matrix as defined above, wherein liquid solution 1 is gelatin or gelatin methacrylate.

[0044] It is another object of the present invention to provide the matrix as defined above, wherein the source of said collagen or collagen methacrylate is human recombinant collagen, human skin source and any combination thereof.

[0045] It is another object of the present invention to provide the matrix as defined above, wherein the liquid solution 1 is based on gelatin or gelatin methacrylate.

[0046] It is another object of the present invention to provide the matrix as defined above, wherein the total thickness of the graft is between 3 and 50 microns.

[0047] It is another object of the present invention to provide the matrix as defined above, wherein the total thickness of the graft is below 25 microns, therefore mimicking native DMEK graft.

[0048] It is another object of the present invention to provide the matrix as defined above, wherein the mechanical properties of said support layer allows positioning and flattening of the graft inside the anterior chamber during implantation.

[0049] It is another object of the present invention to provide the matrix as defined above, wherein said cells remodeled said support layers to form comeal endothelium extra-cellular matrix, by a long maturation period or by additional nutrients to the cells. It is another object of the present invention to provide the matrix as defined above, wherein the support layers are transparent and permeable.

[0050] It is another object of the present invention to provide the matrix as defined above, additionally comprising introduction of EDC and NHS molecules to crosslink said film to thereby result in said scaffold support layer for artificial endothelial keratoplasty graft.

[0051] It is another object of the present invention to provide the matrix as defined above, wherein said introduction of EDC and NHS is obtained within said at least one case or outside thereof.

[0052] It is another object of the present invention to provide a method of drying at least one liquid solution to provide a film for artificial endothelial keratoplasty graft, wherein said method consisting steps of: a. providing an array of at least one case, having a base sized and shaped for enclosing at least one type of liquid solution , wherein said at least one case is rotatable at a predetermined velocity around a main longitudinal axis, being substantially perpendicular to the horizonal plane; b. placing at least one type of liquid solution in at least one said cases; c. rotating said at least one case at said predetermined velocity around said main longitudinal axis thereby drying said liquid solution to provide a film for the generation of a film for artificial endothelial keratoplasty graft .

[0053] It is another object of the present invention to provide the method as defined above, wherein said at least one case comprising at least one cover for sealingly fitting said base.

[0054] It is another object of the present invention to provide the method as defined above, wherein said base is characterized by having a predetermined curvature to shape said artificial endothelial keratoplasty graft accordingly.

[0055] It is another object of the present invention to provide the method as defined above, comprising a plurality of cases.

[0056] It is another object of the present invention to provide the method as defined above, wherein each case is aligned with a neighboring case.

[0057] It is another object of the present invention to provide the method as defined above, additionally comprising at least one alignment element in communication with at least two neighboring cases. It is another object of the present invention to provide the method as defined above, wherein said at least one alignment element is provided so as to facilitate substantially the same rotational velocity or angular velocity to all of said cases.

[0058] It is another object of the present invention to provide the method as defined above, wherein said at least one alignment element is at least one pin (also referred to as a common rotation axis).

[0059] It is another object of the present invention to provide the method as defined above, wherein said predetermined velocity of each of said at least one case is independently controlled.

[0060] It is another object of the present invention to provide the method as defined above, wherein said predetermined velocity of each of said at least one case is changeable as a function of time.

[0061] It is another object of the present invention to provide the method as defined above, wherein said liquid solution is collagen solution comprising at least one selected from a group consisting of collagen or collagen methacrylate and any combination thereof.

[0062] It is another object of the present invention to provide the method as defined above, wherein liquid solution is gelatin or gelatin methacrylate.

[0063] It is another object of the present invention to provide the method as defined above, wherein the source of said collagen or collagen methacrylate is human recombinant collagen, human skin source and any combination thereof.

[0064] It is another object of the present invention to provide the method as defined above, wherein the liquid solution is based on gelatin or gelatin methacrylate.

[0065] It is another object of the present invention to provide the method as defined above, wherein the total thickness of the graft is between 3 and 50 microns.

[0066] It is another object of the present invention to provide the method as defined above, wherein the total thickness of the graft is below 25 microns, therefore mimicking native DMEK graft.

[0067] It is another object of the present invention to provide the method as defined above, wherein the mechanical properties of said support layer allows positioning and flattening of the graft inside the anterior chamber during implantation.

[0068] It is another object of the present invention to provide the method as defined above, wherein said cells remodeled the said support layers to form comeal endothelium extra-cellular matrix, by a long maturation period or by additional nutrients to the cells. It is another object of the present invention to provide the method as defined above, additionally comprising a crosslinking step.

[0069] It is another object of the present invention to provide the method as defined above, wherein the crosslinking step involves in introducing EDC and NHS molecules to the material.

[0070] It is still an object of the present invention to provide the method as defined above, wherein said crosslinking step involves in introducing LAP or Irgacure 2959 molecules to the material and applying light on it.

[0071] It is lastly an object of the present invention to provide the method as defined above, wherein said drying step is performed in controlled environmental conditions.

[0072] BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The various features and advantages of the present invention may be more readily understood with reference to the following detailed description taken in conjunction with the accompanying drawings in which

[0074] Fig. 1 schematically illustrating an array of at least one case, according to one embodiment of the present invention.

[0075] Figs. 2-4 schematically illustrating at least one case for enclosing at least one type of liquid solution (which will eventually be dried and converted to a film and cross linked to provide support layer / scaffold for artificial endothelial keratoplasty graft).

[0076] Fig. 5 schematically illustrating a matrix of 5 arrays, each having 5 cases.

[0077] Fig. 6 schematically illustrating the Precise Vision Endothelial Keratoplasty, PVEK, DMEK- like graft according to one embodiment of the invention;

[0078] Fig. 7 schematically illustrating the Precise vision endothelial keratoplasty, PVEK, DMEK- like graft on a designated carrier according to another embodiment of the invention; and

[0079] Fig. 8 schematically illustrating a comeal graft orientation in the eye, according to yet another embodiment of the invention.

[0080] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In the following description, various aspects of the invention will be described. For the purposes of explanation, specific details are set forth in order to provide a thorough understanding of the invention. It will be apparent to one skilled in the art that there are other embodiments of the invention that differ in details without affecting the essential nature thereof. Therefore, the invention is not limited by that which is illustrated in the figure and described in the specification, but only as indicated in the accompanying claims, with the proper scope determined only by the broadest interpretation of said claims. In some cases, for clarity or conciseness, individual components of the invention disclosed herein may be described without reference to other components of the invention. Nonetheless, any combination of components that is not self-contradictory is considered by the inventors to be within the scope of the invention, even if that specific combination is not described explicitly in the specification. In addition, in cases in which an embodiment of the invention is described as "comprising" certain listed elements, embodiments that consist of the listed elements (i.e. contain those elements and no others) are considered by the inventors to be within the scope of the invention.

[0081] As used herein, the term "corneal graft" refers to a bioengineered construct that is designed to be implantable in a mammalian eye and to have properties of at least part of a cornea and having comeal cell monolayer on it.

