Biosynthetic corneal tissue, production method and use of same

Abstract

The present invention relates to a biosynthetic corneal tissue formed by a single layer of corneal endothelial cells cultured on an ultrathin collagen membrane. The primary purpose of the invention is to provide an efficient alternative to conventional corneal endothelial transplants, which suffer limitations such as corneal tissue availability, risk of rejection and complications associated with the transplant. The invention therefore represents an effective solution to overcome the technical and biological limitations associated with corneal transplants. The corneal tissue of the invention improves tissue regeneration, controls water absorption and graft degradation, and ensures long-term functionality, significantly improving the quality of life of patients needing corneal transplants.

Description

[0001] BIOSYNTHETIC CORNEAL TISSUE, METHOD OF MANUFACTURING AND USE THEREOF

[0002] Field of Invention

[0003] The present invention relates, in general, to a biosynthetic corneal tissue that is an alternative to corneal endothelium transplants, formed from biosynthetic materials and tissue engineering techniques applied to corneal regeneration, with a particular focus on optimizing the use of available corneal tissue, as well as including the manufacture of ultrathin collagen membranes as a support for the culture of corneal endothelial cells, as well as with methods of transplanting regenerated tissue using standard medical devices, such as corneal transplant microinjectors.

[0004] Background of the Invention

[0005] Currently, diseases and injuries affecting the cornea are among the leading causes of vision loss worldwide. The cornea, being the transparent outer layer of the eye, plays a crucial role in refracting light and protecting the internal ocular structures, and it is widely known that any damage or deterioration severely compromises a person's visual capacity, significantly impacting their quality of life.Among the most common conditions requiring specialized medical intervention for the cornea are corneal dystrophy, a hereditary condition that causes the accumulation of material in the layers of the cornea, affecting its transparency and visual function; corneal edema, characterized by the accumulation of fluid in the cornea, which causes inflammation, blurred vision, and, in severe cases, pain; keratopathies, which include a wide spectrum of degenerative or inflammatory diseases that alter the corneal structure; and damage caused by trauma or infection, which can severely compromise the integrity and function of this delicate ocular structure. If left untreated, these conditions can progress to significant vision loss or even blindness.

[0006] The standard treatment for advanced corneal diseases is corneal transplantation, a surgical procedure that involves replacing the damaged cornea with healthy tissue from a human donor. This method, widely recognized and practiced in ophthalmology, has proven effective in restoring vision in numerous cases. However, its success depends heavily on the availability of donated human corneal tissue, which is currently a critical limiting factor. Moreover, this approach faces a critical barrier: the global shortage of corneal tissue for donation. According to recent data, the demand for corneas far exceeds the supply, resulting in long waiting lists and leaving thousands of patients without access to timely treatment. This problem is especially evident in regions with underdeveloped donation systems or a high prevalence of corneal diseases.The global shortage of corneal tissue for donation is one of the biggest obstacles to timely access to this treatment. Despite progress in awareness campaigns and the promotion of tissue donation, the supply of corneas remains insufficient to meet existing demand. According to recent estimates, only a small percentage of people who need a corneal transplant manage to access the procedure, leaving thousands of patients on long waiting lists. This situation not only prolongs the suffering of those who require a transplant but also exacerbates visual impairment, reducing their chances of future functional recovery.

[0007] This problem is even more evident in regions where tissue donation systems are underdeveloped. In many countries, a lack of adequate infrastructure, inconsistent regulations, and cultural or religious barriers hinder tissue donation, resulting in an extremely limited availability of corneas. Furthermore, in areas with a high prevalence of corneal diseases, such as certain tropical regions where eye infections are more common, demand far exceeds any available supply, exacerbating the crisis.

[0008] Consequently, the exclusive reliance on donated human corneal tissue for the treatment of corneal diseases represents an unsustainable barrier in the current context. This underscores the need to explore and develop alternative solutions that can alleviate the burden on the donation system and ensure that more patients have access to effective and timely treatment.

[0009] In response to this situation, scientific research has sought alternatives through the development of synthetic or artificial corneal tissues; such is the case of the scientific article entitled “Regeneration of corneal epithelium utilizing a collagen vitrigel membrane in rabbit models for corneal stromal wound and limbal stem cell deficiency” by J. JEREMY CHAE, ET. AL., published on December 11, 2014, which refers to the use of a collagen vitrigel membrane (CVM) to regenerate the corneal epithelium in rabbit models that present wounds in the corneal stroma and stem cell deficiency in the limbal area.

[0010] Specifically, the aforementioned article employs a collagen matrix that functions as a scaffold for epithelial growth, designed to be applied via fibrin glue. This approach was evaluated in two models: one for corneal stromal injuries and another for limbal stem cell deficiency (LSCD). In the first model, the article uses three experimental groups: one treated with a collagen matrix scaffold (CVM) and fibrin glue (CVM + FG), another with fibrin glue alone, and a control group without treatment. Regeneration was assessed using macroscopic observations and techniques such as immunohistochemistry and electron microscopy. The results suggest that in the group treated with CVM and fibrin glue, the corneal epithelium showed adequate regeneration without signs of hypertrophy, unlike the group treated with fibrin glue alone, which exhibited undesirable epithelial differentiation.

[0011] In the LSCD model, the aforementioned article describes a method in which a CVM membrane containing human limbal epithelial cells (hLECs) was applied to one group, while a control group received no treatment. Evaluation of these models indicated that the CVM-treated group exhibited a transparent corneal surface, low inflammation, and minimal neovascularization compared to the control group, which showed corneal opacity and significant inflammation. The article documents that the CVM is a suitable scaffold for epithelial differentiation and prevents hypertrophy, in addition to serving as a substrate for limbal epithelial cell placement without apparent complications.

[0012] This regeneration device is configured to prevent inflammation and promote transparency in the regenerated tissue, being applied mainly in situations of stromal injury and in conditions of limbal cell deficiency, acting both as a physical barrier and a cellular support structure for the growth of the corneal epithelium, and in general, the mentioned article seeks to evaluate the potential of a collagen-based membrane, called collagen vitrigel (CV), for the reconstruction of the corneal epithelium in stromal injury and limbal stem cell deficiency (LSCD) models.

[0013] Also known in the literature is Japanese patent application No. JP2024060011A, published on May 1, 2024, which describes a reinforced biopolymer. This biopolymer has a structure composed of a synthetic support membrane and biopolymers arranged to allow cell growth and adhesion. The material configuration according to application JP2024060011A includes at least two layers of biopolymer, positioned on either side of the synthetic membrane, or in some cases, a single layer of biopolymer partially impregnated into the membrane. This design aims to make the biopolymer suitable for supporting a cell monolayer, potentially enabling its use in cell therapy applications, such as those requiring stem cells, epithelial cells, or endothelial cells.

[0014] In general, the JP'O11 application presents a reinforced biopolymer with technical characteristics that include the ability to support cells, mechanical properties to resist manipulation and damage, and a structure that allows control over the distribution of the biopolymer layers, giving it versatility in biotechnological applications, such as medical devices or therapeutic systems.

[0015] Additionally, the reinforced biopolymer of application JP'O11 has mechanical properties that give it high optical transparency and resistance to deformation, characteristics that make it suitable for applications where the manipulation and transport of cells must be carried out without compromising the structure of the material, in addition to being resistant to tearing or rupture during implantation and after insertion into a biological environment.

[0016] The synthetic support membrane in the invention of application JP'O11 is biologically inert, making it compatible with the biological environment where it is implanted, ensuring that it does not react adversely once implanted; likewise, application JP'O11 addresses the need for the biopolymer to allow bulk transport, which is essential to ensure that nutrients and other necessary elements can reach the attached cells without obstruction.

[0017] Regarding its structure, the reinforced biopolymer of application JP'O11 can vary in the thickness ratio between the biopolymer layers on each side of the synthetic membrane, seeking to customize its behavior depending on the specific needs of the application, which, according to application JP'O11, facilitates the use of the biopolymer not only as a structural support for cells, but also in the targeted delivery of cell therapies to specific sites of the body, such as those required in cell regeneration procedures or in the treatment of certain types of diseases.

[0018] Additionally, within the prior art, European patent No. 2755598B1, published on July 23, 2014, is known, whose teachings are directed to the manufacture of a gelatin hydrogel sheet, specifically designed for use in biomedical applications; the hydrogel of patent EP'598 is composed mainly of a collagen-derived biopolymer, which provides a base for the cultivation of corneal endothelial cells.

[0019] The hydrogel sheet described in patent EP'598 features a flexible and transparent structure, which seeks to improve compatibility with corneal endothelial cells, allowing these cells to adhere and proliferate on its surface.

[0020] Furthermore, it is observed that the objective of patent EP'598 is to create a scaffold that facilitates the cultivation of corneal endothelial cells for the treatment of eye diseases, such as corneal endothelial dystrophy, said scaffold being a mechanical support to the cultured cells and, in turn, being a graft material in the cornea.

[0021] Specifically, the corneal endothelial cell culture described in patent EP'598 is performed on a hydrogel sheet in a monolayer arrangement, which emulates the natural behavior and function of cells in the cornea. This aims to facilitate the maintenance of corneal transparency and controlled cell proliferation. Furthermore, the collagen biopolymer described in patent EP'598 promotes cell adhesion and proliferation by serving as a three-dimensional scaffold for the corneal endothelial cell culture.

