WO2012085298A1

WO2012085298A1 - Fibrin gel containing sclerocorneal limbus cells and the use thereof in ocular surface bioengineering

Info

Publication number: WO2012085298A1
Application number: PCT/ES2010/070865
Authority: WO
Inventors: Margarita Calonge Cano; Francisco Javier IGLESIAS MUÑOZ; Elena VUELTA LÓPEZ; Manuel GONZALO ORDEN
Original assignee: Universidad De Valladolid (40%); Universidad De Leon (20%); Fundación Clinica San Francisco De León (40%)
Priority date: 2010-12-22
Filing date: 2010-12-22
Publication date: 2012-06-28

Abstract

The invention relates to fibrin gel containing sclerocorneal limbus cells and the use thereof in ocular surface bioengineering. The present invention relates to an autologous fibrin gel that includes stroma cells from the sclerocorneal limbus (CES) and epithelial cells from the sclerocorneal limbus (CEP) (corneal stem cell niche) and a method for the production thereof. Said gel is used in the preparation of medications for use in the treatment and/or prevention of degenerative, inflammatory or genetic diseases or lesions affecting the ocular surface or in respect of diseases for which this technique could also be used for tissue engineering.

Description

Fibrin gel containing sclerocorneal limbus cells and their use in ocular surface bioengineering

The present invention falls within the field of biomedicine, regenerative medicine and tissue engineering with stem cells (advanced therapies) and refers to a fibrin gel comprising stromal cells of the sclerocorneal limbus (preferably fibroblasts). More preferably said gel further comprises epithelial cells of the sclerocorneal limbus (preferably stem cells). Likewise, the present invention also relates to different methods of preparing said gels and their use in the elaboration of advanced therapy medications for the treatment and / or prevention of degenerative, inflammatory or genetic lesions or diseases of the ocular surface. STATE OF THE PREVIOUS TECHNIQUE

In recent years, tissue engineering techniques have acquired great importance in the development of new therapies for the resolution of various types of pathologies. Specifically, in the field of ophthalmology, these techniques have focused mainly on the development of biological substitutes that facilitate the repair of ocular surface defects. This is the case of some of the treatments currently available against various diseases that affect the corneal surface, such as those that are included in the group of pathologies classified as limbic insufficiency syndromes (SIL). Clinically, syndromes characterized by destruction and / or dysfunction of epithelial stem cells or SIL, would need a replacement of the corneal-scleral limbus and not just the current approach, a corneal transplant, since donor buttons lack stem cells, due to what these transplants are bound to fail. One of the most booming treatments for the resolution of this group of Pathologies consists of the transplantation of epithelial cells of the limbus (the niche of the epithelial stem cells of the cornea) cultured or expanded ex vivo. When the limbar niche fails, only subsequent failure of the corneal epithelium occurs, not of the other layers of the cornea, stroma or endothelium. But it is true that a certain pathological process could also affect the entire thickness of the cornea and, in these cases, in addition to needing a reconstruction of the limbar niche, a cornea transplant that replaces the stroma (lamellar keratoplasty) will be needed later , the endothelium (endothelial keratoplasty) or both layers (penetrating keratoplasty). SIL cases cannot be solved with a corneal transplant of any kind if, before, the limbar niche is not reconstructed.

Examples of artificial or biological corneas used as equivalents of the human cornea are those built on substrates of collagen and chondroitin sulfate with glutaraldehyde, containing cell lines developed from cells isolated from the three layers of the cornea, epithelium, stroma and endothelium, which are immortalized by viral transfection (May Griffith, et al., 1999, Science; 286: 2169-2172). Another biological substitute for the cornea is based on fibrin gels with agarose, which requires the cultivation of the three cell layers present in the cornea: epithelial, stromal and endothelial cells. In this method, endothelial cells are seeded at the base of a porous culture, a human fibrin gel is developed with agarose containing stroma cells or fibroblasts cultured therein, which is placed on the endothelial cell layer. Finally, epithelial cells grow on the surface of all this support, from the healthy limbus of the recipient subject. Although in these studies it has been postulated that pure fibrin is not always comparable to the stroma of the cornea in terms of its transparency and consistency, so the use of fibrin and agarose gels is proposed instead (Miguel Alaminos, et al., 2006, Investigative Ophthalmology & Visual Science; 47: 8, 331 1-3317). On other occasions, the stromal stem cells of the human cornea have been cultured in pellets, in the absence of a rigid support. In this case, this cell growth on pellets has been compared with that which occurs on a fibrin gel, from which it has been concluded that these gels are malleable and fragile and do not allow the deposition of a dense matrix (Yiqin Du, et al. , 2007, Investigative Ophthalmology & Visual Science; 48: 1 1, 5038-5045). Furthermore, in these tests it has not been demonstrated that said cells are indeed stem cells.

However, as indicated above, artificial or biological corneas, such as those described in the previous paragraph, require a healthy limbar niche for their success after transplantation, a problem that is currently being solved by transplanting expanded limbal cells in cultivation

Under normal physiological conditions, the stem cells of the corneal epithelium are located in the basal region of the limbar epithelium, in structures known as Vogt palisades. This location is extremely important for the maintenance not only of the phenotype of this cell type but also of its capabilities and functions as an adult stem cell. It is the microenvironment in which said cell population is that determines its potential as an adult stem cell. Said microenvironment or niche is composed of the rest of cell types that are in the vicinity of the population of stem cells of the corneal epithelium, by the surrounding extracellular matrix and, very importantly, by the basal lamina and the underlying stroma in the that are scattered limbal fibroblasts. It has been described that the contact between the epithelial and stromal regions of the limbus, as well as the communications that established between the cell types of both components, they are essential for maintaining the characteristics of the stem cells of the limbar epithelium. During the usual mechanisms of maintenance of the corneal epithelium under normal conditions, or in cases of alterations of the corneal surface, the population of stem cells of the limbar epithelium, which is normally in the quiescent state, is induced to undergo division processes, Migration and differentiation. It is the moment in which the stem cells of the limbar epithelium leave that niche when they begin to differentiate and therefore lose their adult stem cell characteristics.

For this reason, it is of vital importance that during the ex vivo expansion of this cell type (in the culture laboratory or in the cleanrooms, if they are already for human use) the conditions are reproduced, as far as possible, microenvironments in those found in native tissue. Thus, in recent years different types of substrates or supports have been studied on which to expand said cell population. Examples are the amniotic membrane, the 3T3 irradiated fibroblast support layers (Pellegrini G., et al., 1997, The Lancet; 345: 990-993) and the fibrin gels (Han B., et al., 2002, Cornea; 21: 505-510). During the mechanisms of natural regeneration after an injury the epithelial cells migrate through a natural matrix composed of fibrin and fibronectin. In addition, it has been proven that fibroblasts cultured in fibrin gels proliferate, migrate and are capable of synthesizing extracellular matrix components, such as collagen (Tuan T., et al., 1996, Experimental Cell Research; 223: 127-134 ). Therefore, one of the approaches has been the in vitro culture of epithelial cells of the limbus on a basal layer of 3T3 irradiated murine fibroblasts, so that this construction can be subsequently embedded in a fibrin gel. Currently, tissue bioengineering techniques developed in this regard are pursuing the search for an adequate support that allows not only the correct expansion of limbar epithelium cells under in vitro culture conditions, but also the subsequent use of said cell population in the treatment of ocular surface alterations. As support for said cell expansion, a biomaterial that is biocompatible, biodegradable and that allows cell adhesion and proliferation is required.

Generally, the use of biomaterials that act as a support for cell growth for clinical purposes is associated with a possible induction of the inflammatory response. In addition to this drawback, it is necessary that said support be biocompatible, and therefore, allow the proliferation of sclerocorneal limb epithelial cells while maintaining the characteristics that define them as stem cells of the corneal epithelium, and therefore, can fulfill the repair function of the tissue, which is very complex, and can trigger immune responses in the transplant patient.

DESCRIPTION OF THE INVENTION

The present invention relates to a fibrin gel comprising stromal cells of the sclerocorneal limbus (preferably fibroblasts). More preferably said gel further comprises epithelial cells of the sclerocorneal limbus (preferably stem cells). Likewise, the present invention also relates to different methods of preparing said gels and their use in the preparation of therapeutic drugs. advanced for the treatment and / or prevention of degenerative, inflammatory or genetic lesions or diseases of the ocular surface. Any gel of the present invention, constructed by tissue bioengineering techniques, can be used for the reconstruction of the ocular surface, both of the limbus and of the cornea or conjunctiva, by means of its transplantation into the damaged tissue allowing its regeneration, or It can be used in the preparation of medicines for the treatment of those same diseases for which this technique would also be used as tissue engineering.