[0082] As used herein, the term “Precise vision endothelial keratoplasty” and PVEK for short, will be interchangeably herein after refer to a set of solutions found in a few cases being equal to, if not better than the DSEK implant (comparable to DMEK implant).

[0083] As used herein, the term “angular velocity” or “rotational velocity” refers to the angular frequency vector, which is a pseudovector representation of how fast the angular position or orientation of an object changes with time (i.e. how quickly an object rotates or revolves relative to a point or axis). The magnitude of the pseudovector represents the angular speed, the rate at which the object rotates or revolves, and its direction is normal to the instantaneous plane of rotation or angular displacement.

[0084] As used herein, the term "coffee ring effect" refers to a pattern left by a puddle of particleladen liquid after it evaporates. The phenomenon is named for the characteristic ring-like deposit along the perimeter of a spill of coffee. The coffee-ring pattern originates from the capillary flow induced by the evaporation of the drop: liquid evaporating from the edge is replenished by liquid from the interior. The resulting edgeward flow can carry nearly all the dispersed material to the edge. As a function of time, this process exhibits a "rush-hour" effect, that is, a rapid acceleration of the edgeward flow at the final stage of the drying process.

[0085] As disclosed above, it is the object of the present invention to provide a simple, a reliable and efficient device and method for drying collagen solution to be used, e.g., in PVEK, whilst overcoming the coffee effect and providing a homogeneously dried graft. Such is enabled by application of rotational movement to the solution to be dried.

[0086] According to one embodiment, said rotation movement is controllable at a predetermined angular velocity. Such control also controls the thickness of the resulting dried film.

[0087] It is further the object of the present invention to provide a device and method for drying a liquid solution (containing collagen) to a film to be used for, e.g., corneal grafts.

[0088] Thus, it is an object of the present invention to provide means for homogeneously drying a liquid solution being collagen solution comprising at least one selected from a group consisting of collagen or collagen methacrylate and any combination thereof) to a thin film for a comeal graft (e.g., an endothelial keratoplasty graft).

[0089] After drying of said collagen solution to a thin film, cross linking of the same is facilitated to provide a support layer (a scaffold) for a comeal graft (e.g., an endothelial keratoplasty graft).

[0090] The main obstacle to overcome with such support layer is the curved landscape. When drying such a support layer in such curvature the coffee stain effect, which creates a hole in the center of the support layer, occurs. Such an effect is not trivial, it involves surface forces, drying rates, particle dynamics etc.

[0091] The main reason for the hole is in the center of the film (the support layer) is as follows: the drying process evaporates water from the collagen solution drop until eventually the edges of the drop (which are thinner than the center) start getting dry, from the edges toward the center of the film. Eventually, the last wet collagen is somewhere in the center of the film - and only at this point the coffee stain effect takes place, and pushes the collagen molecules sideways, to form a ring around this last wet circle, with almost no collagen at the center of the circle (the ‘hole’).

[0092] To overcome this, the device and method of the present invention forces the collagen to start drying from the center of the support layer (films) outwards . This is the essence of the rotational movement (the spinning) of each case in the array (or matrix) as provided by the present invention. Also, the wetting properties of the casing (also referred to as mold) are important for the detachment of the dried fdm from the case. Therefore, the base of each of the cases (or molds) should be made of a hydrophobic material. Such materials could be selected from a group consisting of polyproline, polypropylene, polystyrene, polycarbonate and any combination thereof.

[0093] According to another embodiment, the surface roughness of the casing has an effect on the fdms and extraction thereof. Therefore, it is within the scope of the present invention to provide a casing where the surface of the base and / or cover is smooth.

[0094] According to another embodiment, the base of the case (or mold) is provided with an envelope (‘walls’). It was found by the inventors of the present invention that such envelope is needed to prevent the coffee-stain effect. Such envelope (see numerical reference no. 13 in figures 3- 4) walls helps the liquid solution (collagen solution) aggregate at the edges. It should be pointed out that the envelope could be an integral part of the base 11 or alternatively as part of the cover 12.

[0095] Reference is now made to figure 1, schematically illustrating an array 100 of at least one case 10, and to figures 2-4 illustrating at least one case 10 for enclosing at least one liquid solution to be dried for providing a thin fdm for artificial endothelial keratoplasty graft, according to one embodiment of the present invention. It should be noted that once said film is cross linked, a scaffold (support layer) is provided for said artificial endothelial keratoplasty graft.

[0096] According to one embodiment, the array 100, comprising a plurality of cases 10.

[0097] According to one embodiment, all cases are aligned to each other by means of at least one alignment element 20.

[0098] According to one embodiment, the array is provided with means (e.g., motor), adapted to apply rotational movement (illustrated via arrow 11) to each of the cases at a predetermined velocity.

[0099] According to one embodiment, the alignment element 20 is provided so as to facilitate substantially the same rotational velocity or angular velocity to all of said cases.

[0100] According to one embodiment, the alignment element 20 is at least one pin threaded throughout all said cases 10. In other words, the alignment element 20 acts as a common rotation axis to at least 2 of the cases. Preferably to all cases.

[0101] According to one embodiment, all cases 10 are in mechanical communication with each other and with a rotating element (e.g., plate, illustrated as numerical ref. 12). Said rotating element is in communication with a motor adapted to rotate the same. Once said rotating element is rotated, all cases on communication therewith are rotated as well.

[0102] It should be noted that such rotating element is not mandatory and it could be that each of the cases is in direct communication with the motor to facilitate the rotational movement thereof, such that each rotational velocity of each case is controlled separately.

[0103] As an alternative embodiment, it could be that a group of the cases (or all cases) are in direct communication with the motor to facilitate the rotational movement thereof, such that the rotational velocity of the group (or all cases) is controlled together.

[0104] According to one embodiment, each case 10 comprising a base 11 (see figures 2-3) sized and shaped for enclosing at least one liquid solution to be drying for (eventually, post cross linking) providing a support layer (refers hereinafter as scaffold) for artificial endothelial keratoplasty graft, wherein said at least one case is rotatable at a predetermined angular velocity around a main longitudinal axis, being substantially perpendicular to the horizonal plane.

[0105] Once the liquid solution containing collagen is dried, a thin film is provided. Should cross linking agent(s) is added (e.g., l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N- hydroxysuccinimide (NHS), and a combination thereof) to the case, the film becomes a scaffold (a support layer) for artificial endothelial keratoplasty graft.

[0106] According to one embodiment, each case comprising at least one cover 12 for sealingly fitting the base 11 (see figures 2-3).

[0107] It should be noted that according to one embodiment and described in figures 1-4, the case is provided in 2 different parts: the base and the cover. However, it is within the scope of the present invention where (a) the base is provided with an envelope and a cover sealingly fitting the base; and, (b) the cover is provided with an envelope sealingly fitting the base.

[0108] According to one embodiment, the base 11 is characterized by having a predetermined curvature (or radius of curvature) to shape said artificial endothelial keratoplasty graft accordingly.

[0109] According to one embodiment, the predetermined rotational velocity of each of said at least one case is independently controlled.