[0022] Another well-known document is US patent application No. US20070092550A1, published on April 26, 2007, which discloses methods and compositions for growing human corneal endothelial cells on biopolymers, in order to produce artificial cornea transplants.

[0023] In particular, it is noted that patent application IIS'550 describes how a monolayer of endothelial cells is grown on the surface of a biopolymer support, developing an artificial transplant structure with functional characteristics similar to a human cornea, especially in terms of its transparency and hydration regulation, essential aspects for vision.

[0024] The cultivation process detailed in patent application IIS'550 includes the application of proteins and growth factors, such as fibronectin, laminin, type IV collagen, and growth factors like bFGF, which are conjugated with compatible polymers such as polyacarbophil. These agents aim to improve cell adhesion and facilitate the orderly growth of endothelial cells on the biopolymer surface, promoting the creation of a cornea that simulates the biological and mechanical structure of human tissue, with the potential for integrating this artificial cornea into transplant procedures.

[0025] Additionally, patent application IIS'550 addresses both the materials and culture conditions necessary to achieve adequate cell growth on the biopolymer matrix. The invention describes the desired properties of the matrix, which must possess sufficient flexibility and transparency to replicate the functionality of corneal tissue. Creating this structure also involves considering mechanical and biological factors that allow the transplant to respond similarly to the human cornea, particularly in terms of intraocular pressure response and light transmission.

[0026] Additionally, patent application US'550 mentions the use of specific biopolymer matrices that, when integrated with cell growth agents, facilitate the creation of a viable artificial cornea for transplantation in humans, where the procedure is aimed at patients who have severe damage to the cornea and require an intervention with artificial tissue that simulates, as far as possible, the natural behavior of corneal tissue.

[0027] Finally, patent application US'550 explores the feasibility of using these biopolymers in a clinical context, highlighting the stability and biocompatibility of these materials when serving as a basis for the growth of endothelial cells, offering an alternative for corneal replacement by structuring ophthalmic tissues from human cells.

[0028] For its part, US patent No. US10052411B2, published on September 16, 2010, discloses a method for treating diseases related to the cornea, specifically those affecting the corneal endothelium, using an endothelial cell preparation. The treatment according to patent IIS'411 focuses on cell regeneration within the cornea through a process in which endothelial cells are cultured on a biocompatible substrate, which in some cases may be collagen, facilitating their growth and subsequent application in the anterior chamber of the eye.

[0029] The process described in US patent 411 includes the preparation of a substrate that provides a suitable environment for endothelial cells to grow and adhere in a monolayer structure. This substrate is a biocompatible material, either flexible or rigid, depending on the treatment requirements and the characteristics of the cells to be cultured. This endothelial cell preparation is placed in the anterior chamber of the eye to allow for the regeneration of lost or damaged cells. The invention is notable for the use of a medium that maintains the viability of the endothelial cells until they are needed for treatment.

[0030] The invention also indicates that this treatment offers an alternative to traditional corneal transplants, eliminating the need to resort to a full graft to resolve problems arising from corneal endothelium dysfunction.

[0031] In particular, the present invention uses endothelial cells from a donor tissue (in particular, from cadaveric donors), with a substantial and advantageous reduction of risks associated with graft rejection, as well as post-surgical complications.

[0032] In terms of application methods, US patent 411 details the conditions under which cultured endothelial cells should be applied, such as the number of cells required, the exposure time, and the appropriate surgical techniques to achieve more suitable integration.

[0033] Additionally, within the prior art, there is known US patent application No. US20080050423A1, published on February 28, 2008, which mentions a biopolymer construct and a bioengineered cell sheet, designed primarily for the treatment of the cornea, specifically in the reconstruction of the corneal endothelium.

[0034] The construct of patent application US'423 comprises a bioresorbable and deformable biopolymer carrier and a bioengineered cell sheet consisting of a monolayer or multilayer of interconnected cells, where said cells have a uniform orientation and are attached to the surface of the carrier; it is also known from patent application US'423 that the biopolymer carrier is preferably made of materials such as gelatin, collagen, chitin, alginate, among others, said carrier having a thickness of between 0.5 and 1.0 mm.

[0035] The cell sheet of the construct in patent application US'423 is composed of cultured human corneal endothelial cells, which can form a uniform layer with a specific structural distribution, which seeks to promote the regeneration of the corneal endothelium, where the design of the construct allows the cells to be distributed in such a way that the basal surfaces of the cells of the sheet are in contact with the posterior surface of the cornea, thus promoting the regeneration of the corneal endothelium after implantation.

[0036] In terms of procedure, patent application IIS'423 describes a method for reconstructing a patient's corneal endothelium, involving the implantation of the aforementioned construct in the anterior chamber of the patient's cornea, thereby offering a technique for transplanting cultured cells, especially those from human corneas, for the restoration of the corneal endothelium without the carriers remaining permanently in the host, thus facilitating the regeneration and functional restoration of the tissue.

[0037] Based on the lessons learned from the state of the art, it is clear that, to date, advances in this field have not fully resolved the technical problems associated with current grafts. First, many of these tissues exhibit an inconsistent degradation rate, hindering their integration with the recipient tissues. Rapid or irregular degradation can prolong postoperative inflammation, impair graft adaptation, and compromise its long-term functionality.

[0038] Furthermore, water absorption in synthetic grafts presents a critical challenge. This parameter is essential to ensure proper fluid balance in the anterior chamber of the eye and maintain intraocular pressure within normal limits. Excessive absorption that is not replenished can result in corneal edema, blurred vision, and prolonged discomfort for the patient, exacerbating the negative impact of the procedure.

[0039] Another significant problem lies in the low cell density of existing artificial tissues. The proliferation of corneal cells, especially in the endothelium, is essential for regenerating this layer and ensuring corneal transparency. Without adequate cell density, grafts fail to effectively restore corneal function, limiting their usefulness as a therapeutic solution.

[0040] Finally, patients who receive synthetic corneal grafts face prolonged recovery times. These extended adaptation periods not only cause physical and psychological discomfort but also increase the costs associated with treatment and follow-up care. This can be particularly problematic for patients in vulnerable situations or with limited resources.

[0041] Despite technological and scientific advances in the field of tissue engineering, current solutions have not yet overcome these technical barriers. Challenges related to degradation, water absorption, cell proliferation, and postoperative recovery underscore the urgent need to develop new technologies that provide effective and sustainable solutions for people affected by corneal diseases. Summary of the Invention

[0042] It is therefore an objective of the present invention to provide a configurable biosynthetic corneal tissue capable of replacing corneal endothelium transplants by culturing endothelial cells on an ultrathin collagen membrane, allowing transplants to be more accessible to patients by maximizing the amount of usable tissue.

[0043] A particular objective is to provide corneal tissue which is a viable and efficient option for large-scale production of corneal grafts with less dependence on human donors, solving the technical problem of the shortage of available corneal tissue.

[0044] Another objective of the present invention is to provide a process for culturing corneal endothelial cells at a controlled concentration, allowing the formation of a cell monolayer with a hexagonal morphological structure similar to healthy corneal tissue.

[0045] In particular, the present invention proposes a process that ensures that cell proliferation in the graft is sufficient for successful regeneration of the corneal endothelium and thus provides an effective solution to improve clinical outcomes by overcoming the limitation of low cell concentration observed in previous synthetic grafts, ensuring the long-term quality and functionality of the graft in terms of adequate cell adhesion and coefficient of variation in cell morphology.

[0046] Furthermore, another objective of the present invention is to provide a configurable method of control over the degradation rate of the collagen membrane used to culture the endothelial cells, such that said control provides a gradual and efficient partial degradation in a medium similar to the human aqueous humor, facilitating the integration of the graft with the patient's corneal tissues.

[0047] The present invention provides a solution that improves the long-term functionality of the graft, ensuring that the membrane partially degrades at an appropriate rate and avoiding problems of premature decomposition, which can interfere with patient adaptation.

[0048] Another objective of the present invention is to provide an improvement in the configurable water absorption capacity of the collagen membrane, which is essential to ensure proper filtration of fluids into the aqueous humor, maintaining adequate eye pressure and avoiding complications such as corneal edema.

[0049] In particular, the present invention provides a graft that improves water absorption, optimizing the functionality of the corneal transplant in both the short and long term, and contributes to the patient's overall ocular health, thereby significantly reducing postoperative recovery times for patients receiving biosynthetic corneal tissue transplantation. By achieving faster integration with the patient's tissues and ensuring greater functionality in the first few days after transplantation, the invention provides a solution that improves the patient's quality of life by reducing the stress, pain, and discomfort associated with long recovery periods.

[0050] Another objective related to the present invention is to provide a tissue with better structural stability compared to the cell monolayer grown on the ultrathin collagen membrane, ensuring that the graft maintains its integrity during the transplantation process and in its post-operative life cycle, thereby providing a robust solution that prevents deformation or loss of functionality of the graft over time, ensuring a more reliable and durable treatment compared to previous grafts.