Preferably, the gel of the invention is a tissue engineering product consisting of two constructs: 1) a substitute for the limbar niche (corneal stem cell niche) made by a fibrin gel, preferably autologous, comprising two cell types from said limbar niche, namely epithelial cells (in undifferentiated monolayer) and stromal cells, preferably fibroblasts, and 2) a corneal epithelial construct, composed of a fibrin gel, preferably autologous, embedded with the same epithelial cell type described above but in differentiated multilayer.

Fibrin, in this sense, is an excellent biomaterial because it does not present toxicity, it is able to promote the expansion not only of the epithelial component, but also of cells of a fibroblastic nature, also allowing the construction of three-dimensional tissue substitutes, and due to that its obtaining from plasma isolated from a blood or cryoprecipitate sample is relatively simple, allows the manufacture of a scaffold of a totally autologous nature, which avoids the possible problems of the appearance of inflammatory reactions after implantation, associated with the use of other types of biomaterials. In addition, the cells included in the gel can also come from the patient himself. Said stromal cells of the sclerocorneal limbus (ESC) or epithelial cells of the sclerocorneal limbus (CEP) are isolated from the niche of sclerocorneal limbus stem cells. As a consequence, said cells may have stem cell characteristics. In this sense, the gel of the invention reproduces the microenvironmental conditions in which said epithelial and fibroblast cells are found in the native tissue, thus allowing the maintenance of their characteristics. Thus, the gel of the invention can be used as artificial tissue to increase, restore or partially or totally replace the functional activity of a sclerocorneal limbus that exhibits destruction, non-existence or disturbance of the functionality of the stem cells, as is the case. of the pathologies encompassed under the name of limbic insufficiency syndrome (SIL).

Thus, a first aspect of the invention relates to a fibrin gel, hereinafter "gel of the invention", which comprises stromal cells of the sclerocorneal limbus (hereinafter the acronym CES can be used to refer to these cells ). In a preferred embodiment, the ESCs are fibroblasts of said stroma. In a more preferred embodiment, the fibroblasts express the beta 1 integrin protein. In even more preferred embodiments, said ESCs come from a human, where preferably their origin is autologous. Hereinafter, reference may be made to any of the fibrin gels defined in this paragraph as "CES gel of the invention".

The integrin beta 1 protein is a known marker of mesenchymal stem cells. As demonstrated in the examples of the present invention, the cells from the stromal of the sclerocorneal limbus expanded in a specific culture medium of fibroblasts, express at least the beta 1 mesenchymal integrin stem cell marker, but they also express typical fibroblast markers and retain their characteristics.

In the present description "fibrin" is understood as the fibrillar protein capable of forming three-dimensional networks, which plays an important role in the coagulation process, is shaped like a cane with three globular areas and the property of forming aggregates with other fibrin molecules forming a soft clot. Normally found in the blood in an inactive form, fibrinogen, which, by the action of thrombin, is transformed into fibrin, which has the ability to polymerize, in the presence of a cofactor, for example, calcium.

"Sclerocorneal limbus" means the circular area, slightly raised above which corresponds to the transition line between the cornea and the conjunctiva-sclera. The sclerocorneal limbus is the corneal stem cell niche. The main cell types of the sclerocorneal limbus are epithelial cells and stromal cells or fibroblasts. The gel of the invention comprises stromal cells of the limbus, preferably fibroblasts, more preferably embedded inside, expanding in the fibrin lattice. The characterization of the cell types of the sclerocorneal limbus can be performed by, for example but not limited to, the identification of surface and / or intracellular proteins, genes, and / or other markers. Methods that can be used for characterization include, but are not limited to: immunocytochemical analysis, northern blot analysis, western blot analysis, RT-PCR, microarray gene expression analysis, or morphological analysis by optical or electron microscopy.

In the present invention, the cells of the sclerocorneal limbus comprised in the gel are characterized, but not limited to, by immunocytochemical analysis. Thus, "fibroblasts" are understood as those cells present in the stromal layer of the limbus that are characterized, but not limited to, being positive for staining with the antibodies against the antigens ALDH1A1, α-SMA (smooth muscle actin alpha), type II collagen, β1 and Cx43 integrin ( connexin 43), but not to staining with the pancytokeratin antibody.

A "stem cell or stem cell" is a cell capable of dividing indefinitely, of self-renewal and of differentiation into specialized cells, not only morphologically but also from a functional point of view. Of the cells present in the sclerocorneal limbus, some of them, both fibroblasts and epithelial, are stem cells and are therefore able to divide and differentiate to replace injured or senescent cells of the ocular surface with new cells, thus maintaining a cell population constant. As demonstrated in the present invention, limbo fibroblasts are very important for the maintenance of the niche in which the stem cells of the corneal epithelium are located. These cells can be obtained by any method known in the state of the art for this purpose, for example, but not limited to, any of the cell digestion and isolation protocols described in the examples of the present invention.

The corneal epithelium has the ability to regenerate rapidly thanks to the sclerocorneal limbus epithelium cells. After an injury, maintenance of the corneal epithelium is achieved by this population of epithelial cells. The stem cells of the sclerocorneal limbus epithelium are located in the basal epithelium of the limbus.

In another preferred embodiment, the gel of the invention comprises, in addition to CES, sclerocorneal limbus epithelium cells (hereinafter the acronym CEP can be used to refer to these cells). A Most preferred embodiment of the present invention relates to the fibrin gel in which the CEPs express the beta 1 integrin protein and the pancytokeratin protein. As can be seen in the examples of the present invention, the recovered and expanded CEP express pancytokeratin, however said protein is not expressed in the recovered and expanded CES. In an even more preferred embodiment the CEPs are stem cells. In another even more preferred embodiment the CEPs come from a human. Even more preferably, the CEPs are of autologous origin. Hereinafter, any of the fibrin gels defined in this paragraph may be referred to as the "CEP gel of the invention".

As demonstrated in the examples of the present invention, cells from the sclerocorneal limbus epithelium expanded in a specific culture medium of epithelial cells express at least the beta 1 mesenchymal integrin stem cell marker.

Preferably the CEPs come from the basal region of the limbar epithelium. These cells can be obtained by any method known in the state of the art for this purpose, for example, but not limited to, any of the cell digestion and isolation protocols described in the examples of the present invention. Thus, in the present invention "epithelial cells of the sclerocorneal limbus" means those cells isolated from the tissue of the limbus that are characterized, but not limited to, being positive for staining with antibodies against the antigens ALDH1A1, α-SMA (smooth muscle actin alpha), desmoplachin, β1 integrin, pancytokeratin and Cx43.

Another preferred embodiment of the present invention relates to the fibrin gel where said gel has a disk shape. Preferably the disk has a diameter of at least the sum of the arc curve length of the central curvature of the cornea and the length of the sclerocorneal limbus of the ocular surface An even more preferred embodiment refers to the fibrin gel wherein said disk comprises:

to. a central fibrin region having the diameter of at least the arc curve length of the central curvature of the cornea, comprising CEP, and

b. a region of peripheral fibrin to the central region of section (a) comprising CES.

According to another preferred embodiment, the peripheral region of the preceding paragraph further comprises CEP. Hereinafter, reference may be made to any of the fibrin gels defined in this paragraph as "gel of the invention comprising the central and peripheral region". Preferably the central region of section (a) of the gel of the invention comprising the central and peripheral region consists of fibrin and CEP (preferably stem cells), and the peripheral region of section (b) consists of fibrin and CES (preferably fibroblasts) . According to another preferred embodiment, the central region of section (a) of the gel of the invention comprising the central and peripheral region consists of fibrin and CEP (preferably stem cells), and the peripheral region of section (b) consists of fibrin, CES (preferably fibroblasts) and CEP (preferably stem cells).