[0110] According to one embodiment, the predetermined velocity of each of said at least one case is changeable as a function of time.

[0111] According to one embodiment, the predetermined velocity is in the range of 200 to 1000 RPM. According to one embodiment, the curvature is designed to fit the normal internal curvature of the eye. For Human eye, the curvature is approx. 13mm diameter as a measure of the best fit sphere of the center of the human cornea.

[0112] According to one embodiment, the curvature (namely, the radius of curvature) of at least one base of at least one of the cases is in the range of 10mm to 17mm. It is known that there are differences in the radius of curvature, ROC, between men, women, and kids. Also, the ROC of testing animals (rabbits, pigs, etc.) is also different. Thus, according to one embodiment of the present invention, a wide variety of curvatures of the case is provided.

[0113] According to another embodiment, overcoming the variance in the cornea curvature is obtained by the elastic properties of the support layer (namely, the collagen film).

[0114] Reference is now made to figure 5, schematically illustrating a matrix 1000 of 5 arrays 100, each having 5 cases 10.

[0115] The angular velocity w is defined by the centrifugal forces according to the following equations: where F is the force in Newtons, m is the mass, w is the angular velocity in rad / sec and R is the radius.

[0116] R, for cornea application could be in the range of 10 -12mm.

[0117] On the other hand, the gravitational force is defined as:

[0118] , - # where g is the gravity acceleration and equals to 9.81 meter / secA2.

[0119] Therefore, the required angular velocity w is approx.:

[0120] However, it is within the scope of the present invention where the angular velocity is in the range of 200 to 1000 RPM. It was also found by the inventors of the present invention that the drying rate of the liquid solution is a function of Temperature (T), Relative Humidity (RH), air velocity (airflow) and surface area (A).

[0121] It is within the scope of the present invention to use the drying method in temperature range of about 15-25 degrees.

[0122] It is within the scope of the present invention to use the drying method in a relative humidity more than 30%.

[0123] According to one embodiment of the present invention, the drying time was 12-96 hours.

[0124] As the rotational velocity (the spinning rate) and the volume of the liquid solution can result in spilling out of the case (the mold) some of the liquid solution, it is within the scope of the present invention to provide walls (or envelope) 13 (see figures 3-4) to the cover of the base.

[0125] According to one embodiment of the present invention, the base of the case is made of hydrophobic material.

[0126] According to one embodiment of the present invention, the base of the case is made of material selected from a group consisting of polyproline, polypropylene, polystyrene, polycarbonate and any combination thereof.

[0127] According to one embodiment of the present invention, the cover of the case is made of hydrophobic material.

[0128] According to one embodiment of the present invention, the cover of the case is made of material selected from a group consisting of polyproline, polypropylene, polystyrene, polycarbonate and any combination thereof.

[0129] According to another embodiment of the present invention, the angular velocity is alterable during the drying process. As the viscosity of the solution is changeable during the drying process. In the last stages, the material behaves more like a paste than a liquid, so the diffusion of water through the same is limited. Therefore, the velocity is amended accordingly.

[0130] It is within the scope of the present invention where the comeal graft is to be used in Descemet Membrane Endothelial Keratoplasty, DMEK, applications, where drying process induce connection between collagen molecules, in a process sometimes known as “vitrification”. It is mainly known in production of gelatin films. In gelatin films the vitrification is done after or during the crosslinking, not before it. Thus, it is within the scope of the present invention where the verification (the drying step) is performed before the cross-linking stage which facilitate the provision of ultra-thin (2-7 microns thick) films.

[0131] According to one embodiment of the present invention, the artificial Endothelial Keratoplasty graft is made of Descemet membrane-like support layer (which is the dried liquid solution containing collagen; thereby providing a thin film; and, then cross linked to provide the support layer), and a layer of comeal endothelial cells (CEC) layer on top. The endothelial layer is made of CEC cells obtained from donors’ corneas and proliferated, or by stem-cells differentiation, by methods known in the art.

[0132] The support layer is made of a thin collagen or gelatin layer, which was designed to have mechanical properties, permeability, transparency and dimensions. It allows light and nutrients flow through it and allows endothelial cell attachment; and thus, to improve patients’ health condition.

[0133] To produce the endothelial keratoplasty implant, a layer of low concentration collagen solution is spread on a surface and dehydrated. After drying out, a crosslinker solution is introduced to the dried material which form a crosslinked film / membrane in thicknesses between 2 to 50 micrometers. The excess and un-crosslinked reagents are washed away using PBS and comeal endothelial cells are seeded / printed on top of the film / membrane, forming a two-layer construct, similarly to DMEK cadaver grafts.

[0134] In some cases, the comeal graft is implanted onto the posterior cornea using a designated DMEK tools or carrier. In preferred embodiments, prior to the implantation a carrier layer is attached to the comeal graft using biocompatible adhesive material, forming a 4-layers implant: An Endothelial keratoplasty graft made of a collagen / gelatin layer and a cells layer, an adhesive layer, and a carrier. After implantation, the adhesive and the carrier layer are possibly detached from the graft and pulled out or dissolved in the eye.

[0135] In other cases, the comeal graft is implanted similarly to cadaver-sourced DMEK grafts, by methods known in the art.

[0136] The present invention hence discloses, inter alia, an artificial endothelial keratoplasty graft consisting a support layer made of rehydrated crosslinked film / membrane and comeal endothelial cells on top or within said support layer.

[0137] Reference is made to figure 6, schematically illustrating a DMEK-like graft according to one embodiment of the invention. Upper layer is comeal endothelial cells (3) and below is a support layer (4). Reference is now made to figure 7, schematically illustrating a DMEK-like graft on a designated carrier according to another embodiment of the invention, upper layer in this illustration is a carrier (1); and below are adhesive (2), Corneal endothelial cells (3) and support layer (4).

[0138] Reference is now made to figure 8, schematically illustrating a comeal graft orientation in the eye, according to yet another embodiment of the invention. Upper illustration is a comeal graft with adhesive and carrier layers attached to the posterior cornea (not in scale) (10). Cornea (20) and anterior chamber (30) are also illustrated. Figure is adapted from a currently available public draw https: / / www.iconspng.com / image / 92595 / eye-3.

[0139] It is according to a few embodiments of the invention wherein the graft comprises two main layers, namely (i) a support (Descemet membrane-like) layer, made of materials selected from a group consisting of collagen, gelatin, collagen methacrylate, gelatin methacrylate and a combination thereof; and (it), a comeal endothelial cells layer, seeded and / or printed on top of the support layer.

[0140] It is according to a few embodiments of the invention wherein the geometry of the support layer is ranging between about 2 and about 50 microns thick; diameter ranging between about 7to about 9.5 mm.

[0141] It is further according to a few other embodiments of the invention wherein the geometry of the cells layer is ranging between about 2 and about 20 microns in thickness, cells density of about 1,500 to about 5,500 cells / mmA2; diameter of about 7 to about 9.5 mm.