[0051] Furthermore, another objective of the present invention is to provide a configurable preclinical model that allows for the validation of the results of the biosynthetic corneal graft under controlled conditions, providing a detailed evaluation of the graft's functionality in animals, thus ensuring the effectiveness of the invention before its clinical application. The invention thereby provides reliable support for the viability and efficacy of the graft, demonstrating that it meets the necessary standards for use in humans.

[0052] Brief Description of the Figures

[0053] Figure 1 is an overview of the process for producing a construct that functions as a biosynthetic tissue for transplantation to replace the corneal endothelium.

[0054] Figure 2 describes the process of isolating corneal endothelium and culturing the cells for expansion.

[0055] Figure 3 shows an example of a process modality for the production of a vitrified collagen membrane that serves as a scaffold in the formation of a construct.

[0056] Figure 4 shows the specific conditions in the assembly of a construct between corneal endothelial cells and vitrified collagen membrane.

[0057] Figure 5 illustrates the assembly of the construct within a microinjector that enables intraocular transplantation.

[0058] Figure 6 illustrates the ex vivo model, where surgical manipulability tests of the construct were performed.

[0059] Figure 7 shows micrographs of corneal endothelial cells (CECs) obtained by the peeling and dumping peeling methods. A. Peeling of the corneal endothelium; the orange arrow shows the folded endothelium along with Descemet's membrane, while the green arrow shows the stroma. B. Full layer of corneal endothelium concentrated in the center by dumping peeling. C. Fragmented corneal endothelium after enzymatic digestion. D. Primary CEC culture after 24 h of digestion. Figure 8 shows comparative micrographs of CECs cultured in MEM and Optimem media (10X, scale bar 100µm).

[0060] Figure 9 shows an analysis of the cell area observed in CEC cultured in MEM and Optimem medium.

[0061] Figure 10 shows CEC micrographs positive for ZO-1 and Na / K-ATPase by immunofluorescence at 40X and 20X magnifications.

[0062] Figures 11a and 11b illustrate the absorption of water (edema) and degradation (mass loss) of CVMs under accelerated conditions (47°C) in aqueous humor (HA) and phosphate-buffered saline (PBS).

[0063] Figures 12a and 12b illustrate the absorption of water (edema) and degradation (mass loss) of CVMs under physiological conditions (37°C) in aqueous humor (HA) and PBS.

[0064] Figure 13A shows the proliferation rate of bovine CEC (BCEC) when cultured at 3 different initial densities (a. low: 2,500 cells / mm³ 2 , b. average: 5,000 cells / mm 2 , c. high: 7000 cells / mm 2 ).

[0065] Figure 13B shows the proliferation of BCEC on membrane (CVM1) at an initial density of 2,500 cells / mm 2 compared to its non-membrane control (C1).

[0066] Figure 13C shows the proliferation of BCEC on membrane (CVM2) at an initial density of 5,000 cells / mm² 2compared to its non-membrane control (C2).

[0067] Figure 13D shows the proliferation of BCEC on membrane (CVM3) at an initial density of 7,000 cells / mm³ 2 compared to its control without membrane (C3). (***p < 0.001, **P < 0.01, *P < 0.05).

[0068] Figure 14 shows the results of the CEC adhesion strength tests at low concentrations (500 cells / mm²). 2 ) and high (5,000 500 cells / mm³ 2 ) already different centrifugation forces in culture without substrate, on the CVMs and on culture plates coated with poly-D-lysine.

[0069] Figure 15 illustrates the analysis of the area and coefficient of variation (COV) in um 2 of the CECs grown without substrate and on CVM.

[0070] Figures 16 and 17 illustrate comparisons between post-transplant corneal thickness of the construct vs injection group and intraocular pressure (IOP - Figure 17) between the same groups.

[0071] Figure 18 depicts an eye treated with CEC injection after descemetorrhexis (top) and a transplanted eye with a CEC / CVM construct, after 90 days. On the right are histological micrographs of a cornea with signs of inflammation in the posterior stroma and a disorganized endothelium (top panel). The histological micrographs in the bottom panel correspond to a transplanted cornea with a construct showing an organized structure similar to that of a healthy corneal endothelium. Figure 19 shows an example of a preferred embodiment of the methodology of the present invention, schematically described in steps.

[0072] Detailed Description of the Invention

[0073] Some aspects of the present invention will now be described in more detail, also using reference to the accompanying drawings which show some embodiments and advantages of the present invention.

[0074] It will be evident to a person skilled in the art that various embodiments of the invention can be expressed in different ways and should not be interpreted as being limited to the embodiments described herein; rather, these exemplary embodiments are provided to make this invention clear and complete and to fully convey the scope of the invention to those skilled in the art. For example, unless otherwise indicated, something described as first, second, or the like should not be interpreted as a particular order. As used in the description and in the appended claims, the singular forms "a," "an," "the," and "a" include plural referents unless the context clearly indicates otherwise.

[0075] The various aspects of the present invention relate to a biosynthetic corneal tissue composed of a monolayer of corneal endothelial cells cultured on an ultrathin collagen membrane. Its primary objective is to provide an efficient alternative to traditional corneal endothelial transplants, which face limitations such as corneal tissue availability, the risk of rejection, and transplant-related complications. This biosynthetic corneal tissue represents an effective solution to overcome the technical and biological limitations associated with corneal transplants. The corneal tissue of the present invention improves tissue regeneration, controls water absorption and graft degradation, and ensures long-term functionality, significantly improving the quality of life for patients requiring corneal transplants.

[0076] Each of the components and features of the proposed invention - which will be described in detail within this application later on - has been carefully selected and tested by the applicant to ensure that the generated biosynthetic corneal graft is both effective and safe in its clinical application.

[0077] Corneal endothelium transplantation has been one of the most widely used solutions for treating various corneal diseases, such as Fuchs' dystrophy, but difficulties regarding donor availability and the need to preserve the tissue for a long period have limited its applicability. In this regard, the proposed invention addresses these problems by creating a biosynthetic graft that provides greater accessibility to transplants without depending on the shortage of donors. One of the biggest challenges in corneal transplant medicine is the limited availability of human corneas suitable for transplantation.The invention described in this application addresses this need by offering an alternative that not only utilizes a synthetic membrane for cell support but also optimizes the use of the limited number of available grafts. This is achieved by creating a graft composed of an ultrathin collagen membrane, minimizing the amount of tissue required for effective corneal function restoration. In this way, dependence on human cornea donors is reduced, contributing to more efficient resource management.

[0078] The use of a biosynthetic membrane as a basis for the culture of corneal endothelial cells allows overcoming the shortage of donor tissue, since the collagen membrane is biocompatible and offers a suitable environment for cell growth, not only reducing the demand for human tissue, but also accelerating the cell regeneration process, which in turn improves the transplant success rate.

[0079] Furthermore, the present invention improves corneal tissue regeneration, a crucial aspect in corneal transplants. Corneal endothelial cells cultured on the collagen membrane of the present invention adhere effectively to the base and, moreover, proliferate and organize their structure in a way that mimics the natural cornea, resembling the coefficient of variation in its morphology.

[0080] This controlled regeneration process, which is not performed by any other technology, allows the restoration of corneal function, contributing to the restoration of vision. Furthermore, the monolayer of corneal endothelial cells ensures the restoration of essential corneal functions, such as protection against infections and the barrier against dehydration.

[0081] One of the most outstanding features of the present invention is the ability of the ultrathin collagen membrane to absorb water and then secrete it, which improves the graft's ability to integrate with the tissues of the patient's eye.

[0082] The membrane according to the present invention has a high capacity for water absorption and secretion, which allows it to remain hydrated during the regeneration process and facilitates the distribution of nutrients between the endothelial cells, a property that is essential to prevent complications such as corneal edema, which can affect visual function.

[0083] Controlled water absorption also contributes to the stability of the graft in the patient's eye, ensuring that it remains in place during the healing process.

[0084] On the other hand, the present invention provides a tissue with minimal, controlled degradation of the collagen membrane, which is another essential aspect, since it integrates completely as a graft with the natural tissues. This controlled degradation process is crucial to prevent the graft from causing adverse reactions in the patient's eye, such as inflammation or rejection.

[0085] The membrane of the proposed invention does not show significant decomposition over time, in ex vivo and in vivo systems, allowing the cultured corneal endothelial cells to establish themselves stably and permanently in the patient's eye, without affecting the functionality of the graft, since the regeneration of the corneal tissue is advanced enough to assume the functions of the graft without interference.

[0086] In addition to its immediate benefits, the present invention is designed to provide a long-term solution; in particular, the cellular regeneration of the tissue generated by the present invention not only occurs in the first weeks of the transplant, but the graft structure continues to function efficiently for a long period (90 days), demonstrating effectiveness in preclinical models for more than 60 days, suggesting that said graft will continue to provide a stable and functional solution for an advantageously long period, implying a significant advance over traditional transplants, which can face long-term complications such as rejection or graft dysfunction.

[0087] Current technologies in corneal transplants face several challenges, such as limited tissue availability, immune rejection, vulnerability to infections, and postoperative complications. The proposed invention addresses these challenges by creating a biosynthetic graft that eliminates the need for donor tissue entirely or significantly reduces its quantity. By using a collagen membrane that integrates seamlessly with the patient's eye and promotes the regeneration of corneal endothelial cells, the likelihood of rejection or long-term complications is reduced.