Another preferred embodiment relates to the fibrin gel wherein said disk comprises: (a) a layer of CEP or a layer of fibrin comprising CEP, and (b) a layer of fibrin comprising CES. According to a more preferred embodiment, the layer of section (b) comprises: (i) a hollow central region having the diameter of the curve arc length of the central curvature of the cornea, and (ii) a region of peripheral fibrin to the central region of section (i) comprising CES. According to another preferred embodiment, the fibrin layer of section (b) further comprises CEP. Another preferred embodiment relates to the fibrin gel in which said disk further comprises a fibrin layer located between the section layer (a) and section layer (b). According to another preferred embodiment of the present invention, the CEPs are stem cells and the ESCs are fibroblasts. Hereinafter, reference may be made to any of the fibrin gels defined in this paragraph as "gel of the invention comprising the CEP-CES layers". Preferably the layer of section (a) of the gel of the invention comprising the CEP-CES layers consists of fibrin and CEP (preferably stem cells), and the layer of section (b) consists of fibrin and CES (preferably fibroblasts). According to another preferred embodiment, the layer of section (a) of the fibrin gel of the invention comprising the CEP-CES layers consists of fibrin and CEP (preferably stem cells), and the layer of section (b) consists of fibrin, CES (preferably fibroblasts) and CEP (preferably stem cells). In another preferred embodiment of this aspect of the invention, the gel fibrin of the invention is derived from blood or cryoprecipitate plasma, more preferably from a human. In an even more preferred embodiment the blood plasma or cryoprecipitate is of autologous origin. In the present description "blood plasma" means the liquid and acellular fraction of blood, composed of water and multiple substances dissolved in it, of which the most abundant are proteins. It also contains carbohydrates and lipids, as well as metabolic waste products. Fibrin could be obtained from plasma by, for example, but not limited to, the action of thrombin on the fibrinogen present in the plasma; or it could be obtained from cryoprecipitate (product obtained from plasma by centrifugation and cryopreservation) by thawing the cryoprecipitate, dissolving it and calculating the amount of fibrinogen to later obtain the fibrin matrix as explained above. The advantage of using cryoprecipitate as a source of fibrin is that it is obtained with identical concentrations of its components, since while in the plasma there are "undesirable products" that can be trapped inside the fibrin matrix, the cryoprecipitate allows to obtain the fibrin almost in its purest form. However, since the cryoprecipitate does not have platelets (which could imply a slowdown in cell growth), it would be advisable to add platelets to the culture medium or even to the fibrin matrix itself obtained from it.

Although CES or CEP (and the respective fibroblasts or stem cells), as well as the blood plasma or cryoprecipitate from which the fibrin is obtained, comprised in the gel of the invention could proceed, for example, but not limited to, from any mammal, preferably from a human, the fact that all the components of the gel come from the patient himself who could be transplanted, or who could be given a medication that understands it, eliminates the possibility that he suffers an immune rejection and / or an inflammatory response. Therefore, preferably the gel fibrin of the present invention is autologous, that is, it is derived from the blood plasma or cryoprecipitate of the patient himself.

In the present description, "autologous origin" means any origin of the sample taken from the tissues, fluids or cells of an individual or patient that is the same in a donor and in the recipient thereof when they are administered after treatment or transplanted after modification.

Another aspect of the invention relates to a method of preparing the CES gel of the invention, from now on the term "first method of the invention" can be used to refer to this method, which comprises: (a) putting CES contact with a composition comprising fibrin; and (b) polymerize the mixture from step (a) in a container of sterile culture. In a preferred embodiment, the first method of the invention further comprises, between steps (a) and (b), adding an antifibrinolytic agent to the mixture of step (a). The ESCs (or, where appropriate, later, the CEPs) are obtained, for example, but not limited, by means of digestion protocols of the limbar tissue, as described in the examples of the present invention.

The cells obtained with digestion are grown under in vitro conditions to achieve an expansion of the cell types sufficient to be able to make the gels. Examples of culture media could be, but not limited to, DMEM (Dulbecco ^{^} s Modified Eag / es Medium), RPMI 1640, Ham's F12, Ham's F10, MCDB 131, MEM {Minimum Essential Media) or DMEM / F12. The culture media may be supplemented with other components suitable for cell growth, such as, but not limited to, serum, serum substitute, amino acids, antibiotics or growth factors, in appropriate proportions. Primary cultures are those from cells that have been broken down from an original tissue taken from an organ of an animal. Secondary cultures are those that come from the reseeding or subculture of one or several types of cells of a primary culture, and which are preferably carried out when the primary cultures are in a preconfluence state by performing an expansion pass or subculture In these secondary cultures the cellular heterogeneity is lower than in the primary ones due, among others, to nutritional factors.

To prepare the fibrin gel containing embedded CES (preferably fibroblasts), the cells, previously cultured, are resuspended in a composition comprising fibrin (preferably the solution is plasma or cryoprecipitate). As explained above, fibrin preferably comes from a human, and more preferably it is of autologous origin, whereby blood plasma or cryoprecipitate, which is where fibrin is obtained, also preferably comes from a human and more preferably is of autologous origin.

Next, an antifibrinolytic agent is optionally added to the mixture, to prevent fibrinolysis of the gel. "Antifibrinolytic agent" means any agent that decreases or eliminates fibrinolytic activity that degrades fibrin networks formed during the blood coagulation process. Examples of antifibrinolytic agents in the present invention are, without limitation, tranexamic acid, PEPH (p-hydroxybenzoic acid propyl ester) or aprotinin. The antifibrinolytic agent of the first method of the invention is preferably tranexamic acid, being understood as such, the synthetic compound used to neutralize fibrinolysis, whose mechanism of effect involves the inhibition of clot dissolution.

In order for the mixture thus obtained to begin to polymerize, it is necessary to add an agent capable of inducing said polymerization, such as, but not limited to, a source of calcium. Said calcium source is preferably a calcium salt, such as, but not limited to, calcium chloride, calcium gluconate or a combination of both. In a more preferred embodiment, the calcium salt is calcium chloride or CaCI ₂ . "Polymerization" means the chemical process by which reagents or monomers (compounds of low molecular weight) are chemically grouped together, giving rise to a molecule of greater molecular weight, called polymer. Preferably, the polymerization of step (b) of the first method of the invention is carried out at a temperature of 37 ° C. The mixture is then deposited in a sterile culture vessel, preferably in a culture plate, such as It is shown in the examples of the present invention, to allow its solidification and gel formation.

In another preferred embodiment, the composition comprising fibrin of step (a) is blood plasma or cryoprecipitate, more preferably blood plasma. Even more preferably the blood plasma comes from a human. In an even more preferred embodiment, the blood plasma is of autologous origin. Another aspect of the invention relates to a method of preparing the CEP gel of the invention that comprises the steps of the first method of the invention and further comprises sowing CEP on the fibrin gel obtained in step (b). Hereinafter the term "second method of the invention" can be used to refer to this method. Sowing of CEP on the fibrin gel obtained in step (b) is carried out in a sterile culture vessel, preferably a culture plate. Seeding of the second method of the invention can be carried out directly on the fibrin gel obtained in step (b) of the first method of the invention once it has polymerized or on another fibrin gel which, once polymerized, is polymerize the fibrin gel obtained in step (b) of the first method of the invention once both gels show stability. Limb epithelial cells, previously cultured in any of the culture media described above, but not limited to, can be seeded on the surface of this fibrin structure with embedded fibroblasts for expansion and proliferation. "Proliferation" means the processes of growth, expansion, adhesion, division, multiplication or differentiation that cells undergo. Another aspect of the invention relates to a method of preparing the gel of the invention comprising the central and peripheral region, which comprises:

to. contacting CES, or said CES and CEP, with a composition comprising fibrin,

b. contacting CEP with a composition comprising fibrin, and

C. Polymerize in a sterile culture vessel the mixtures of steps (a) and (b) so that the mixture comprising CEP occupies a central region having the diameter of at least the length of the arc of curve of the central curvature of the cornea, and the mixture comprising CES occupies the peripheral region to said central region.

Hereinafter the term "third method of the invention" can be used to refer to this method. In a preferred embodiment, the third method of the invention further comprises, between steps (b) and (c), adding an antifibrinolytic agent to the mixture of step (a) and the mixture of step (b). Another aspect of the invention relates to a method of preparing the gel of the invention comprising the CEP-CES layers, comprising the steps of the third method of the invention, where the polymerization of step (c) is carried out by such that the mixture comprising the ESCs, or comprising said ESCs and CEP, and the mixture comprising the CEPs form two layers. Hereinafter the term "fourth method of the invention" can be used to refer to this method.