[0142] It is according to a few embodiments of the invention wherein the materials of the support layer are selected from a group consisting of Collagen, Collagen methacrylate, Gelatin, Gelatin from about 1 to about 50% w / v., whereas native stroma has 13% collagen.

[0143] It is according to a few embodiments of the invention wherein additional materials of the support layer are selected from a group consisting of photo-initiators and crosslinkers: LAP, Irgacure 2959, APS-TEMED, l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N- hydroxysuccinimide (NHS), and a combination thereof.

[0144] It is according to a few embodiments of the invention wherein additional materials of the support layer are selected from a group consisting of biocompatible dyes for easy handling such as trypan blue. It is according to a few embodiments of the invention wherein the materials of the cells layer are selected from a group consisting (i) human comeal endothelial cells from human donors, with or without expansion of the cells by a proliferation step; (ii) human comeal endothelial cells derived from stem cells such as induced pluripotent stem cells (iPSC) or human embryonic stem cells (hESC); (iii) cells own ECM, produced by the cells during the maturation process and any combination thereof.

[0145] It is according to a few embodiments of the invention wherein the preparation methods comprise drying step(s). Such a drying steps comprises steps of spreading or molding about 0.1 to about 15% (w / v) materials selected from collagen, collagen methacrylate, gelatin, and gelatin methacrylate on a surface in the casing in the array. The spread / molded layer thickness may vary between about 0.1 and about 10 mm. Then, the materials are let to dry at specific range of temperatures, between about 4 and 60 degrees Celsius, and relative humidity levels. Total drying period time is ranging from Ih to about 10 days. The drying process may be consisted of several different environmental condition steps.

[0146] It is according to a few embodiments of the invention wherein the preparation methods comprise crosslinking step(s). After drying, crosslinking the dried sheet by washing it with one or more of the following: EDC, NHS, LAP, Irgacure 2959 or other crosslinker solution, or a mixture of the above. Using photo-initiating light source is possible to induce the process. Crosslinking time between about 1 minute and about 48 hours. Afterwards, washing the crosslinked gel to remove residues of crosslinker and un-crosslinked polymer and immersing it in water / PBS / media. Then, possibly repeating the drying and crosslinking steps multiple times.

[0147] The present invention discloses a method of manufacturing an artificial endothelial keratoplasty graft, wherein the method consisting of a step of drying support layer material followed by a crosslinking step.

[0148] It is according to a few embodiments of the invention wherein the preparation methods comprise seeding (and / or printing) step(s). Seeding (and / or printing) comeal endothelial cells on top of the gel. Maturing for about 1 to about 28 days in an incubator.

[0149] The PVEK-implant of the present invention is very thin, but is still stiff enough to handle with standard ophthalmic tools. Also, native DMEK graft tend to fold due to the contraction of the cells on the Descemet membrane (or due to the elastin content of the Descemet membrane). This makes the implantation much more difficult as the surgeon has to flatten the graft in the eye. Here however, the membrane does not tend to fold and the graft opens more easily after insertion.

[0150] The PVEK-grafting by the present technology comprises, inter alia, step(s) of drying out a solution of collagen and / or gelatin on a surface, and crosslinking it with EDC / NHS solution, that bond the collagen and / or gelatin polymers and make it a scaffold. Since the crosslinking process occurs while the material is not fully wetted, the resulting scaffold is very thin and with high polymer concentration. These layers can be implanted in a similar way to DMEK, but have an advantage that they do not tend to fold after implantation like DMEK.

[0151] The present invention hence discloses, inter alia, an artificial endothelial keratoplasty graft consisting a support layer made of rehydrated crosslinked hydrogel and comeal endothelial cells on top or within said support layer.

[0152] The present invention also discloses an artificial endothelial keratoplasty graft as defined above, wherein the rehydrated crosslinked hydrogel is based on collagen or collagen methacrylate.

[0153] The present invention also discloses an artificial endothelial keratoplasty graft as defined in any of the above, wherein the source of said collagen or collagen methacrylate is human recombinant collagen, human skin source and any combination thereof.

[0154] The present invention also discloses an artificial endothelial keratoplasty graft as defined in any of the above, wherein the rehydrated crosslinked hydrogel is based on gelatin or gelatin methacrylate.

[0155] The present invention also discloses an artificial endothelial keratoplasty graft as defined in any of the above, wherein the total thickness of the graft is between 3 and 50 microns.

[0156] The present invention also discloses an artificial endothelial keratoplasty graft as defined in any of the above, wherein the total thickness of the graft is below 25 microns, therefore mimicking native DMEK graft.

[0157] The present invention also discloses an artificial endothelial keratoplasty graft as defined in any of the above, wherein the mechanical properties of said support layer allows positioning and flattening of the graft inside the anterior chamber during implantation.

[0158] The present invention also discloses an artificial endothelial keratoplasty graft as defined in any of the above, wherein the cells remodeled the said support layers to form comeal endothelium extra-cellular matrix, by a long maturation period or by additional nutrients to the cells.

[0159] The present invention further discloses a method of manufacturing an artificial endothelial keratoplasty graft, wherein the method consisting of a step of drying support layer material followed by a crosslinking step.

[0160] The present invention also discloses the method as defined above, wherein the support layer material is collagen or collagen methacrylate.

[0161] The present invention also discloses the method as defined in any of the above, wherein the source of said collagen or collagen methacrylate is human recombinant collagen, or human skin source.

[0162] The present invention also discloses the method as defined in any of the above, wherein the support layer material is gelatin or gelatin methacrylate.

[0163] The present invention also discloses the method as defined in any of the above, wherein the crosslinking step involves in introducing EDC and NHS molecules to the material.

[0164] The present invention also discloses the method as defined in any of the above, wherein the crosslinking step involves in introducing LAP or l-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy- 2-methyl-l -propane- 1 -one (a commercially available as Irgacure 2959 trademark by Ciba) molecules to the material and applying light on it.

[0165] The present invention also discloses the method as defined in any of the above, wherein the drying step is performed in controlled environmental conditions.

[0166] The present invention also discloses the method as defined in any of the above, wherein the final thickness of said graft is below 25 microns.

[0167] The present invention also discloses the method as defined in any of the above, wherein the mechanical properties of said graft allow pulling and pushing it during implantation, therefore allowing positioning and flattening inside the anterior chamber of the eye.

[0168] The present invention also discloses the method as defined in any of the above, wherein the water content in said support material is between 30 and 90 percent.

[0169] Thus, it is one object of the present invention to provide an array of at least one case, having a base sized and shaped for enclosing at least one type of liquid solution, wherein said at least one case is rotatable at a predetermined angular velocity around a main longitudinal axis, being substantially perpendicular to the horizonal plane, such rotatable movement of said at least one case results in drying of said liquid solution to provide a fdm for the generation of a scaffold support layer for artificial endothelial keratoplasty graft.

[0170] It is another object of the present invention to provide the array as defined above, wherein said at least one case comprising at least one cover for sealingly fitting said base.

[0171] It is another object of the present invention to provide the array as defined above, wherein said predetermined velocity is in the range of 200 to 1000 RPM.