[0088] The ultrathin collagen membrane fully integrates with the recipient tissue as the patient's natural corneal cells take over the function of the graft, and its high water absorption capacity ensures that the graft maintains adequate hydration without causing swelling or intraocular pressure. This integration not only improves recovery but also ensures the functional integrity of the graft without compromising the patient's ocular health.

[0089] In one embodiment, as can be seen in Figures 1 to 17, the present invention comprises:

[0090] A) At least one membrane,

[0091] In one form it is an ultrathin membrane.

[0092] "Ultrathin membrane" within the context of the present invention shall be understood as a structure with an extremely reduced thickness, which in one embodiment is in the range of 3 to 7 micrometers. In one embodiment, the at least one membrane is any selected from the group comprising a transparent, biocompatible, biosynthetic, manipulable membrane, combinations thereof and / or similar membranes.

[0093] At least one membrane is configured to serve as a base for the culture of corneal endothelial cells, its main function being to provide structural support that facilitates the formation of a cell monolayer that mimics the structure of the natural corneal endothelium, which is crucial for the success of a corneal transplant.

[0094] More specifically, at least one membrane serves as a support for the culture of corneal endothelial cells, said membrane being, in one modality, biocompatible to allow efficient integration with the corneal tissue of the recipient patient, thus providing a structural substrate that facilitates the regeneration of the corneal endothelium without interfering with the natural functionality of the eye, and its ability to degrade in a controlled manner ensures that it does not remain in the body longer than necessary, allowing the patient's tissue to gradually recover its functions.

[0095] In one modality, the membrane is a collagen membrane.

[0096] In this modality, collagen is present in the membrane in a range from 10% to 50% and is type I or type III collagen.

[0097] Collagen is selected due to its biocompatible properties and its ability to integrate with human body tissues without inducing rejection; however, collagen should not be considered a limiting factor, and on the contrary, it should be understood that in an alternative modality, the membrane is a membrane with any selected from the group comprising hyaluronic acid, chitosan, fibrinogen, elastin, polylactic acid (PLA), polyglycolic acid (PGA), hydrogels, and synthetic materials based on biocompatible polymers such as polyethylene glycol (PEG) or polyester, combinations of the same and / or similar materials that present biocompatibility characteristics and properties that favor integration with human tissues.

[0098] In an optional modality, the membrane is a collagen membrane combined with any selected from the group comprising hyaluronic acid, chitosan, fibrinogen, elastin, polylactic acid (PLA), polyglycolic acid (PGA), hydrogels, and synthetic materials based on biocompatible polymers such as polyethylene glycol (PEG) or polyester, combinations thereof and / or similar.

[0099] As previously mentioned, the membrane is biocompatible, meaning its integration with the recipient's tissues should be efficient and free of complications such as rejection. Additionally, the fact that at least one membrane is configurable to degrade slowly under controlled ex vivo conditions is fundamental for graft durability and to ensure that, as the patient's corneal tissue regenerates, the membrane material integrates without adverse effects. In this sense, "degrading slowly" means that the collagen membrane decomposes in a controlled and progressive manner over a period ranging from 21 to a maximum of 60 days under specific ex vivo conditions. During this time, the membrane gradually loses its structure and mechanical properties without negatively affecting the functionality of the corneal graft or the regeneration of the host tissue.Once transplanted, its degradation is minimal, allowing its integration into the recipient tissue.

[0100] B) at least one cell layer,

[0101] The present invention comprises at least one cell layer, which in a particular embodiment is a monolayer of corneal endothelial cells grown on the membrane (A).

[0102] The at least one cell layer aims to restore damaged corneal tissue by replacing lost corneal endothelial cells, thus enabling regeneration and functional restoration of the patient's cornea. Specifically, this at least one cell layer is configurable to functionally couple corneal endothelial cells and allow for their culture (growth).

[0103] The corneal endothelial cells used come from donated corneal tissue, which is obtained from remnant transplant tissue (sclerocorneal rings), corneal buttons (central part of donated corneas) and enucleated eyeballs.

[0104] Through “ocular enucleation process 1 'should be understood as a surgical intervention by which the eyeball of a patient is removed, usually as a result of a serious eye condition, such as an eye tumor, a severe infection, or a traumatic injury, performed to preserve the patient's health, and the removal is carried out carefully to avoid damage to surrounding structures.

[0105] In one modality, corneal tissue for isolation of corneal endothelial cells is preferably obtained by enucleation, improving the cell density obtained for culture.

[0106] In one modality, cultivation is carried out under controlled conditions to ensure its viability and proliferation.

[0107] The culture (CEC culture) conveniently comprises an adequate cell density as well as functional biomarkers as a result of the conditions and parameters of the different modalities of the present invention.

[0108] It should be noted that the culture is performed according to a specific method, isolating the corneal endothelium from the donor cornea using a "dumpling" technique. In this technique, the edges of the endothelial layer are dissected from the periphery toward the center, ensuring that the tissue is obtained with the greatest possible integrity. In this sense, the "dumpling technique" should be understood as a specific surgical procedure used to isolate the corneal endothelium from the donor cornea, in which the edges of the endothelial layer are carefully dissected from the periphery toward the center of the cornea. This process allows for the controlled extraction of endothelial tissue with the greatest possible integrity, minimizing damage to the endothelial cells and preserving their viability for subsequent culture or transplantation.The term “dumpling” refers to the appearance of the tissue, which resembles a bag or “dumpling” after isolation, facilitating the manipulation and transplantation of the corneal tissue.

[0109] Having said that, it is imperative that the expert in the field understand that the present application does not seek to protect in any way a surgical procedure, on the contrary, what is sought is to fully describe all the characteristics that are involved or related to the invention.

[0110] This method ensures the highest possible cell density. The corneal endothelium layer isolated by this method is enzymatically digested using collagenase II (2 mg / mL) in 0.36 mM calcium chloride for 2 hours at 37°C with agitation at 30 rpm. The endothelial cells released by this digestion method are cultured in a medium composed of Optimem medium supplemented with 8% fetal bovine serum, 1% antibiotic (streptomycin / ampicillin), calcium chloride (200 mg / mL), ascorbic acid (20 µg / mL), chondroitin sulfate (0.08%), HEPES solution (6 g / L), sodium bicarbonate (2.5 g / L), neural growth factor (20 ng / mL), and fibroblast growth factor (5 ng / mL).

[0111] The culture is maintained by changing the medium every 3 days until confluence is achieved (more than 80% of the culture is populated). Once confluence is reached, the cell culture is divided into 3 new culture plates (cell passage) using "basal" culture medium (Optimem with 8% fetal bovine serum and 1% antibiotic). Upon reaching confluence, a second passage is performed, and the cells are analyzed for the presence of functional markers (ZO-1 and Na / K-ATPase).

[0112] This results in the formation of an organized and functional monolayer of cells, which facilitates the recovery of the structure and function of the corneal endothelium.

[0113] Specifically, the culture method according to the present invention ensures that the cells are arranged in an organized monolayer with a hexagonal morphology and the presence of tissue-specific molecular markers, such as the sodium / potassium-ATP pump (Na / K-ATPase) and zonula occludens (ZO-1) tight intercellular junctions, which advantageously resembles that of a healthy cornea. This type of cellular organization is essential to ensure that corneal function, such as adequate hydration to maintain transparency for light transmission, which enables vision, is properly restored.

[0114] Furthermore, the present invention comprises a methodology for generating the ultrathin membrane with at least one cell layer; said methodology comprises the steps of: Formation of the construct

[0115] Endothelial cells cultured according to the methodology described above are grown at a density of between 4000 and 6000 cells / mm 2 on circular collagen membranes between 6 and 12 mm in diameter for 24 to 48 h to allow the formation of a monolayer of hexagonal cells with a minimum density of 2,500 cells / mm 2 a uniform coefficient of variation, such as is required in a corneal endothelium for a transplant.

[0116] The term “uniform coefficient of variation” refers to the statistical measure that expresses homogeneity in cell morphology, calculated as the ratio between the standard deviation and the mean value of a morphological parameter (for example, cell area), expressed as a percentage, so that less variation indicates a more homogeneous distribution of values.

[0117] Likewise, by “coefficient of variation similar to that required in a healthy corneal endothelium” it will be understood that said coefficient of variation is close to the characteristic value obtained in healthy corneal endothelia, which has been experimentally determined to be around 40%.

[0118] Therefore, in one modality, the coefficient of variation is in a range from approximately 35% to approximately 45%.

[0119] The construct, in one modality, is a monolayer of hexagonal cells with adequate coefficient of variation, sufficient cell adhesion, both sufficient for the construct to be adapted for surgical manipulation.

[0120] As shown in Figure 1, in one modality, the construct is trepanned to obtain a button (a), subsequently, the button is prepared in a “double roll” (b) ensuring that the cells are inside the roll; and finally, the “double roll” is placed in an injector device (c), at this point the construct is ready to proceed to transplantation.

[0121] In particular, the coefficient of variation of cell morphology is a health parameter in the corneal endothelium associated with its proper function. Cells cultured on the collagen membrane are able to proliferate while maintaining a coefficient of variation of around 40%, which is within the range observed in a healthy corneal endothelium.