According to a preferred embodiment of the fourth method of the invention, the preparation of the gel of the invention comprising the CEP-CES layers comprises: (a) polymerizing the mixture comprising CES, or which comprises said ESC and CEP, in the peripheral region (3) of a well without the polymerized gel exceeding the upper part of the central elevation (2); and (b) polymerizing the mixture comprising the CEPs above the polymerized fibrin gel layer according to step (a). The term "above" refers to the polymerized mixture comprising the CEPs according to step (b) being in a position higher than the polymerized fibrin gel layer according to step (a), having as reference the bottom of the well . That is, in contact with the bottom of the well is the layer of the passage (a) and above it is the layer of the passage (b). Both layers can be in contact or not, in the case of not being directly in contact, they are separated by a layer of fibrin. According to a preferred embodiment, a fibrin layer is polymerized on the polymerized layer according to step (a) and on this polymerized fibrin layer CEP is deposited, or on said fibrin layer the mixture comprising fibrin and CEP is deposited and polymerized. More preferably, the CEPs are stem cells and the ESCs are fibroblasts. Hereinafter the term "fifth method of the invention" may be used to refer to any of the methods in this paragraph. Another preferred embodiment of the present invention relates to the first, second, third, fourth or fifth method of the invention, wherein the antifibrinolytic agent is tranexamic acid. In another preferred embodiment of the first, second, third, fourth or fifth method of the invention, polymerization is carried out by adding CaCl ₂ to the respective mixtures.

Another aspect of the present invention relates to a well for carrying out the fifth method of the invention, which comprises a central elevation (2) closed in its upper part, which has the diameter of the curvature curve arc length central cornea, and a peripheral region (3) to the central elevation (2), where the height of the central elevation (2) is less than the wall height of the well (1) and where the well has a diameter of at least the sum of the arc curve length of the central curvature of the cornea and the length of the sclerocorneal limbus of the ocular surface. A graphic representation of said well can be seen in Fig. 8A and 8 B. According to a preferred embodiment, the well has the diameter corresponding to the sum of the arc curve length of the central curvature of the cornea and the length of the sclerocorneal limbus of the ocular surface. Another aspect of the present invention relates to a solid support comprising at least one well described in the preceding paragraph. The solid support is preferably a multiwell plate.

Another aspect of the invention relates to the use of the well described in the preceding paragraphs, or of the solid support comprising it, for the preparation of the gel of the invention comprising the CEP-CES layers.

A further aspect of the present invention relates to the use of the CES gel of the invention as a support for the proliferation of CEP. Preferably the CEPs are stem cells.

Fibrin is a biocompatible material that is reabsorbed once it is implanted in vivo, allowing the formation of extracellular matrix and tissue regeneration. Therefore, the transplantation of any gel of the invention involves the proliferation of the corneal epithelium and, as a consequence, tissue regeneration. Thus, preferably, the gel of the invention is used for transplantation on a dysfunctional ocular surface.

Insufficient contribution, absence or dysfunction of limb epithelial cells causes alterations in corneal epithelial repair, resulting in persistent epithelial defects, corneal vascularization, scarring, chronic inflammation, ulceration, growth of the conjunctival epithelium on the cornea (conjunctivalization) or a probable corneal perforation, known as limbic insufficiency syndrome. The normal replacement of the corneal epithelial cells and the healing of lesions or diseases that affect the ocular surface, such as but not limited to, corneal ulcers or superficial keratitis, depends on the proper functioning of the sclerocorneal limbus, since it is here where the stem cells of the corneal epithelium are housed. The transplantation of the gel of the invention containing CEP (preferably stem cells of said epithelial tissue of the limbus) and / or CES (preferably fibroblasts), involves the restoration of epithelial cells of the limbus, which contributes to the regeneration of lesions or diseases of the ocular surface Therefore, another aspect of the invention relates to the use of any fibrin gel of the invention for the manufacture of a medicament. According to a preferred embodiment, any fibrin gel of the invention is used for the preparation of a medicament for use in the treatment and / or prevention of ocular surface lesion, degenerative disease of the ocular surface, inflammatory disease of the ocular surface or genetic disease of the ocular surface. In the present invention, "ocular surface" means the surface of the eye that corresponds to the sclerocorneal limbus, the cornea or the conjunctiva. Preferably the ocular surface is the ocular surface that corresponds to the cornea.

The medicament referred to in the present invention can be for human or veterinary use. The "medicine for human use" is any substance or combination of substances that is presented as having properties for the treatment or prevention of diseases in humans or that can be used in humans or administered to humans in order to restore, correct or modify physiological functions by exerting a pharmacological, immunological or metabolic action, or establishing a medical diagnosis. The "medicine of veterinary use "means any substance or combination of substances that is presented as having curative or preventive properties with respect to animal diseases or that can be administered to the animal in order to restore, correct or modify its physiological functions by exercising a pharmacological, immunological action or metabolic, or to establish a veterinary diagnosis.

The term "treatment" as understood in the present invention refers to combating the effects caused as a result of the disease or pathological condition of interest in a subject (preferably mammal, and more preferably a human) that includes:

(i) inhibit the disease or pathological condition, that is, stop its development;

(ii) alleviate the disease or the pathological condition, that is, cause the regression of the disease or the pathological condition or its symptomatology; (iii) stabilize the disease or pathological condition.

The term "prevention" as understood in the present invention consists in preventing the onset of the disease, that is, preventing the disease or pathological condition from occurring in a subject (preferably mammal, and more preferably a human), in particularly, when said subject has a predisposition for the pathological condition, but has not yet been diagnosed as having it.

Another preferred embodiment of the present invention relates to the use of any fibrin gel of the invention for the preparation of a medicament for the treatment and / or prevention of a limbic insufficiency syndrome (SIL). "Limbic insufficiency syndrome or SIL" is the term used to encompass all pathologies that occur with the disruption of functionality, destruction or non-existence of stem cells of the sclerocorneal limbus. Among the pathologies encompassed under this term are, for example, but not limited to, those caused by chemical or thermal burns, autoimmune healing conjunctivitis (such as, but not limited to, Steven-Johnson syndrome or scarring penfigoid), those caused by surgery or cryotherapy, contact lens-induced keratopathy, microbial infections, ectasia, those caused by ionizing or ultraviolet radiation, those caused by the use of medication such as anti-metabolites or 5-fluorouracil or mitomycin c (iatrogenic) ); those congenital pathologies such as, for example, but not limited to, aniridia, coloboma, neoplasia, corneal dystrophy, hormonal deficiencies, peripheral ulcerative diseases of the cornea, neurotrophic keratopathy, chronic bullous keratopathy, chronic limbitis, pterigium or idiopathic limbar insufficiency, or in general, corneal blindness.

Another preferred embodiment of the present invention relates to the use of any fibrin gel of the invention for the preparation of a medicament for the treatment and / or prevention of corneal blindness.

Another aspect of the invention relates to the use of any gel of the invention for in vitro evaluation of the behavior of CEP cells and / or CES. Preferably the CEPs are stem cells and the ESCs are fibroblasts.

Any gel of the present invention can be useful for studying, for example but not limited to, the growth, proliferation, interaction or phenotypic or genotypic evolution of CEPs (preferably epithelial cells) and / or CES ( preferably of the fibroblasts), so that the analysis of their behavior in vitro can provide evidence of how they behave in I live in the sclerocorneal limbus. In addition, said gel could be used, for example but not limited to, for the search for drugs, pharmacological studies, toxicological studies, pharmacogenomic studies and / or genetic studies. Such assays can be used for the identification and / or characterization of biological targets, bioactive compounds and / or pharmacological agents.

Another aspect of the invention relates to a pharmaceutical composition, hereinafter "pharmaceutical composition of the invention", which comprises any gel of the invention. In a preferred embodiment, the pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier. In a more preferred embodiment, the pharmaceutical composition of the invention comprises, in addition to the pharmaceutically acceptable carrier, another active ingredient.

As used herein, the term "active substance", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological activity or other different effect on the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals.

The pharmaceutical compositions of the present invention can be formulated for administration in a variety of ways known in the state of the art.