[0172] It is another object of the present invention to provide the array as defined above, wherein said base is characterized by having a predetermined curvature (radius of curvature) to shape said artificial endothelial keratoplasty graft accordingly.

[0173] It is another object of the present invention to provide the array as defined above, wherein said radius of curvature of said at least one base of at least one of the cases is in the range of 10 mm to 17mm.

[0174] It is another object of the present invention to provide the array as defined above, wherein at least one selected from a group consisting of said base, said cover and any combination thereof comprising an envelope adapted to facilitate sealingly fitting said base and said cover.

[0175] It is another object of the present invention to provide the array as defined above, wherein said base of the case is made of hydrophobic material.

[0176] It is another object of the present invention to provide the array as defined above, wherein said hydrophobic material is selected from a group consisting of polypropylene, polystyrene, polycarbonate and any combination thereof.

[0177] It is another object of the present invention to provide the array as defined above, wherein said cover of said case is made of hydrophobic material.

[0178] It is another object of the present invention to provide the array as defined above, wherein said hydrophobic material is selected from a group consisting of polypropylene, polystyrene, polycarbonate and any combination thereof.

[0179] It is another object of the present invention to provide the array as defined above, wherein said envelope is made of hydrophobic material. It is another object of the present invention to provide the array as defined above, wherein said hydrophobic material is selected from a group consisting of polypropylene, polystyrene, polycarbonate and any combination thereof.

[0180] It is another object of the present invention to provide the array as defined above, comprising a plurality of cases.

[0181] It is another object of the present invention to provide the array as defined above, wherein each case is aligned with a neighboring case.

[0182] It is another object of the present invention to provide the array as defined above, additionally comprising at least one alignment element in communication with at least two neighboring cases.

[0183] It is another object of the present invention to provide the array as defined above, wherein said at least one alignment element is provided so as to facilitate substantially the same rotational velocity or angular velocity to all of said cases.

[0184] It is another object of the present invention to provide the array as defined above, wherein said at least one alignment element is at least one pin (also referred to as a common rotation axis) being in mechanical communication with said at least two neighboring cases.

[0185] It is another object of the present invention to provide the array as defined above, wherein said predetermined velocity of each of said at least one case is independently controlled.

[0186] It is another object of the present invention to provide the array as defined above, wherein said predetermined velocity of each of said at least one case is changeable as a function of time.

[0187] It is another object of the present invention to provide the array as defined above, wherein said case is hingly connected to the array.

[0188] It is another object of the present invention to provide the array as defined above, wherein said liquid solution comprising hydrogel.

[0189] It is another object of the present invention to provide the array as defined above, wherein said hydrogel is collagen solution comprising at least one selected from a group consisting of collagen or collagen methacrylate and any combination thereof.

[0190] It is another object of the present invention to provide the array as defined above, wherein hydrogel is gelatin or gelatin methacrylate. It is another object of the present invention to provide the array as defined above, wherein the source of said collagen or collagen methacrylate is human recombinant collagen, human skin source and any combination thereof.

[0191] It is another object of the present invention to provide the array as defined above, wherein the hydrogel is based on gelatin or gelatin methacrylate.

[0192] It is another object of the present invention to provide the array as defined above, wherein the total thickness of the graft is between 3 and 50 microns.

[0193] It is another object of the present invention to provide the array as defined above, wherein the total thickness of the graft is below 25 microns, therefore mimicking native DMEK graft.

[0194] It is another object of the present invention to provide the array as defined above, wherein the mechanical properties of said support layer allows positioning and flattening of the graft inside the anterior chamber during implantation.

[0195] It is another object of the present invention to provide the array as defined above, wherein said cells remodeled the said support layers to form comeal endothelium extra-cellular matrix, by a long maturation period or by additional nutrients to the cells.

[0196] It is another object of the present invention to provide the array as defined above, additionally comprising introduction of EDC and NHS molecules to crosslink said film to thereby result in said scaffold support layer for artificial endothelial keratoplasty graft.

[0197] It is another object of the present invention to provide the array as defined above, wherein said introduction of EDC and NHS is obtained within said at least one case or outside thereof.

[0198] It is another object of the present invention to provide a matrix of x arrays, each comprising at least one case, each case having a base sized and shaped for enclosing at least one type of liquid solution, wherein said at least one case is rotatable at a predetermined velocity around a main longitudinal axis, being substantially perpendicular to the horizonal plane, such rotatable movement of said at least one case results in drying of said liquid solution to provide a film for the generation of a scaffold support layer for artificial endothelial keratoplasty graft; where x is an integer being equals to or greater than 0.

[0199] It is another object of the present invention to provide the matrix as defined above, wherein at least one of said cases comprising at least one cover for sealingly fitting said base.

[0200] It is another object of the present invention to provide the matrix as defined above, wherein said predetermined velocity is in the range 200 to 1000 RPM. It is another object of the present invention to provide the matrix as defined above, wherein said case is hingly connected to the array

[0201] It is another object of the present invention to provide the matrix as defined above, wherein at least one of said bases is characterized by having a predetermined curvature to shape said artificial endothelial keratoplasty graft accordingly.

[0202] It is another object of the present invention to provide the matrix as defined above, wherein said radius of curvature of said at least one base of at least one of the cases is in the range of 10mm to 17mm.

[0203] It is another object of the present invention to provide the matrix as defined above, wherein at least one selected from a group consisting of said base, said cover and any combination thereof comprising an envelope adapted to facilitate sealingly fitting said base and said cover.

[0204] It is another object of the present invention to provide the matrix as defined above, wherein said base of the case is made of hydrophobic material.

[0205] It is another object of the present invention to provide the matrix as defined above, wherein said material is selected from a group consisting of polypropylene, polystyrene, polycarbonate and any combination thereof.

[0206] It is another object of the present invention to provide the matrix as defined above, wherein said cover of said case is made of hydrophobic material.

[0207] It is another object of the present invention to provide the matrix as defined above, wherein said material is selected from a group consisting of polypropylene, polystyrene, polycarbonate and any combination thereof.

[0208] It is another object of the present invention to provide the matrix as defined above, wherein said envelope is made of hydrophobic material.

[0209] It is another object of the present invention to provide the matrix as defined above, wherein said material is selected from a group consisting of polypropylene, polystyrene, polycarbonate and any combination thereof.

[0210] It is another object of the present invention to provide the matrix as defined above, wherein at least one of said arrays comprising a plurality of cases.

[0211] It is another object of the present invention to provide the matrix as defined above, wherein each case is aligned with a neighboring case. It is another object of the present invention to provide the matrix as defined above, additionally comprising at least one alignment element in communication with at least two neighboring cases.

[0212] It is another object of the present invention to provide the matrix as defined above, wherein said at least one alignment element is provided so as to facilitate substantially the same rotational velocity or angular velocity to all of said cases.

[0213] It is another object of the present invention to provide the matrix as defined above, wherein said at least one alignment element is at least one pin (also referred to as a common rotation axis).

[0214] It is another object of the present invention to provide the matrix as defined above, wherein said predetermined velocity of each of said at least one case is independently controlled.