[0122] The resulting construct is stained with trypan blue after loading onto the device for visualization and manipulation.

[0123] The tissue is inserted into a double-roll microinjector device using a surgical viscoelastic fluid, with the cells facing inward. This allows the tissue to be inserted into the anterior chamber of the eye through a peripheral incision in the cornea, with the cells oriented toward the aqueous humor, as found in healthy tissue. This parameter is of vital importance, since corneal endothelium with high morphological variation in cells is associated with loss of function and future development of opacities. Additionally, corneal endothelial cells cultured at the described density (4000 to 6000 cells / mm³) 2They adhere with sufficient force to ensure that after 24 to 48 hours the density obtained in the construct is at least that required for a healthy corneal endothelium. In an analysis performed by subjecting the cells cultured on the collagen membrane to different centrifugation forces, it was observed that with the described density, the cell detachment fraction is less than 50%. This ensures that the cells can remain adhered to the membrane to the greatest extent possible during culture, before and after surgical manipulation.

[0124] Derived from the strategic combination of at least one membrane (A) and at least one cell layer (B) in proportions determined according to the different modalities of the present invention, as well as derived from the parameters and conditions of each of the steps that characterize the methods described in the present application, the present invention provides efficient control of the degradability of the membrane, ensuring that it degrades in a controlled manner, allowing efficient integration with the patient's corneal tissues without interfering with the recovery of the recipient tissue, guaranteeing that the membrane remains in the patient's eye for the time necessary for the patient's natural corneal tissue to fully assume the functions of the graft.

[0125] In this case, the applicant has found that, advantageously, the membrane degradation rate is approximately 40% after 21 days under laboratory conditions. And that once transplanted, it remains intact after 90 days.

[0126] This behavior is crucial since it allows the graft to remain long enough to facilitate cell integration and regeneration without causing prolonged interference.

[0127] The degradation rate has been tested in a medium simulating human aqueous humor, providing precise data on how the membrane will behave once implanted—as seen in the examples included later in this application—ensuring gradual degradation, which also reduces the risk of side effects such as graft rejection or inflammation. Animal model tissues analyzed 90 days post-transplant show that the membrane remains intact and fully integrated with the corneal tissue, correlating with the restoration of corneal clarity.

[0128] Now, as a result of all the above, the proposed invention also adds the membrane's ability to absorb water advantageously, which is crucial to maintain adequate eye pressure and facilitate corneal regeneration, substantially favoring the patient's vision restoration process.

[0129] In particular, the membrane has been shown to have a water absorption capacity between 1050% and 1167%, which is then secreted over a prolonged period, facilitating its integration while contributing to maintaining graft hydration. In tissues analyzed 90 days post-transplant, the integrated membrane was found to be approximately 10 microns thick, which is consistent with the absorption and secretion observed in the laboratory system and also represents the thickness of the natural Descemet's membrane upon which healthy corneal endothelial cells align.

[0130] The at least one membrane according to the present invention is highly absorbent, allowing it to remain hydrated during the tissue regeneration process, which advantageously facilitates the proper filtration of fluids into the aqueous humor, a process that is critical for the health and proper functioning of the eye.

[0131] More specifically, at least one membrane has an absorption capacity that varies between 1050% and 1167% of water during the first 21 days, subsequently secreting it gradually, which ensures that the graft maintains its functionality without causing swelling or edema in the eye, in addition to facilitating the adaptation of the graft to the conditions of the patient's eye, which accelerates the recovery process, as well as contributing to the stability of the ocular pressure, thus preventing postoperative complications such as blurred vision or ocular hypertension, which are common in traditional corneal transplants.

[0132] Figure 19 shows a schematic summary of the methodology of the present invention.

[0133] EXAMPLES

[0134] 1. Isolation and culture of CECs

[0135] All procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals, Eighth Edition (2011) and considering the ARVO Statement on the Use of Animals in Ophthalmic and Visual Research. Corneas were obtained from 3-month-old New Zealand rabbits weighing 3 kg. The rabbits were euthanized following a protocol approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL) of Tecnológico de Monterrey (Folio 2023-010), using a lethal dose (200 mg / kg) of pentobarbital administered intracardiacly (Penta-Hypnol, Agrovet Market, Lima, Peru).

[0136] Corneas intended for subsequent isolation of corneal endothelial cells were obtained using two different procurement methods: i) ocular enucleation and ii) sclero-corneal ring grafting, with the aim of comparing both methods in terms of cell culture yield, adhesion, and growth time. A group of four corneas was evaluated for endothelial isolation to compare maximum tissue recovery, significant differences in handling, and efficiency in obtaining primary cell cultures between the two methods: corneal procurement versus extraction by ocular enucleation.

[0137] Ocular enucleation

[0138] A wire eyelid speculum (Rumex, Buenos Aires, Argentina) was inserted to retract the eyelids and expose the eye. Enucleation was initiated by performing a 360-degree peritomy. The rectus and oblique muscles were identified and divided at their base. While gentle anterior traction was applied to expose the optic nerve, additional traction was applied to allow its division. Any remaining adherent conjunctival-Tenon's membrane was sectioned with surgical scissors to fully release the eyeball. The isolated eyeball was disinfected by immersion in 5% povidone-iodine solution for 2–5 minutes and then washed with balanced saline. It was subsequently stored in 50 mL conical tubes lined with sterile gauze to maintain moisture.Inside the laminar flow chamber, the sclero-corneal ring graft was isolated by making a scalpel incision 4 mm from the cornea and completing the ring cut with surgical scissors.

[0139] Sclero-corneal ring graft

[0140] The procedure used to place the tissue generated by the present invention will now be described. However, this description should not be interpreted as seeking to protect the surgical methodology in any way. In fact, as anyone with ordinary knowledge in the field would know, the procedure involving the detachment or removal of the corneal endothelium (descemethorhexis) (d) and the subsequent injection (e), the placement of the construct (f), resulting in the recipient stroma with the constructed unfolded (g) (as shown in Figure 1) is a widely known and used methodology. Therefore, it should be reiterated that this application does not seek to protect in any way a surgical method applied to a human being and / or animal.

[0141] To place the tissue generated by the present invention, an ophthalmic surgical microscope (Rumex, Buenos Aires, Argentina) was used. Corneoscleral buttons with the iris and lens attached were obtained by cutting the sclera with surgical scissors 5 mm from the limbus, within the first hour after the donor's death. The iris and lens were then removed to isolate the sclero-corneal graft, preserving the location of Schwalbe's line. The corneal endothelium was stained with trypan blue (Sigma-Aldrich, Merck, St. Louis, USA) and subsequently isolated from the corneal stroma using a detachment technique with a curved scalpel (Rumex, Buenos Aires, Argentina). Finally, the corneal endothelium samples were stored in a balanced saline solution until use.

[0142] All procedures were performed inside a biosafety cabinet. Corneoscleral rings were treated with 1% streptomycin / penicillin antibiotic and placed in a sterile tissue culture dish. Corneas were stained with a drop of Tympano Blue for 1 minute to identify endothelial margins, using a VE-S3 stethoscope (Velab, TX, USA).

[0143] The endothelium was isolated, maintaining its adhesion to Descemet's basement membrane, by gently scraping from various points on the periphery toward the center, a technique we call "dumpling-peeling." The complete endothelium was then transferred to 12-well culture plates using basal medium (OPTIMEM, 8% SBF, and 1% penicillin / streptomycin). Finally, they were incubated overnight under standard conditions (37°C, 5% CO2). The Descemet-endothelium slides were briefly exposed to a collagenase II solution (2 mg / mL) and CaCl2 (0.36 mM in 2 mL of 10 mM PBS) for 1–2 hours at 37°C, with shaking at 30 rpm to digest Descemet's membrane (DM), resulting in the formation of compact clusters of corneal endothelial cells.

[0144] Subsequently, the samples were centrifuged at 400g for 6 minutes, the supernatant was decanted, and the cell pellet was resuspended in 1 mL of fresh OptiMEM proliferative medium. Then, it was transferred to a 12-well culture plate and brought up to a final volume of 2 mL of OptiMEM proliferative medium per well, following the two-phase cell culture method described by Montalvo-Parra et al., 2020.

[0145] Result

[0146]

[0147] Optimization of CEC culture with different media

[0148] After optimizing the endothelium recovery method, two groups of four endothelia from independent rabbit corneas were evaluated. Rabbit corneal endothelial cell (RCEC) cultures were tested using two different culture media: OptiMEM vs. MEM a (Gibco-Thermo Fisher Scientific, New York, USA), supplemented according to Table 1.

[0149] Table 1. Comparison of the OptiMEM vs MEMc culture media formulation

[0150]

[0151] Cell growth was monitored for 20 days, performing two cell passages. The objective was to compare cell morphology, growth rate, and cell size between the two culture media.