Such compositions and / or their formulations can be administered to an animal, including a mammal and, therefore, to man, in a variety of ways, including, but not limited to, intralesional, intraorbital, intracapsular or surgical implant. The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as age, weight, sex or tolerance, of the mammal, preferably human. In the sense used in this description, the term "therapeutically effective amount" refers to the amount of the pharmaceutical composition of the invention that produces the desired effect and, in general, will be determined, among other causes, by the characteristics of said pharmaceutical composition and the therapeutic effect to be achieved. The "adjuvants" and "pharmaceutically acceptable carriers" that can be used in said compositions are the vehicles known in the state of the art.

The pharmaceutical compositions of the present invention can be used in a treatment method in isolation or in conjunction with other pharmaceutical compounds.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1. It shows the cell growth of sclerocorneal limbus fibroblasts in primary cultures (PO) and in the first pass or secondary culture (P1). Limbo fibroblasts seeded at an initial density (IN) of 1 x 10 ⁴ cells / cm ² on culture surfaces of plastic, show a good proliferation in both cultures, primary culture (P0) and secondary culture (P1), without differences between the cells obtained by the inverted digestion protocol (Pinv) and the standard (PE) (p> 0, 05).

Fig. 2. Shows the growth of fibroblasts in fibrin gels that only contain fibroblasts (GFFs) for 14 days. The number of cells growing in these gels is determined on days 3, 5, 7, 10, 12 and 14 of culture, in 20 GFFs with an initial cell density of 1 x 10 ⁴ cells / GFF.

Fig. 3. Shows the expression of differentiation markers in the central cornea (A-G) and in the sclerocorneal limbus (H-N). The markers studied are ALDH1A1 (A, H), α-SMA (B, I), type II collagen (C, J), connexin 43 (D, K), desmoplachin (E, L), integrin β1 (F, M ) and pancytokein (G, N) (at 20 magnifications).

Fig. 4. It shows the expression of differentiation markers in P0 (AG) and P1 (HN) cultures of limbus fibroblasts and in P0 epithelial cells (OU). The markers studied are ALDH1A1 (A, H, O), α-SMA (B, I, P), type II collagen (C, J, Q), connexin 43 (D, K, R), desmoplachin (E, L , S), integrin β1 (F, M, T) and pancytokeratin (G, N, U) (at 20 magnifications). Fig. 5. Represents cell growth in fibrin gels containing fibroblasts (GFFs). Hematoxylin-eosin stained sections of these gels for 14 days under in vitro culture conditions, 10 increases (A) and 20 increases (B). Electron microscopy images of a fibrin gel after cell seeding (C), and of the fibroblasts adhered and growing in the fibrin lattice (D). Fig. 6. It shows the hematoxylin-eosin staining of a paraffin section of the fibrin gel containing limbus cells (GFCL), specifically fibroblasts and epithelial cells of the sclerocorneal limbus (A), where a layer of epithelial cells growing is observed on the construction surface. Electron microscopy image of epithelial cell growth in fibrin gels containing fibroblasts (B) forming a layer with a typical paving stone morphology.

Fig. 7. It shows the corneal region and corneoescleral limbus of the human ocular surface. The circle with the largest diameter indicates the corneoescleral limbus region. The smaller circle that is located within the larger circle indicates the corneal region.

Fig. 8. Shows the adapted well of the present invention. Plan view of the well (A) and the gel components in the well (C). Profile view of the well (B) and the gel components in the well (D). CME: Epithelial Stem Cells of the sclerocorneal limbus. F: Fibroblasts of the sclerocorneal limbus. I: Intermediate fibrin layer. The well parts are: well wall (1), central elevation (2), peripheral region (3).

Fig. 9. Stages of the fibrin gel preparation process of the invention comprising the CEP-CES layers. CME: Epithelial Stem Cells of the sclerocorneal limbus. F: Fibroblasts of the sclerocorneal limbus. I: Intermediate fibrin layer. The well parts are: well wall (1), central elevation (2), peripheral region (3). In A the plan view of the well is shown. B comprises the addition and polymerization of the mixture with F or with F + CME to the peripheral region (2). C comprises the addition and polymerization of the intermediate layer. D comprises the addition of the epithelial stem cell layer of the sclerocorneal limbus or the addition of the mixture comprising fibrin and epithelial stem cells of the sclerocorneal limbus. EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which show the effectiveness of the fibrin gel containing the epithelial cells of the sclerocorneal limbus (CEP) and EStromal cells of the sclerocorneal limbus (CES), as well as the method of preparation of said gel. These specific examples provided serve to illustrate the nature of the present invention and are included for illustrative purposes only, and therefore should not be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.

Example 1. Preparation of the fibrin gel containing two cell types of the sclerocorneal limbus.

PROCESSING OF THE NICHE OF MOTHER CELLS OF THE ESCLEROCORNEAL LIMBO The biological samples used in the examples of the present invention came from pigs, males and females, 6 months old, approximately 80 kg.

Once fresh pig eyes were obtained, for example from any slaughterhouse, they were transported in sterile containers at 4 ° C to the laboratory, immersed in a mixture of DMEM supplemented with penicillin and streptomycin. Once in the laboratory, and under a laminar flow cabinet, the sclerocorneal limbus was removed from each eyeball by cutting the sclera and cornea to 1.5 mm from the limbus. Next, all the uveal tissue was removed with a cotton swab and the limbus was sterilized using a standard protocol (Allamby D, et al., 1997, IOVS, 38: 2064-2072): in First, the samples were immersed for 2 minutes in DMEM supplemented with antibiotics (10 units of penicillin / 10 μg streptomycin / mL), then washed in a 96% alcohol mixture in PBS (Phosphate Buffered Saline) (1: 1 v / v) for 2 more minutes, and finally the samples were washed 3 times in fresh DMEM with a solution of antibiotics for 2 minutes each. The sclerocorneal limbs were trimmed to obtain 1 x 3 mm limbal samples. DIGESTION PROTOCOLS TO OBTAIN THE CELLS OF THE STROMA AND EPITELIALS OF LIMBO

To isolate the two main cell types present in limbar tissue (stromal cells [CES] and epithelial cells [CEP]), limbo samples were digested. In the present example two different enzymatic digestion protocols were carried out, to compare them and finally select the most effective: a) Standard protocol (PE): n = 50 (where n is the number of samples). The samples were digested using a previously described protocol, consisting of depositing the limbus pieces in 35 mm Petri dishes containing 2.5 mL of 0.25% trypsin / 0.02% EDTA and incubating at 37 ° C and 5% of C0 ₂ for 2 hours to achieve dissociation of the CEP. The enzyme activity was neutralized every 30 minutes by adding an equivalent volume of DMEM with 10% fetal bovine serum (FBS). Subsequently, the solution was removed and fresh trypsin was added. Finally, the four solutions obtained were mixed and centrifuged at 1,400 rpm for 10 minutes at room temperature. The precipitate was resuspended in an epithelial cell culture medium (MCE), the cells obtained were counted and their viability was determined using the trypan blue staining method (Phillips HJ., 1973, New York: Academic Press; 406-408). The remaining stromal tissue, not digested during trypsinization, was introduced into 50 ml_ centrifuge tubes and digested with type I collagenase at a concentration of 2 mg / mL in DMEM, at 37 ° C and 5% CO2 for 18- 24 hours, until a complete disintegration of the tissue was observed. The cell suspension obtained was centrifuged at 1,400 rpm for 10 minutes. The precipitate was resuspended in a fibroblast growth medium (MCF), the cells were counted and viability tests were carried out. b) Inverted protocol (Pinv): n = 50. Alternative method to the standard protocol. It consists of the inversion of the order of action of the enzymes used in the previous protocol. First, limbo samples were incubated in a type I collagenase solution (2 mg / mL) for approximately 24 hours, to release CES from limbar tissue. After this incubation, the undigested tissue was removed and deposited in culture plates to be trypsinized, as described above, until complete digestion of the sample. Both cell suspensions obtained in the two steps of digestion were centrifuged at 1,400 rpm and the precipitates were resuspended, counted, and cell viability was determined.