[0215] It is another object of the present invention to provide the matrix as defined above, wherein said predetermined velocity of each of said at least one case is changeable as a function of time.

[0216] It is another object of the present invention to provide the matrix as defined above, wherein said liquid solution comprising hydrogel.

[0217] It is another object of the present invention to provide the matrix as defined above, wherein said hydrogel is collagen solution comprising at least one selected from a group consisting of collagen or collagen methacrylate and any combination thereof.

[0218] It is another object of the present invention to provide the matrix as defined above, wherein hydrogel is gelatin or gelatin methacrylate.

[0219] It is another object of the present invention to provide the matrix as defined above, wherein the source of said collagen or collagen methacrylate is human recombinant collagen, human skin source and any combination thereof.

[0220] It is another object of the present invention to provide the matrix as defined above, wherein the hydrogel is based on gelatin or gelatin methacrylate.

[0221] It is another object of the present invention to provide the matrix as defined above, wherein the total thickness of the graft is between 3 and 50 microns.

[0222] It is another object of the present invention to provide the matrix as defined above, wherein the total thickness of the graft is below 25 microns, therefore mimicking native DMEK graft. It is another object of the present invention to provide the matrix as defined above, wherein the mechanical properties of said support layer allows positioning and flattening of the graft inside the anterior chamber during implantation.

[0223] It is another object of the present invention to provide the matrix as defined above, wherein said cells remodeled the said support layers to form comeal endothelium extra-cellular matrix, by a long maturation period or by additional nutrients to the cells.

[0224] It is another object of the present invention to provide the matrix as defined above, additionally comprising introduction of EDC and NHS molecules to crosslink said film to thereby result in said scaffold support layer for artificial endothelial keratoplasty graft.

[0225] It is another object of the present invention to provide the matrix as defined above, wherein said introduction of EDC and NHS is obtained within said at least one case or outside thereof.

[0226] It is another object of the present invention to provide a method of drying at least one hydrogel support layer for artificial endothelial keratoplasty graft, wherein said method consisting steps of: a. providing an array of at least one case, having a base sized and shaped for enclosing at least one type of liquid solution, wherein said at least one case is rotatable at a predetermined velocity around a main longitudinal axis, being substantially perpendicular to the horizonal plane; b. placing at least one type of liquid solution in at least one said cases; c. rotating said at least one case at said predetermined velocity around said main longitudinal axis thereby drying said liquid solution to provide a film for the generation of a scaffold support layer for artificial endothelial keratoplasty graft.

[0227] It is another object of the present invention to provide the method as defined above, wherein said at least one case comprising at least one cover for sealingly fitting said base.

[0228] It is another object of the present invention to provide the method as defined above, wherein said predetermined velocity is in the range of 200 to 1000 RPM.

[0229] It is another object of the present invention to provide the method as defined above, wherein said base is characterized by having a predetermined curvature to shape said artificial endothelial keratoplasty graft accordingly. It is another object of the present invention to provide the method as defined above, wherein said radius of curvature of said at least one base of at least one of the cases is in the range of 10mm to 17mm.

[0230] It is another object of the present invention to provide the method as defined above, wherein at least one selected from a group consisting of said base, said cover and any combination thereof comprising an envelope adapted to facilitate sealingly fitting said base and said cover.

[0231] It is another object of the present invention to provide the method as defined above, wherein said base of the case is made of hydrophobic material.

[0232] It is another object of the present invention to provide the method as defined above, wherein said material is selected from a group consisting of polypropylene, polystyrene, polycarbonate and any combination thereof.

[0233] It is another object of the present invention to provide the method as defined above, wherein said cover of said case is made of hydrophobic material.

[0234] It is another object of the present invention to provide the method as defined above, wherein said material is selected from a group consisting of polypropylene, polystyrene, polycarbonate and any combination thereof.

[0235] It is another object of the present invention to provide the method as defined above, wherein said envelope is made of hydrophobic material.

[0236] It is another object of the present invention to provide the method as defined above, wherein said material is selected from a group consisting of polypropylene, polystyrene, polycarbonate and any combination thereof.

[0237] It is another object of the present invention to provide the method as defined above, comprising a plurality of cases.

[0238] It is another object of the present invention to provide the method as defined above, wherein each case is aligned with a neighboring case.

[0239] It is another object of the present invention to provide the method as defined above, additionally comprising at least one alignment element in communication with at least two neighboring cases. It is another object of the present invention to provide the method as defined above, wherein said at least one alignment element is provided so as to facilitate substantially the same rotational velocity or angular velocity to all of said cases.

[0240] It is another object of the present invention to provide the method as defined above, wherein said at least one alignment element is at least one pin (also referred to as a common rotation axis).

[0241] It is another object of the present invention to provide the method as defined above, wherein said predetermined velocity of each of said at least one case is independently controlled.

[0242] It is another object of the present invention to provide the method as defined above, wherein said predetermined velocity of each of said at least one case is changeable as a function of time.

[0243] It is another object of the present invention to provide the method as defined above, wherein said hydrogel is collagen solution comprising at least one selected from a group consisting of collagen or collagen methacrylate and any combination thereof.

[0244] It is another object of the present invention to provide the method as defined above, wherein hydrogel is gelatin or gelatin methacrylate.

[0245] It is another object of the present invention to provide the method as defined above, wherein the source of said collagen or collagen methacrylate is human recombinant collagen, human skin source and any combination thereof.

[0246] It is another object of the present invention to provide the method as defined above, wherein the hydrogel is based on gelatin or gelatin methacrylate.

[0247] It is another object of the present invention to provide the method as defined above, wherein the total thickness of the graft is between 3 and 50 microns.

[0248] It is another object of the present invention to provide the method as defined above, wherein the total thickness of the graft is below 25 microns, therefore mimicking native DMEK graft.

[0249] It is another object of the present invention to provide the method as defined above, wherein the mechanical properties of said support layer allows positioning and flattening of the graft inside the anterior chamber during implantation.

[0250] It is another object of the present invention to provide the method as defined above, wherein said cells remodeled the said support layers to form comeal endothelium extra-cellular matrix, by a long maturation period or by additional nutrients to the cells. It is another object of the present invention to provide the method as defined above, additionally comprising a crosslinking step.

[0251] It is another object of the present invention to provide the method as defined above, wherein the crosslinking step involves in introducing EDC and NHS molecules to the material.

[0252] It is another object of the present invention to provide the method as defined above, wherein said crosslinking step involves in introducing LAP or Irgacure 2959 molecules to the material and applying light on it.

[0253] It is another object of the present invention to provide the method as defined above, wherein said drying step is performed in controlled environmental conditions.

[0254] It is another object of the present invention to provide a method of manufacturing an artificial endothelial keratoplasty graft, wherein said method consisting of a step of drying a support layer material according to any of the defined above followed by a crosslinking step.

[0255] All references cited throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application.