[0152] Cell Morphology Analysis

[0153] Morphological changes in corneal endothelial cells cultured in different media were evaluated by measuring cell area using Fiji software (updated version 2024). Each phase of the culture was photographically documented, taking four images of different samples per cell passage using the microscope camera and ZenBlue software. The image scale bar was configured in ZenBlue or Fiji by loading the .czi image format and accessing the option: Analyze > Tools > Scale bar, adjusting the thickness and font size. In total, eight images of cells cultured in MEM and eight in OptiMEM were analyzed. For each image, 40 cells were delineated using the Freehand tool, and their area was calculated using the Measure Analysis function in Fiji (Ctrl+M to measure, Ctrl+F to fill area). The average area of ​​the 40 cells per image was used as a representative value for each sample.For statistical analysis, a multiple unpaired t-test was applied, using the Holm-Sidak multiple comparison method to assess significant differences between groups.

[0154] Result

[0155] A significant difference (p = 0.0061) was observed in the areas of cells cultured with the two different media (Optimem and MEM), as shown in Figures 8 and 9.

[0156] Maximum Proliferation Assay

[0157] The Rabbit Corneal Endothelial Cell Maximal Proliferation Assay aims to measure the cell division rate of rabbit corneal endothelial cells (RCECs) obtained by ocular enucleation and evaluate their maximum proliferative capacity, minimizing the number of cell passages and preserving their hexagonal morphology. The purpose of this assay was to generate a high concentration of cells for the creation of an RCEC cryopreservation bank for future applications in bioengineered corneal transplants. For the experiment, four rabbit corneal endothelium samples, obtained by ocular enucleation, were used. The cells were cultured exclusively in proliferative OptiMEM medium. Each whole endothelium sample was seeded in a 12-well culture plate, using one well per whole endothelium, and incubated at 37°C with 5% CO2 in a humidified incubator for 12 days.Cell passages were performed when at least 80% confluence was reached, using 0.25% trypsin-EDTA (Corning, New York, USA) to detach the cells from the culture plate. Subsequently, the cells were counted using the trypan blue exclusion method (Sigma-Aldrich, Merck, St. Louis, USA) before being subcultured, adding one additional well per cell passage to expand the culture area. Results.

[0158] Table 2. Cell proliferation record of isolated CECs using the enucleation, dumpling peeling and two-phase culture method with Optimem medium.

[0159]

[0160] *P: Passage, CryoP: cryopreservation**Considering that 1 well of the 12-well plate has a surface area of ​​3.5 cm 2

[0161] Analysis of functional marker expression in cultured cells

[0162] To analyze the presence of the specific markers ZO-1 and Na+ / K+-ATPase, an immunocytochemical assay was performed on sterile glass coverslips coated with Poly-D-lysine (Gibco-Thermo Fisher Scientific, New York, USA). Approximately 10,000 cells were seeded and incubated overnight until reaching 80% confluence. The medium was removed, and the cells were fixed with 4% paraformaldehyde (2 mL per well) and incubated for 30 min at 4°C. Subsequently, the cells were washed twice with 10 mM PBS for 5 min each time and permeabilized with Triton X-100 (0.5–1%) or Tween 20 (0.5%), incubating for 5 min at room temperature (~23–25°C). Next, the samples were washed twice with 1% PBS (5 min each) and the nonspecific binding sites were blocked with 5% BSA for 30 min at 37°C. Finally, the samples were washed twice with 10 mM PBS for 5 min each.Fifty milliliters of primary antibody (Na+ / K+-ATPase ab7671, 1:100; ZO-1 Invitrogen 617300, 5 pg / ml) were added, and the plate was sealed and incubated overnight at 4°C. For secondary antibody detection, samples were washed twice with PBS for 5 min with 0.5% Tween. Subsequently, 50 milliliters of Alexa fluorescent secondary antibody (Alexa Fluor 488 ab150077, 1:200 for ZO-1 and AlexaFluor 568 ab175473, 1:200 for Na+ / K+-ATPase) (Abeam, Cambridge, UK) were added, and the plates were incubated for 1 h at room temperature in the dark.

[0163] Cell nuclei were stained with DAPI (4',6-diamidino-2-phenylindole dihydrochloride) (Invitrogen, Thermo Fisher Scientific, MA, USA) at a 1:1000 dilution (50 pL) and incubated in the dark at 4°C for 5 min. For mounting preparation, a drop of Vectashield mounting medium (Vector Laboratories, California, USA) was added to the cells and a coverslip was placed over them. The samples were analyzed by fluorescence microscopy using the AXIO Imager.ZI (Zeiss, Germany) with a 20x objective, observing the fluorescence of Alexa Fluor 488 (Rabbit) for ZO-1 and Alexa Fluor 568 for Na+ / K+-ATPase.

[0164] Results

[0165] CECs isolated by enucleation and dumpling peeling method cultured in Optimem medium by the two-step system express the functional markers ZO-1 and Na / N-ATPase, detected by immunofluorescence.

[0166] 2. Membrane production

[0167]

[0168] Production of

[0169]

[0170] Membrane assembly

[0171]

[0172] Vitrigel collagen membranes were produced according to a protocol previously described by the research group (Montalvo-Parra et al., 2022). The process involved three main stages: (1) preparation of the collagen gel, (2) drying, and (3) rehydration in a Matryoshka system.

[0173] The Matryoshka system consisted of an incubator that maintained a constant temperature (T) with a desiccator containing a saturated potassium carbonate (K2CO3) solution to control relative humidity (RH). First, 50 mL of a solution were prepared using 1.15 g / mL of K2CO3 in double-distilled water. This solution was placed in a 200 mL beaker and secured inside a 330 x 246 x 262 mm desiccator. A closed system was established by placing the sealed desiccator inside an incubator with a shaking plate. RH and temperature were monitored with a Traceable Hygrometer (4040CC; Traceable® Products, Webster, Texas, USA) placed inside the desiccator. The system maintained a temperature of 40°C and an RH of 40%. The incubator was set to 37°C with a stirring speed of 30 rpm.

[0174] Dulbecco's Modified Eagle Medium (DMEM) was prepared with 1% penicillin-streptomycin and 10% fetal bovine serum, kept on ice. CellAdhere™ Type I Collagen (6 mg / mL, STEMCELL Technologies, Vancouver, Canada) was diluted to 5 mg / mL with sterile distilled water or a sterile solution, following the product instructions. HEPES 22 mM was then added and mixed with the chilled Type I collagen (5 mg / mL) in a 1:1 ratio until a uniform yellow mixture was obtained by gentle resuspension with a micropipette, avoiding bubble formation. Six-well plates were used as molds, each containing 4.4 mL of the collagen mixture. The plates containing the collagen mixture were incubated at 37°C with 5% CO2 for 2 h to promote gelation. The plates were then transferred to the Matryoshka system. The incubator was set to 40°C and 30 rpm. During 10-13 days, the collagen gels decreased in volume, forming membranes.The Matryoshka system was then opened and the membranes were rinsed with double-distilled water until the phenol red was removed from the medium. The samples were returned to the system for a total of 37 days of incubation.

[0175] The Matryoshka system plates were then removed. Double-distilled water was added to each well containing collagen membranes, allowed to stand for approximately 20 minutes, and then removed. The edges of each membrane were lifted using water pressure. Round-tipped forceps were used to gently detach the remaining membranes from the bottom of the wells.

[0176] Characterization of Biomaterial

[0177] Membrane Transparency: Transmittance

[0178] The membrane's transmittance was measured using a spectrophotometer. Absorbance was recorded across the UV-VIS spectrum (340–700 nm) in 10 nm increments using a Synergy HT spectrophotometer (BioTek, Winooski, VT, USA). The Beer-Lambert law equation was used to convert absorbance values ​​to transmittance. Triplicate samples (4 mm diameter circles) of CVM were placed on a p96 plate containing 200 pL of sterile 1% PBS or sterile water per well. PBS solution was used as a blank. The experiment followed international guideline D1003-21: Standard Test Method for Haze and Light Transmittance of Transparent Plastics.

[0179] Result

[0180] The transmittance of the membrane was 95.8%. This is similar to the transmittance of the cornea (https: / / pubmed.ncbi.nlm.nih.gov / 2338122 / ).

[0181] Membrane Degradation Test

[0182] The membrane degradation test was performed in accordance with ASTM International Standard Guide D1042-22 and ISO 10993-9: Framework for the identification and quantification of potential degradation products. CVMs were tested in two different solutions: PBS (10 mM) and Artificial Aqueous Humor (AH), composed as follows:

[0183] . NaCl 0.9% (w / v) - 100 mL

[0184] . KCI 1.15% (w / v) - 5 mL

[0185] . CaCI21.1% (w / v) - 1 mL

[0186] . MgSO4*7H2O 3.82% (w / v) - 0.6 mL

[0187] . KH2PO42.1% (w / v) — 1 mLNaHCO31.3% (w / v) - 42 mL

[0188] Glucose-Dextrose 12% (w / v) - 1 mL

[0189] Total final volume: 150.6 mL (Kaye & Pappas, 1962).

[0190] Two experimental conditions were tested:

[0191] • Normal conditions: Monitoring for 35 days at 37°C.

[0192] • Accelerated conditions: Monitoring for 21 days at 47°C.

[0193] The objective was to evaluate the membrane's behavior and lifespan over time and temperature under normal and accelerated conditions, simulating physiological environments. Two main parameters were used:

[0194] • Mass loss (%) where m_0 is the initial mass and m_d is the mass after degradation.