Both digestion protocols differ in the number of total cells obtained per limb tissue sample, so that more cells were obtained with Pinv (p <0.05), although there were no significant differences in cell viability (p = 0 , 3634) (Table 1). In addition, more cells of epithelial origin (after using trypsin / EDTA) and less fibroblasts (after using collagenase) were obtained with Pinv than with PE. Cellular density (mean ± SD) / Cell viability (mean ± SD) (%)

PE digestion (n = 50) Pinv (n = 50) P value (PE vs

Pinv)

Treatment 1, 924 ± 1, 477 / 56,817 ± 33,479 <0.05 / 0.14

with 87.76 ± 28.04 / 95.97 ± 8.01

Trypsin / EDTA

Digestion with 38,476 ± 27,666 1 1,007 ± 8,863 / <0.05 / 0.94 collagenase / 95.81 ± 10.21 95.38 ± 13.79

Trypsin / EDTA 39,005 ± 27,135 62,825 ± 36,554 <0.05 / 0.36 + Collagenase / 93.30 ± 11, 12 / 95.67 ± 8.40

Table 1. Mean ± Standard deviation (SD) (electron microscopy) of cell density and viability after subjecting pig limbo tissues to two different digestion protocols: standard protocol (PE), 1 ^o : trypsin / EDTA , 2 ^o : collagenase; inverted protocol (Pinv), 1 ^or :

collagenase, 2 ^o : trypsin / EDTA.

IN VITRO CELLULAR EXPANSION AND CHARACTERIZATION The CEP and CES obtained from the limb stem cell niche by means of the two digestion protocols described above, were expanded in primary cultures, and the percentage of crop success was calculated. To obtain the primary cultures (P0), the cells of epithelial origin obtained after trypsinization in either of the two digestion protocols were seeded in 1x10 ⁴ plastic culture plates. cells / cm ² and MCE was added. This culture medium consisted of a mixture of DMEM / Ham ^' s F12 (1: 1) with 5% FBS, 10 ng / mL EGF, 5 mg / mL insulin, 100 ng / mL choleric toxin, penicillin / streptomycin (100 units / 100 Mg / mL) and 2 mM L-Glutamine.

To obtain the P0 culture of CES, the stromal cells obtained from samples of limbar tissue after collagenase digestion (second and first step of PE and Pinv, respectively), were plated in plastic culture plates at a density of 1 x10 ⁴ cells / cm ² and were cultured in MCF consisting of DMEM supplemented with 10% FBS, penicillin / streptomycin (100 units / 100 Mg / mL) and 2 mM L-Glutamine.

Both the P0 of cells of epithelial origin and the P0 of cells of stromal origin were maintained at 37 ° C, 5% CO2 and 95% humidity. The medium was changed every 2 or 3 days and the cultured cells were checked daily by inverted microscopy, until they reached the confluence state. Subsequently, the cells were separated from the culture surface using a 0.05% trypsin / 0.02% EDTA solution, counted and used to establish the secondary cultures (P1) and / or for the gel construction of fibrin containing limbus cells (GFCL).

The cell expansion ratio (ER) of the cells in culture was determined at the confluence state of the epithelial and stromal cells in P0 and of the cell cultures in P1. This ratio is calculated as: RE = final cell density / initial cell density.

For characterization, the stromal cells P0 and P1 and the epithelial cells P0 obtained in the Pinv were cultured in cell culture chambers at a cell density of 1x10 ⁴ cells / cm ² until reaching the preconfluence state. The cells are washed twice in PBS and fixed in a mixture of acetone-methanol (1: 1) at -20 ° C for 20 minutes. Samples were stored at this temperature until use.

As control tissue, porcine eyeballs frozen in liquid nitrogen, embedded in OCT (compound of optimum cutting temperature) (Tissue-Tek) were used. 5 to 8 fine sections (μηπ) were fixed in cold acetone at 4 ° C for 10 minutes, mounted on slides, air dried and stored at -20 ° C until stained. Immunofluorescent staining of the fixed sections and cell cultures was carried out as follows: the samples are washed 3 times with PBS, 5 minutes each, and incubated in the 1% Morpho Save solution (Window Mediacal Systems) in water deionized for 15 minutes. After 3 more washes with PBS, the samples were permeabilized with 0.3% Triton X-100 in PBS for 10 minutes and washed again. Non-specific binding sites were blocked with 5% donkey serum in PBS for 1 hour. The samples were incubated with the primary antibodies (Table 2) for 1 hour at room temperature in a humid chamber. After three washes with PBS, the Alexa Fluor 488 conjugated secondary antibody (donkey immunoglobulin G prepared against mouse or rabbit at 1: 300 dilution) was added and incubated for 1 hour in the dark at room temperature. The nucleus was stained with propidium iodide (1: 100) for 10 minutes, for viewing under confocal microscopy (Radiance 2000, BIORAD). For the negative controls, 5% donkey serum in PBS was used instead of the primary antibodies. For immunofluorescence studies in control tissues, only the central cornea and limbal regions were analyzed for the expression of the aforementioned antigens. To ensure the Skill of the results, all immunofluorescence studies were carried out in three specimens. The two digestion protocols used differed in the success rate in P0. Of the 50 limbo biopsies digested with PE, only 24 (48%) produced successful P0 cultures of both cell types, CEP and CES. This ratio increased significantly (p <0.005) when Pinv was used, as the results show, in which 48 biopsies of the 50 digested limbo (96%) were successful in primary crops (P0).

As for the results of the expansion ratio, the cells obtained after collagenase treatment of the tissue samples submitted to the two protocols and seeded for P0 in plastic wells, adhered to the culture surface after 24 hours and resulted in viable cell cultures in 62% of cases for PE and in 96% of cases for Pinv (p <0.005). These crops took the form of spindle and starry when they were grown in MCF and formed a connection network that reached the confluence at 6 or 10 days, showing an expansion rate of 6.76 (± 1, 92, n = 31) for the PE and 6.09 (± 1.74, n = 48) for the Pinv (p = 0.12). The final cell density of the isolated cells is approximately 67,649 (± 19,232, n = 31) and 60,973 (± 17,476, n = 48) cells / cm ² for PE and Pinv, respectively (Figure 1). The P1 cells obtained from the digestion with the two protocols showed the typical morphology of the fibroblasts and the expansion ratio was significantly higher (p <0.05) than in P0 with values of 9.44 (± 2.55, n = 31) and 10.31 (± 2.93, n = 48) for PE and Pinv respectively, showing no significant differences between both digestion protocols. Cell proliferation increased and populations completely colonized the surface of the culture in 3 or 5 days, faster than in P0, indicating that in this time interval the subcultured cells proliferated more slowly compared to the cells of the primary cultures. Between days 6 and 15, cell cultures reached a population number of more than 5x10 ⁵ cells per cm ² of culture surface for stromal cells isolated by PE or Pinv (Figure 1). The percentage of samples that were successful in PO cultures of epithelial cells (after trypsinizing the tissues of the limbus) is 48% for PE and 96% for Pinv (p <0.005). In the PO, the cells adhered to the surface of the culture 24 hours after planting and small colonies of CEP were observed in the early stage of growth (2 days) showing a polygonal morphology. The average time for PO cells, which were at an initial cell density of 1 x 10 ⁴ cells / cm ² , to reach the confluence in a well of 1.9 cm ² was between 10 to 16 days, showing ratio values expansion of 6.03 (± 1, 41, n = 24) and 5.79 (± 1, 44, n = 48) for PE and Pinv, respectively (p = 0.5), reaching a population density cell of approximately 5x10 ⁴ cells / cm ² at the end of P0, for both protocols.

For all this, and since the Pinv provided superior results regarding the establishment of P0, the following steps of this invention, the studies of cellular characterization and the construction of GFFs gels and GFCLs gels, were carried out using only the Expanded cell suspensions obtained from Pinv. CONSTRUCTION AND CHARACTERIZATION OF THE SUBSTITUTES OF THE MOTHER OF LIMBO CELL NICHE: FIBRINE GELS CONTAINING LIMBO CELLS (GFCLs)

CEP and CES cells from cultures P0 and P1, respectively, isolated with the inverted digestion protocol were used for the construction of fibrin gels containing limbus cells (GFCLs).