[0256] The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. The specific embodiments provided herein are examples of useful embodiments of the present invention and it will be apparent to one skilled in the art that the present invention may be carried out using a large number of variations of the devices, device components, and method steps set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods can include a large number of optional composition and processing elements and steps. When a group of substituents is disclosed herein, it is understood that all individual members of that group and all subgroups, including any isomers, enantiomers, and diastereomers of the group members, are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and sub-combinations possible of the group are intended to be individually included in the disclosure. When a compound is described herein such that a particular isomer, enantiomer or diastereomer of the compound is not specified, for example, in a formula or in a chemical name, that description is intended to include each isomer and enantiomer of the compound described individually or in any combination. Additionally, unless otherwise specified, all isotopic variants of compounds disclosed herein are intended to be encompassed by the disclosure. For example, it will be understood that any one or more hydrogens in a molecule disclosed can be replaced with deuterium or tritium. Isotopic variants of a molecule are generally useful as standards in assays for the molecule and in chemical and biological research related to the molecule or its use. Methods for making such isotopic variants are known in the art. Specific names of compounds are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same compounds differently.

[0257] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. As well, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. It is also to be noted that the terms “comprising”, “including”, and “having” can be used interchangeably. The expression “of any of claims XX- YY” (wherein XX and YY refer to claim numbers) is intended to provide a multiple dependent claim in the alternative form, and in some embodiments is interchangeable with the expression “as in any one of claims XX-YY.”

[0258] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0259] Whenever a range is given in the specification, for example, a range of integers, a temperature range, a time range, a composition range, or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. As used herein, ranges specifically include the values provided as endpoint values of the range. As used herein, ranges specifically include all the integer values of the range. For example, a range of 1 to 100 specifically includes the end point values of 1 and 100. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the claims herein. The term “about” refers to any value being lower or greater than 20% of the defined measure.

[0260] As used herein, “comprising” is synonymous and can be used interchangeably with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of’ excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms “comprising”, “consisting essentially of’ and “consisting of’ can be replaced with either of the other two terms. The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

[0261] One of ordinary skill in the art will appreciate that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Claims

CLAIMS1. An array of at least one case, each case having a base sized and shaped for enclosing at least one type of liquid solution, wherein said at least one case is rotatable at a predetermined angular velocity around a main longitudinal axis, being substantially perpendicular to the horizonal plane, such rotatable movement of said at least one case results in drying of said liquid solution to provide a film for the generation of a scaffold support layer for artificial endothelial keratoplasty graft.

2. The array of claim 1, wherein said at least one case comprising at least one cover for sealingly fitting said base.

3. The array of claim 1, wherein said predetermined velocity is in the range of 200 to 1000 RPM.

4. The array of claim 1 , wherein said base is characterized by having a predetermined radius of curvature to shape said artificial endothelial keratoplasty graft accordingly.

5. The array of claim 1, wherein said radius of curvature of said at least one base of at least one of the cases is in the range of 10 mm to 17mm.

6. The array of claim 1, wherein at least one selected from a group consisting of said base, said cover and any combination thereof comprising an envelope adapted to facilitate sealingly fitting said base and said cover.

7. The array of any one of claims 1-6, wherein said base of the case is made of hydrophobic material.

8. The array of any one of claims 1-7, wherein said hydrophobic material is selected from a group consisting of polypropylene, polystyrene, polycarbonate and any combination thereof.

9. The array of any one of claims 1-8, wherein said cover of said case is made of hydrophobic material.

10. The array of any one of claims 1-9, wherein said hydrophobic material is selected from a group consisting of polypropylene, polystyrene, polycarbonate and any combination thereof.

11. The array of any one of claims 1-10, wherein said envelope is made of hydrophobic material.

12. The array of any one of claims 1-11, wherein said hydrophobic material is selected from a group consisting of polypropylene, polystyrene, polycarbonate and any combination thereof.

13. The array of claim 1, comprising a plurality of cases.

14. The array of claim 13, wherein each case is aligned with a neighboring case.

15. The array of claim 14, additionally comprising at least one alignment element in communication with at least two neighboring cases.

16. The array of claim 15, wherein said at least one alignment element is provided so as to facilitate substantially the same rotational velocity or angular velocity to all of said cases.

17. The array of claim 15, wherein said at least one alignment element is at least one pin, acting as a common rotation axis, being in mechanical communication with said at least two neighboring cases.

18. The array of any one of claims 1-17, wherein said predetermined velocity of each of said at least one case is independently controlled.

19. The array of any one of claims 1-18, wherein said predetermined velocity of each of said at least one case is changeable as a function of time.

20. The array of any one of claims 1-19, wherein said case is hingly connected to the array.

21. The array of any one of claims 1-20, wherein said liquid solution comprising hydrogel.

22. The array of any one of claims 1-21, wherein said hydrogel is collagen solution comprising at least one selected from a group consisting of collagen or collagen methacrylate and any combination thereof.

23. The array of any one of claims 21 -22, wherein hydrogel is gelatin or gelatin methacrylate .

24. The array of claims 21-23, wherein the source of said collagen or collagen methacrylate is human recombinant collagen, human skin source and any combination thereof.

25. The array of any one of claims 21-24, wherein the hydrogel is based on gelatin or gelatin methacrylate.

26. The array any one of claims 21-251, wherein the total thickness of the graft is between 3 and 50 microns.

27. The array of claims 21-26, wherein the total thickness of the graft is below 25 microns, therefore mimicking native DMEK graft.

28. The array of any one of claims 21-27, wherein the mechanical properties of said support layer allows positioning and flattening of the graft inside the anterior chamber during implantation.

29. The array of any one of claims 21-28, wherein said cells remodeled the said support layers to form comeal endothelium extra-cellular matrix, by a long maturation period or by additional nutrients to the cells.

30. The array of any one of claims 1-29, additionally comprising introduction of EDC and NHS molecules to crosslink said film to thereby result in said scaffold support layer for artificial endothelial keratoplasty graft.

31. The array of claim 30, wherein said introduction of EDC and NHS is obtained within said at least one case or outside thereof.

32. A matrix of x arrays, each comprising at least one case, each case having a base sized and shaped for enclosing at least one type of liquid solution, wherein said at least one case is rotatable at a predetermined velocity around a main longitudinal axis, being substantially perpendicular to the horizonal plane, such rotatable movement of said at least one case results in drying of said liquid solution to provide a film for the generation of a scaffold support layer for artificial endothelial keratoplasty graft; where x is an integer being equals to or greater than 0.

33. The matrix of claim 21, wherein at least one of said cases comprising at least one cover for sealingly fitting said base.

34. The matrix of claim 32, wherein said predetermined velocity is in the range 200 to 1000 RPM.

35. The matrix of any one of claims 32-34, wherein said case is hingly connected to the array36. The matrix of claim 32, wherein at least one of said bases is characterized by having a predetermined radius of curvature to shape said artificial endothelial keratoplasty graft accordingly.

37. The matrix of claim 32, wherein said radius of curvature of said at least one base of at least one of the cases is in the range of 10mm to 17mm.

38. The matrix of claim 32, wherein at least one selected from a group consisting of said base, said cover and any combination thereof comprising an envelope adapted to facilitate sealingly fitting said base and said cover.