[0195] • % Mass loss = m ° md x 100%

[0196] m0

[0197] • Water absorption (%) where m_w is the wet weight and m_d is the dry weight.

[0198] • % Water absorption = mw md x 100%

[0199] -d

[0200] Each experiment was performed in triplicate under sterile conditions. Two groups were established:

[0201] • Group A (Normal conditions): Three CVMs immersed in 2 mL of PBS 1x and three in 2 mL of AH at 37°C for 35 days (agitation at 30 rpm).

[0202] • Group B (Accelerated conditions): Three CVMs immersed in 2 mL of 1% PBS and three in 2 mL of AH at 47°C for 21 days (agitation at 30 rpm).

[0203] Weight measurements and photographic documentation were performed weekly for Group A and twice a week for Group B using an Analytical Balance (A&D-GR Series, Thomastown VIC 3074, Australia).

[0204] Each CVM was removed from its solution, placed on a slide, and excess liquid was removed using Kimtech tissue paper (Kimberly-Clark de México, Mexico City, Mexico).

[0205] The wet weight was recorded and the drying time was monitored at room temperature (~23°C - 25°C). After 10 minutes, the dry weight was recorded. The CVMs were then returned to their respective solutions, sealed with Parafilm to prevent evaporation, and placed back on the shaker at the appropriate temperature.

[0206] Results

[0207] Under accelerated conditions (47°C), the maximum mass loss was observed in the aqueous humor after 10 days. It was not possible to record the loss after 15 days due to fragmentation of the samples. The percentage of absorption increased by 1050% when the CVMs were treated with PBS after 21 days, as shown in Figures 11a and 11b.

[0208] Under physiological conditions (37°C), the mass loss of the CVMs in PBS stabilized after 14 days at approximately 43%. However, in aqueous humor, the mass doubled. Water absorption was more pronounced in the CVMs immersed in PBS (approximately 800%), as clearly shown in Figures 12a and 12b.

[0209] 2. Production of the construct

[0210] Proliferation Essay in Constructs

[0211] The proliferation assay was performed following the recommendations of Standard Guide F2739-08 of the American Society for Testing and Materials (ASTM) International. Different cell densities of the BCEC C / D-1b cell line (ATCC, CRL-1446) were seeded: low density (2,500 cells / mm³). 2 ), medium density (5,000 cells / mm³) 2 ) and high density (7,000 cells / mm³) 2) on CVMs with a diameter of 6 mm in 96-well culture plates. The constructs were monitored daily for 5 days using a cell viability assay with neutral red (Merck KGaA, Darmstadt, Germany), cell density, confluence, and time-series cell imaging. The neutral red assay was performed according to the product instructions.

[0212] The membranes were pre-disinfected by adding Microdacyn (Sanfer, Monterrey, Mexico) for 10 minutes, rinsed three times with sterile water, and dried at room temperature (~23°C–25°C). Cell densities—low, medium, and high—were plated in triplicate onto CVM and without CVM (control), with 200 µL of DMEM (10% SBF, 1% antibiotic) per well and incubated for 1, 2, 3, 4, and 5 days. After the incubation period, the cell culture medium was removed, gently rinsed with sterile 10 mM PBS, and 100 µL of neutral red preparation medium were added according to the product data sheet protocol and incubated for 3 hours. After incubation, the neutral red medium was discarded and rinsed with 150 pL of PBS, and 150 pL of neutral red distinguishing solution was added; 50% 96% ethanol, 49% deionized water and 1% glacial acetic acid (Sigma-Aldhch, Merck, St. Louis USA).The plates were measured by absorbance at 540 nm using a Synergy HT microplate spectrophotometer (BioTek, Winooski, VT, USA). Wells containing DMEM and CVM + DMEM were used as blanks. All samples were tested in triplicate, using a 96-well plate for daily measurements.

[0213] Standard curve

[0214] To determine the cell density per day, a standard curve was performed using the neutral red technique by seeding different known cell densities (250, 500, 1000, 2500, 5000, 7000 and 10,000 cells / mm²). 2 ) in triplicate. The results were plotted as cells vs. absorbance. The equation for the standard curve for BCEC-CVM is y = 7e A (-5x) + 0.2209, r 2 = 0.9652 and for BCEC it is y = 7e A (-5x) + 0.19258, r 2 = 0.9571.

[0215] Results

[0216] Bovine corneal endothelial cell proliferation occurred at all three densities analyzed, showing a decreasing pattern between the third and fourth day, with a significant increase towards day 5. The intention of this analysis was to choose the ideal initial density for culturing cells on the CVM that would produce a uniform monolayer with a minimum density of 2,500 cells / mm² in the shortest time. 2 The density is 2,500 cells / mm³ 2 It requires a period of 4 days to reach the desired density, which would translate into a greater risk for the production of constructs for human use in clinical trials. The density of 5,000 cells / mm³ 2 The desired density was achieved after 2 days with a monolayer conformation of hexagonal cells. The density was 7000 cells / mm³. 2It allows obtaining constructs with high cell densities from 24 h, however, the monolayer conformation is lost when colony-forming units are observed on the membrane that give rise to the dedifferentiation of cells towards fibroblasts, cells associated with scarring processes.

[0217] Therefore, the initial density of 5,000 cells / mm 2 It represents a viable option for the production of constructs, in terms of time, use of resources (few corneas available, making the most of the minimum amount required to produce more transplantable tissue) and quality of tissue for transplantation, as shown in Figure 13.

[0218] 4.4.2 Cell Adhesion Strength Test on CVM

[0219] This assay measures the fraction of cells that remain attached to the surface after applying a specific centrifugal force to assess adhesion strength. Cell-mediated cells (CMCs) were tested and compared to a culture surface in a dish as a negative control against a common commercial cell coating such as Laminin (23017015, Gibco-Thermo Fisher Scientific, New York, USA). The CMCs were seeded at a cell concentration of 5,000 cells / mm³. 2on CVMs with a diameter of 3.2 cm, laminin-coated (23017015, Gibco), and plate-only surfaces, the latter two being used as comparable controls. Samples were tested in triplicate, using a fluorometer-compatible, opaque-walled, clear-bottom tissue culture plate (CLS3603, Corning, Inc., Germany) for each centrifugal force group (200 g, 400 g, and 800 g). After seeding the cells, the plates were centrifuged at 1 g for 10 min to settle all cells to the bottom and incubated for 2 h. After incubation, each well was carefully aspirated to remove floating cells, and fresh DMEM medium and 20 pL of Cell Titer Blue (Promega, Madison, W1, USA) were added.), with 100 pL of DMEM medium per experimental, control, and sample well, according to the manufacturer's instructions, for a 1 h incubation to obtain an initial fluorescence reading (560 ex / 590 nm) and determine the cell density before detachment (Cell adheredmax). Fluorescence was recorded on a Synergy HT spectrophotometer (BioTek, Winooski, VT, EE. Ull.). After the fluorescence reading, the lid was removed. The wells were filled with medium (maximum volume 200 pL) and covered with Parafilm to prevent medium loss and air bubbles, gently running a finger over the covered Parafilm to help seal it.The plates were then inverted and centrifuged for 10 min in a Thermo Sorvall Legend XTR Refrigerated Centrifuge, 155 mm rotor (Thermo Fisher, Swinging Bucket) at different acceleration fields of 200g, 400g, and 800g at 25°C (corresponding to 1,073, 1,518, and 2,147 rpm, respectively) to detach the cells. The remaining medium in the wells was carefully aspirated with a multichannel micropipette at the lowest speed, gently rinsed with 100 pL of sterile 1x PBS, and topped up with fresh medium plus Cell Titer Blue for fluorescence. The plates were incubated for 1 h for the final reading to determine the density of remaining adherent cells.

[0220] The detachment force imposed on the adhered cell layer is proportional to these relative centrifugal forces (RCF) and is calculated according to the following equation:

[0221] FD = (p_cell - p_media) x V_cell x RCF (4)

[0222] where FD is the detachment force per cell, G is the centrifugal acceleration, V_cell is the cell volume (-1700 pm 3 ), p_cell is the cell density (typically 1.07 g / cm³). 3 ), p_medio is the density of the medium (typically 1.00 g / cm³). 3 ).

[0223] Results

[0224] At low cell density, cells cultured on CVM showed a higher adhesion fraction at an intermediate centrifugation force (greater than 60%). The density was 5000 cells / mm³. 2This allowed cells to maintain approximately 50% adhesion fraction at an intermediate centrifugation speed. These results showed no significant difference compared to cells cultured on the plastic of the culture plates or coated with poly-D-lysine. In general, it was observed that higher cell density is associated with lower cell adhesion fraction. This assay allows for predicting the cell survival rate on the biomaterial in surgical manipulation environments. Therefore, a density of 5000 cells / mm³ is recommended. 2 chosen for producing a construct with desirable density at 24-48 h with a monolayer of hexagonal cells, it also shows an appropriate adhesion fraction (around 50% when centrifuged at medium speed).; the above is visualized in Figure 14.