To prepare the fibrin gels of porcine origin, 9 mL of pig blood were obtained and introduced into plasma collecting tubes containing 0.129 M of sodium citrate (BD Vacutainer Systems, UK). The blood was centrifuged at 1,800 rpm for 10 minutes, the plasma separated, it was introduced into sterile centrifuge tubes and stored at -20 ° C until use. To produce the fibrin gel containing CES, preferably fibroblasts (GFFs) a suspension of 1x10 ⁴ fibroblasts from the P1 limbus was resuspended in 400 µΙ of fresh frozen pork plasma per ml_ of final volume of the fibrin gel. To prevent fibrinolysis of the gel, the mixture was supplemented with 4 mg / mL tranexamic acid (Amchafibrin, Rottapharm SL, Spain), and the final volume was adjusted with MCF. Next, 40 μg / mL of CaCl ₂ (Braun Medical SA) was added to initiate the polymerization of the fibrin. Finally, the mixture was seeded in 24-well culture plates, 1 ml_ per well, for solidification to take place at 37 ° C for 15-30 minutes. As the matrix coagulated in the plastic wells of the culture, the cells spread through the fibrin framework. These fibrin gels containing fibroblasts are 1.9 cm ² in size and approximately 4 mm thick.

After 24 hours, during which the fibrin gel stabilization containing fibroblasts occurred, the cells became spindle-shaped and began to proliferate (Fig. 2), spread and migrate, projecting their dendritic processes in a three-dimensional pattern and establishing intercellular contacts that eventually formed a dense cellular network.

Next, the P0 limbo CEPs were seeded on top of this structure or GFF, at a cell density of 5x10 ⁴ cells / cm ² , to construct the fibrin gel containing these two cell types of the limbo or GFCL, and maintained with MCE under in vitro conditions until the cells had completely covered the construction surface, for approximately 14 days, being monitored daily with an inverted microscope. For characterization studies, frozen sections of porcine eyeballs, CEP and CES of pig limbo from cell cultures were stained by indirect immunofluorescence to study the expression of the following markers: pancytokeratin, ALDH1A1, α-SMA (a-smooth muscle actin), type II collagen, connexin 43 (Cx43), desmoplachin and integrin β1 (Table 2). The results of the expression of these proteins are shown in Table 3.

Antibody Origin Dilution Specificity

Mouse monoclonal antibody - All cells

Abcam 1: 50

Epithelial pancytokein (80)

Mouse monoclonal antibody-

Sigma 1: 50 Myofibroblasts (A-5228) a-SMA

Rabbit Polyclonal Antibody

Sigma 1: 500 Gap Connections Connection 43 (C-6219)

Rabbit polyclonal antibody - Proteins

Serotec 1: 200

Desmoplaquina-1, -2 (AHP320) Desmosome

Rabbit Polyclonal Antibody

Aldehyde keratocytes dehydrogenase type I Abcam 1: 50

the cornea

(ab23375) ALDH1A1

Cell surface receptor

Rabbit Polyclonal Antibody

Scbt 1: 50

lntegrin-βΐ (M-106)

Mesenchymal cells

Monosan 1:15

Rabbit Polyclonal Antibody - Differentiation Type II collagen (PSX1060) cell

Synthesis of extracellular matrix

Table 2. Primary antibodies used for immunofluorescence characterization of the two cell types isolated from the two digestion protocols.

Table 3. Summary of immunofluorescence results of cell cultures and tissue sections (++, very high detection; +, detection; -, no detection). ¹ Basal expression of cytoplasmic actin in all stromal cell cultures. ² Detection in basal and superficial epithelial cells in the region of the central cornea. No expression is detected in the basal epithelial cells of the limbus. ³ Typical expression pattern of corneal keratocytes or fibroblasts.

In the ocular tissue sections, the presence of these antigens in the epithelium and stroma of the central cornea and the limbal regions was analyzed (Figure 3). All porcine cornea keratocytes and limbar stromal fibroblasts were positive for staining with antibodies against ALDH1A1, α-SMA, type II collagen, connexin 43 (Cx43) and β1 integrin, with different levels of expression depending on the marker. So antibodies against α-SMA, type II collagen, connexin 43 (Cx43) and integrin β1 showed a stronger staining than ALDH1A1. The presence of the desmoplachin antigen in the stroma of tissue sections was only located at the conjunctive level. No staining was observed in the stroma for pancytokeratin, of which only signal was obtained in the epithelial cells, which exhibited a different expression pattern in the superficial and basal cells, showing a more marked fluorescence in most of the superficial layers of the epithelium . The epithelial cells stained with all the antibodies used, showing a clear expression of ALDH1A1, α-SMA, integrin β1, desmoplaquine and Cx43, both in the central cornea and in the limbus. In the epithelial regions a weaker staining of type II collagen was observed, which does not appear to represent a significant difference when the central cornea and limbar region were compared, except for Cx43. The basal cells of the cornea were stained for this marker but not the cells of the basal region of the limb epithelium. For immunofluorescence studies in cultured cells, P0 and P1 limbo fibroblasts and P0 epithelial cells were used (Figure 4). CES cultured isolated from the stroma showed staining for all antibodies used except for pan-cytokeratin. The expression of differentiation markers such as ALDH1A1, type II collagen, β1 integrin and α-SMA, revealed that these cells maintained their fibroblast phenotype under in vitro culture conditions.

The cells in culture showed a strong staining for desmoplaquine and β1 integrin, which indicated the existence of adhesion structures to the cell culture surface. Integrin β1 is a known marker of mesenchymal stem cells. The epithelial cells in P0 showed a pattern of antigen expression similar to that of stroma cells, differing only in that the former are positive for the pancytokeratin marker.

Example 2. Evaluation of fibrin gels.

EVALUATION OF CELLULAR GROWTH IN FIBRINE GELS

The ESCs embedded in the fibrin gels were determined by counting the number of cells in each gel at days, 3, 5, 7, 10, 12 and 14. Fibrin gels containing CES were digested to determine cell proliferation. Culture medium was removed, fibrin gels were collected and trimmed before being introduced into sterile centrifuge tubes. Then, the gels were digested by adding 5 mL of a 1 mg / mL solution of type I collagenase for 3 hours at 37 ° C. The samples were carefully pipetted and the separated cells were recovered by centrifugation and counted. Epithelial cells grown in fibrin gels containing CES were analyzed daily by inverted microscopy until the cell population formed a confluent monolayer. The ESCs embedded in the fibrin matrix show a high proliferation capacity reaching a cell population of approximately 1 x 10 ⁶ cells / GFF, after 14 days under in vitro culture conditions (Figure 5), showing an expansion rate of 1 19 , 43 (± 34.67, n = 20).

EVALUATION OF THE CONTRACTION OF FIBRINE GELS

To determine the possible contraction of the fibrin gels containing CES, the thickness of the fibrin gels cultured for 0 and 14 days in wells of 1, 9 cm ² , was measured before and after recovering the gels from the wells (n = twenty). 20 fibrin gels of 15 days in culture containing fibroblasts in pure ethanol were introduced for 30 minutes, then washed 3 times for 30 minutes with PBS. Next, the gels were embedded in fresh MCF for 30 minutes. The diameter and thickness of the gels were measured before and after being embedded in MCF to determine their possible contraction.

No retraction of fibrin gels containing fibroblasts or GFFs was observed in the culture wells, but maintained their initial diameter of 1.6 cm and capillary-mediated adhesion to the wall of the culture well. Although there was a slight narrowing of the gels of approximately 1.5 mm in the central region, reaching a final thickness of 1 mm. When these GFFs were separated from the wells and treated with ethanol, they showed a diameter of 1,117 (± 0,09546, n = 28) cm, which indicated that they suffered a contraction percentage of 30,1339% (± 5.96662%, n = 28), if compared with the initial diameter of the gel. When the GFFs were rehydrated with MCF, they showed no variations in the gel diameter, maintaining a diameter of 1,128 (± 0.09546, n = 28) cm, so that there are no significant differences between the dried and rehydrated gel (p <0.05). In terms of thickness, the GFFs were separated from the wells and treated with ethanol and with MCF, maintaining the depth of 1 mm that they showed in the culture wells after 14 days.

The gels easily separated from the surface of the culture wells, demonstrating their elastic capacity, so that they were able to spread without tearing.

MICROSCOPIC EVALUATION Optical microscopy

For histological evaluation, fibrin gels containing fibroblasts or GFFs of 0, 3, 5, 7, 10, 12 and 14 days and fibrin gels containing the two cell types of limbus (CEP and CES) or GFCLs of 14 days in culture, they were fixed in 4% paraformaldehyde. The samples were dehydrated in increasing concentrations of alcohol and Xylene solutions previously embedded in paraffin. Sections of 6 to 8 μηι were stained with hematoxylin-eosin and visualized in the optical microscope (Optiphot 2, Nikon) at 10, 20 and 40 magnifications.