39. The matrix of any one of claims 32-38, wherein said base of the case is made of hydrophobic material.

40. The matrix of any one of claims 32-39, wherein said material is selected from a group consisting of polypropylene, polystyrene, polycarbonate and any combination thereof.

41. The matrix of any one of claims 32-40, wherein said cover of said case is made of hydrophobic material.

42. The matrix of any one of claims 32-41, wherein said material is selected from a group consisting of polypropylene, polystyrene, polycarbonate and any combination thereof.

43. The matrix of any one of claims 32-42, wherein said envelope is made of hydrophobic material.

44. The matrix of any one of claims 32-43, wherein said material is selected from a group consisting of polypropylene, polystyrene, polycarbonate and any combination thereof.

45. The matrix of claim 44, wherein at least one of said arrays comprising a plurality of cases.

46. The matrix of claim 45, wherein each case is aligned with a neighboring case.

47. The matrix of claim 46, additionally comprising at least one alignment element in communication with at least two neighboring cases.

48. The matrix of claim 47, wherein said at least one alignment element is provided so as to facilitate substantially the same rotational velocity or angular velocity to all of said cases.

49. The matrix of claim 47, wherein said at least one alignment element is at least one pin, acting as a common rotation axis.

50. The matrix of any one of claims 32-49, wherein said predetermined velocity of each of said at least one case is independently controlled.

51. The matrix of any one of claims 32-50, wherein said predetermined velocity of each of said at least one case is changeable as a function of time.

52. The matrix of any one of claims 32-51, wherein said liquid solution comprising hydrogel.

53. The matrix of any one of claims 32-52, wherein said hydrogel is collagen solution comprising at least one selected from a group consisting of collagen or collagen methacrylate and any combination thereof.

54. The matrix of claims 52, wherein hydrogel is gelatin or gelatin methacrylate.

55. The matrix of any one of claims 52-54, wherein the source of said collagen or collagen methacrylate is human recombinant collagen, human skin source and any combination thereof.

56. The matrix of any one of claims 52-55, wherein the hydrogel is based on gelatin or gelatin methacrylate.

57. The matrix of any one of claims 52-56, wherein the total thickness of the graft is between 3 and 50 microns.

58. The matrix of any one of claims 52-57, wherein the total thickness of the graft is below 25 microns, therefore mimicking native DMEK graft.

59. The matrix of any one of claims 52-58, wherein the mechanical properties of said support layer allows positioning and flattening of the graft inside the anterior chamber during implantation.

60. The matrix of any one of claims 52-59, wherein said cells remodeled the said support layers to form comeal endothelium extra-cellular matrix, by a long maturation period or by additional nutrients to the cells.

61. The matrix of any one of claims 52-60, additionally comprising introduction of EDC and NHS molecules to crosslink said film to thereby result in said scaffold support layer for artificial endothelial keratoplasty graft.

62. The matrix of any one of claims 52-61, wherein said introduction of EDC and NHS is obtained within said at least one case or outside thereof.

63. A method of drying at least one hydrogel support layer for artificial endothelial keratoplasty graft, wherein said method consisting steps of: a. providing an array of at least one case, having a base sized and shaped for enclosing at least one type of liquid solution, wherein said at least one case is rotatable at a predetermined velocity around a main longitudinal axis, being substantially perpendicular to the horizonal plane; b. placing at least one type of liquid solution in at least one said cases; c. rotating said at least one case at said predetermined velocity around said main longitudinal axis thereby drying said liquid solution to provide a film for the generation of a scaffold support layer for artificial endothelial keratoplasty graft.

64. The method of claim 61, wherein said at least one case comprising at least one cover for sealingly fitting said base.

65. The method of claim 61, wherein said predetermined velocity is in the range of 200 to 1000 RPM.

66. The method of claim 61, wherein said base is characterized by having a predetermined radius of curvature to shape said artificial endothelial keratoplasty graft accordingly.

67. The method of claim 61, wherein said radius of curvature of said at least one base of at least one of the cases is in the range of 10mm to 17mm.

68. The method of claim 61, wherein at least one selected from a group consisting of said base, said cover and any combination thereof comprising an envelope adapted to facilitate sealingly fitting said base and said cover.

69. The method of any one of claims 61-66, wherein said base of the case is made of hydrophobic material.

70. The method of any one of claims 61-67, wherein said material is selected from a group consisting of polypropylene, polystyrene, polycarbonate and any combination thereof.

71. The method of any one of claims 61-68, wherein said cover of said case is made of hydrophobic material.

72. The method of any one of claims 61-69, wherein said material is selected from a group consisting of polypropylene, polystyrene, polycarbonate and any combination thereof.

73. The method of any one of claims 61-70, wherein said envelope is made of hydrophobic material.

74. The method of any one of claims 61-71, wherein said material is selected from a group consisting of polypropylene, polystyrene, polycarbonate and any combination thereof.

75. The method of claim 61, comprising a plurality of cases.

76. The method of claim 73, wherein each case is aligned with a neighboring case.

77. The method of claim 74, additionally comprising at least one alignment element in communication with at least two neighboring cases.

78. The method of claim 75, wherein said at least one alignment element is provided so as to facilitate substantially the same rotational velocity or angular velocity to all of said cases.

79. The method of claim 75, wherein said at least one alignment element is at least one pin, acting as a common rotation axis.

80. The method of any one of claims 61-77, wherein said predetermined velocity of each of said at least one case is independently controlled.

81. The method of any one of claims 61-78, wherein said predetermined velocity of each of said at least one case is changeable as a function of time.

82. The method of any one of claims 61-79, wherein said hydrogel is collagen solution comprising at least one selected from a group consisting of collagen or collagen methacrylate and any combination thereof.

83. The method of claim 61, wherein hydrogel is gelatin or gelatin methacrylate.

84. The method of claim 81, wherein the source of said collagen or collagen methacrylate is human recombinant collagen, human skin source and any combination thereof.

85. The method of claim 81, wherein the hydrogel is based on gelatin or gelatin methacrylate.

86. The method of claim 61, wherein the total thickness of the graft is between 3 and 50 microns.

87. The method of claim 61, wherein the total thickness of the graft is below 25 microns, therefore mimicking native DMEK graft.

88. The method of claim 61, wherein the mechanical properties of said support layer allows positioning and flattening of the graft inside the anterior chamber during implantation.

89. The method of claim 61, wherein said cells remodeled the said support layers to form comeal endothelium extra-cellular matrix, by a long maturation period or by additional nutrients to the cells.

90. The method of claim 61, additionally comprising a crosslinking step.

91. The method of claim 88, wherein the crosslinking step involves in introducing EDC and NHS molecules to the material.

92. The method of claim 88, wherein said crosslinking step involves in introducing LAP or Irgacure 2959 molecules to the material and applying light on it.

93. The method of claim 61, wherein said drying step is performed in controlled environmental conditions.

94. A method of manufacturing an artificial endothelial keratoplasty graft, wherein said method consisting of a step of drying a support layer material according to any of the claims 61-91 followed by a crosslinking step.

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