[0225] Coefficient of variation analysis: To determine the coefficient of variation (COV), corneal endothelium grown with rabbit CEC seeded on CV in basal medium for 72 h was analyzed using micrographs with ImageJ software, tracing approximately 50 randomly selected cell borders. Cell area and circularity were determined. Statistical analysis was performed using multiple unpaired t-tests, employing the Holm-Sidak multiple comparisons method.

[0226] Results

[0227] As shown in Figure 15, the COV of rabbit CECs cultured on CVM was 47.1 ± 1.8%. While for cells cultured without CVM, it was 29.6 ± 2.8%. This difference was statistically significant (p = 0.69), however, the COV recorded in the CEC on CVM is close to the normal range for a healthy human corneal endothelium (https: / / www.reviewofoptometry.com / article / under-the-specular-microscope#:~:text=CV%20represents%20the%20coeffic¡ent%2C%20or,less%20than%2040% 20¡s%20normal) and is much lower than the COV reported for rabbits between 12 and 48 months of age, corresponding to 64 to 71% (https: / / www.researchqate.net / publication / 321108420 Corneal endothelial cell density and morphology in rabbits' eyes using contact specular microscopy).

[0228] 3. Ex vivo model

[0229] To test the ease of surgical manipulation of the corneal membrane (CM) before performing transplants in animal models, an ex vivo test was used. Bovine and porcine eyes obtained from a local butcher shop were mounted on polystyrene plates. The CMs were stained with thpano blue by immersion for 1 minute and transplanted into the anterior chamber, similar to Descemet's endothelial keratoplasty with epithelialization, the surgical procedure for corneal endothelium transplantation. The surgical procedures were performed by two ophthalmic surgeons to determine if the CM: (1) withstood manipulation with common surgical instruments to prevent breakage, (2) could be introduced folded through an injector via an incision in the periphery of the cornea, and (3) could expand once inside the anterior chamber.

[0230] Results

[0231] Ex vivo manipulation of the constructs revealed that they can be surgically manipulated using a procedure similar to that used for corneal endothelium transplantation. Furthermore, the cells remained adhered to the CVM after the surgical procedure.

[0232] 4. Transplantation in a preclinical model and analysis For this protocol, 18 male New Zealand rabbits between 12 and 18 months of age were used, which were divided into 2 groups, to have a total of 9 eyes per group: Group 1: Injection of corneal endothelial cells

[0233] Group 2: Transplantation of collagen-vitrigel membranes with corneal endothelial cells. Anesthesia was administered with zolazepam / tiletamine / xylazine (5-9 mg / kg). In both groups, the procedure was performed on the right eye, while the contralateral eye served as a control. General anesthesia was used according to the CICUAL procedure manual, and topical anesthesia with tetracaine eye drops was administered, one drop per eye before surgery. In Group 1, descemetorrhexis was performed on the right eye through a transcorneal port. A volume of 10 pL containing 200,000 CEC was injected, and the port was closed with a single suture. In the contralateral eye, a transcorneal port was created, sterile PBS was injected, and the port was closed with sutures. In Group 2, descemetorrhexis was performed on the right eye and the construct was transplanted with an approximate cell density of 2,500 cells / mm². 2Using a microinjector, the construct was pre-stained with trypan blue and rolled in a "double roll" with the cells facing inward. Once inside, the construct was unrolled with surgical forceps, creating an air bubble in the presence of viscoelastic fluid. The contralateral eye was maintained as a healthy control. Postoperative care included the administration of topical anti-inflammatory and antibiotic medication for 48 hours, with one drop in each eye three times daily. Corneal thickness was measured weekly by pachymetry, intraocular pressure was recorded, and the evolution of corneal opacity was photographed. Adverse signs such as edema, neovascularization, and / or infection were also recorded. After 90 days, corneal tissue was harvested following euthanasia and histological analysis was performed.

[0234] Results

[0235] After 90 days post-transplant, a significant difference in central corneal thickness (CCT) was observed between the injection and construct transplant groups, with CCT being greater in the injection group. This correlated with the findings of greater opacity in the injection group, as shown in Figure 16.

[0236] Eyes that received cell injection showed the formation of a monolayer of disorganized cells in the corneal endothelium without Descemet's membrane and with an inflammatory reaction in the posterior part of the stroma. Eyes that received transplants showed clear corneas and an intact structure in the histology without alterations. The corneal endothelium was observed as a monolayer of organized cells with the collagen membrane integrated and oriented towards the stroma, as a Descemet's membrane is normally found in a healthy cornea, as shown and illustrated in Figure 17. Many modifications and other embodiments of the invention will occur to a person skilled in the art to which the invention pertains, having the benefit of the teachings presented in the preceding descriptions and associated drawings.Therefore, it should be understood that the invention is not to be limited to the specific embodiments and examples described, but that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are used herein, they are used only in a generic and descriptive sense and not for limiting purposes. Likewise, it should be understood that the materials from which the various components comprising the invention described herein may be manufactured, the geometries, dimensions, arrangements, and other elements may vary without departing from the scope and spirit of the invention, and therefore, the embodiments referred to herein should not be considered limiting.

Claims

CLAIMS 1. A method for generating biosynthetic corneal tissue, using: at least one configurable membrane as structural support for at least one cell layer, said at least one cell layer being further configurable to functionally couple core endothelial cells allowing their culture (growth), the method comprising the steps of: (a) cultivating endothelial cells on at least one membrane, wherein the culture is carried out at a density in the range of 4000 to 6000 cells / mm 2 ; (b) maintain the culture forming at least a monolayer of cells, generating a construct, where, The culture is maintained for a period of 24 to 48 hours, At least one cell monolayer is hexagonal in shape with a minimum density of 2500 cells / mm³ 2and a coefficient of variation that is in a range from 35% to 45%; (c) perform trepanation of the resulting construct to obtain a “button”; (d) prepare the “button” by forming a “double roll”, where the cells of at least one cell layer are oriented towards the interior of the roll; and (e) insert the construct into an injector device, Where, the construct is inserted into the injector device by means of a surgical viscoelastic liquid.

2. The method according to claim 1, wherein the at least one membrane is an ultrathin membrane, having a thickness in the range of 3 to 7 micrometers.

3. The method according to claim 1, wherein the at least one membrane is any one selected from the group comprising a transparent, biocompatible, biosynthetic, manipulable membrane, combinations thereof and / or similar membranes.

4. The method according to claim 1, wherein the at least one membrane is a collagen membrane.

5. The method according to claim 1, wherein the at least one membrane is a membrane of any material selected from the group comprising hyaluronic acid, chitosan, fibrinogen, elastin, polylactic acid (PLA), polyglycolic acid (PGA), hydrogels, and synthetic materials based on biocompatible polymers such as polyethylene glycol (PEG) or polyester, combinations thereof and / or similar materials.

6. The method according to claim 1, wherein the at least one cell layer is a monolayer of corneal endothelial cells grown on the membrane.

7. The method according to claim 1, wherein the corneal endothelial cells are derived from donated corneal tissue, which is obtained from transplant remnant tissue (sclerocorneal rings), corneal buttons (central part of donated corneas) and enucleated eyeballs.

8. The method according to claim 4, wherein the at least one membrane is a collagen membrane combined with any selected from the group comprising hyaluronic acid, chitosan, fibrinogen, elastin, polylactic acid (PLA), polyglycolic acid (PGA), hydrogels, and synthetic materials based on biocompatible polymers such as polyethylene glycol (PEG) or polyester, combinations thereof and / or similar.

9. The method according to claim 4, wherein the collagen is present in the membrane in a range from 10% to 50% and is type I or type III collagen.

10. The method according to claim 1, wherein the monolayer obtained has an adequate coefficient of variation and sufficient cell adhesion, ensuring that the cell detachment fraction is less than 50% under centrifugation tests; 11. The method according to claim 1, wherein the construct is pre-stained with trypan blue to facilitate its visualization and manipulation; 12. The method according to claim 1, wherein the at least one membrane has a degradability control, degrading approximately 40% after 21 days under laboratory conditions and remaining intact for up to 90 days post-transplant.

13. The method according to claim 1, wherein the at least one membrane has a water absorption capacity of between 1050% and 1167%.

14. The method according to claim 1, wherein the endothelial cells cultured on the at least one membrane have a diameter of between 6 and 12 mm.

15. The method according to claim 1 includes the step of staining the construct with trypan blue before loading it into the injector device, to facilitate visualization and manipulation of the graft.

16. The method according to claim 1, wherein the injector device is a microinjector device.

17. The method according to claim 1 further includes introducing the construct into the anterior chamber of the eye with the cells oriented towards the aqueous humor.

18. The method according to claim 1, wherein the at least one membrane has a water absorption capacity between 1050% and 1167% during the first 21 days.

19. Use of the ultrathin membrane generated from the method according to claims 1 to 19, for corneal endothelium regeneration, in corneal transplant therapies for endothelium regeneration.

20. Use according to claim 19, wherein the at least one membrane, when implanted in the anterior chamber of the eye by means of an injector device, enables: efficient integration with the patient's corneal tissues; a control of membrane degradability, degrading in a controlled manner (approximately 40% after 21 days and remaining intact up to 90 days post-transplant); and Maintaining adequate water absorption (between 1050% and 1167%) promotes hydration and stability of eye pressure, contributing substantially to the restoration of corneal clarity and the regeneration of endothelial tissue.

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