Hematoxylin-eosin analysis of fibrin gels containing CES showed the expansion of fibroblasts through the gels (Figure 5).

Hematoxylin-eosin staining of the paraffin embedded sections of the fibrin gels containing the two cell types of the limbus or GFCLs grown for 14 days showed the growth of the epithelial cells in fibrin gels containing fibroblasts (Figure 6A).

Electron microscopy

Fibrin gels containing CES (preferably fibroblasts (n = 5)) and fibrin gels containing the two cell types of limbus (epithelial cells and fibroblasts) (n = 5) grown for 14 days were fixed in glutaraldehyde at 4 %, then fixed in 2% osmium tetraoxide for 2 hours, and passed through an increasing gradient of alcohol for dehydration. The samples were dried (Balzers CPD 030), mounted on an aluminum matrix and fired with gold-coated particles (Balzers SCD 004) before being examined in an electron microscope (JEOL 6100).

The observation in the electron microscope showed that the surface morphology of the fibrin gels containing CEP and CES had a wavy structure with numerous polygonal cells that showed similar features to those of the epithelial surface of the native epithelial tissue (Figure 6B).

STATISTIC ANALYSIS

To statistically compare the recovery efficiency of the two cell types of interest from both digestion protocols (PE and Pinv) and the value of the crop expansion ratio, its use a two-sided test ("two tailed tesf") and the test t-Student independent of the sample after reviewing the normal distribution of the samples A p value equal to or less than 0.05 is considered statistically significant To measure the differences in the success rate of the primary cultures a Chi ² test is used . Example 3. Preparation of the fibrin gel of the invention comprising the CEP-CES layers by adapted wells.

For the preparation of the fibrin gels, the well described in Fig. 8 A and B was used. In this figure the plan view of the well (A) and profile of the well (B) having the following parts are shown: wall of the well (1), central elevation (2), peripheral region (3). The well can be part of a multiwell plate. Fig. 8 C and D shows the well comprising CEP, CES and fibrin. The figure shows as an illustrative example that the components are: CME, Epithelial Stem Cells of the sclerocorneal limbus; F, Fibroblasts of the sclerocorneal limbus; I, Intermediate fibrin layer. Said well has the dimensions suitable for the cornea and for the sclerocorneal limbus indicated in Fig. 7. The fibrin gel of the invention made by using the described well comprises the following steps, graphically indicated in Fig. 9:

Step 1: The fibrin gel with the stromal cellular component embedded inside was added to the peripheral region (3) of the well for polymerization without covering the upper surface of the central elevation (2).

Step 2: When the previous mixture polymerized a new layer of fibrin was added that covered the entire well without exceeding the well wall (1). Step 3: After complete polymerization of the second fibrin layer, the epithelial component was added.

In this way, it was achieved that in the peripheral region of the well there was a stroma with CES (preferably fibroblasts) and CEP (preferably epithelial stem cells) on it and that in the central region there was only epithelial component, analogously to what happens on the ocular surface in the limbus and cornea respectively.

Claims

one . Fibrin gel comprising stromal cells of the sclerocorneal limbus (CES).

2. Fibrin gel according to claim 1, wherein the ESCs are fibroblasts.

3. Gel according to claim 2, wherein the fibroblasts express the beta 1 integrin protein.

4. Fibrin gel according to any one of claims 1 to 3, wherein the ESCs come from a human.

5. Fibrin gel according to any of claims 1 to 4, wherein the ESCs are of autologous origin.

6. Fibrin gel according to any one of claims 1 to 5 which further comprises epithelial cells of the sclerocorneal limbus (CEP).

7. Fibrin gel according to claim 6, wherein the CEPs express the beta 1 integrin protein and the pancytokeratin protein.

8. Fibrin gel according to claim 6, wherein the CEP are stem cells.

9. Fibrin gel according to any of claims 6 to 8, wherein the CEPs come from a human.

10. Fibrin gel according to any of claims 6 to 9, wherein the CEPs are of autologous origin.

eleven . Fibrin gel according to any one of claims 1 to 10, wherein said gel is disk-shaped.

12. Fibrin gel according to claim 1, wherein said disk has a diameter of at least the sum of the arc curve length of the central curvature of the cornea and the length of the sclerocorneal limbus of the ocular surface.

13. Fibrin gel according to claim 12, wherein said disk comprises:

14. Fibrin gel according to claim 13, wherein the peripheral region further comprises CEP.

15. Fibrin gel according to claim 12, wherein said disk comprises:

to. a layer of CEP or a layer of fibrin comprising CEP, and

b. a fibrin layer comprising CES.

16. Fibrin gel according to claim 15, wherein the layer of section (b) comprises: i. a hollow central region having the diameter of the curve arc length of the central curvature of the cornea, and

ii. a region of peripheral fibrin to the central region of section (i) comprising CES.

17. Fibrin gel according to any of claims 15 or 16, wherein the fibrin layer of section (b) further comprises CEP.

18. Fibrin gel according to any of claims 15 to 17, wherein said disk further comprises a fibrin layer located between the layer of section (a) and the layer of section (b).

19. Gel according to any of claims 13 to 18, wherein the CEPs are stem cells and the ESCs are fibroblasts.

20. Method of preparing the fibrin gel according to any one of claims 1 to 5 comprising:

to. contacting CES with a composition comprising fibrin, and

b. Polymerize the mixture from step (a) in a sterile culture vessel.

twenty-one . Method according to claim 20 further comprising, between steps (a) and (b), adding an antifibrinolytic agent to the mixture of step (a).

22. A method according to any of claims 20 or 21 wherein the composition comprising fibrin of step (a) is blood plasma.

23. Method according to claim 22, wherein the blood plasma is derived from a human.

24. Method according to any of claims 22 or 23, wherein the blood plasma is of autologous origin.

25. Method of preparing the fibrin gel according to any of claims 6 to 10 comprising the steps of the method according to any of claims 20 to 24, and further comprising sowing CEP on the fibrin gel obtained in step (b).

26. Method of preparing the fibrin gel according to any of claims 13 or 14 comprising:

to. contacting CES, or said CES and CEP, with a composition comprising fibrin,

b. contacting CEP with a composition comprising fibrin, and

C. polymerize in a sterile culture vessel the mixtures of steps (a) and (b) so that the mixture comprising CEP occupies a central region described in claim 13, and the mixture comprising CES occupies the peripheral region described in said claim 13.

27. A method according to claim 26, further comprising, between steps (b) and (c), adding an antifibrinolytic agent to the mixture of step (a) and the mixture of step (b).

28. Method of preparing the fibrin gel according to any of claims 15 to 17 comprising the steps of the method according to any of claims 26 or 27, wherein the polymerization of step (c) is carried out such that the mixing which comprises the ESCs, or which comprises said ESCs and CEP, and the mixture comprising the CEP forms two layers.

29. Method according to claim 28, wherein the fibrin gel preparation of any of claims 16 or 17 comprises: a. polymerizing the mixture comprising CES, or comprising said CES and CEP, in the peripheral region (3) of a well without the polymerized gel exceeding the upper part of the central elevation (2), and

b. Polymerize the mixture comprising the CEPs above the polymerized fibrin gel layer according to step (a).

30. A method according to claim 29, wherein a fibrin layer is polymerized on the polymerized layer according to step (a) and on this polymerized fibrin layer CEP is deposited, or on said fibrin layer the mixture comprising is deposited and polymerized fibrin and CEP.

31. Method according to any of claims 20 to 30, wherein the CEPs are stem cells and the ESCs are fibroblasts.

32. Method according to any of claims 20 to 31, wherein the antifibrinolytic agent is tranexamic acid.

33. Method according to any one of claims 20 to 32, wherein the polymerization is carried out by adding CaCl ₂ to the respective mixtures.

34. Well for carrying out the method described in any of claims 29 or 30, comprising a central elevation (2) closed at its upper part, having the diameter of the length of the curve arc of the central curvature of the cornea, and a peripheral region (3) to the central elevation (2), where the height of the central elevation (2) is less than the wall height of the well (1) and where the well has a diameter of at least the sum of the arc curve length of the central curvature of the cornea and the length of the sclerocorneal limbus of the ocular surface.

35. The well according to claim 34, wherein said well has the diameter corresponding to the sum of the arc curve length of the central curvature of the cornea and the length of the sclerocorneal limbus of the ocular surface.

36. Solid support comprising at least one well described in any of claims 34 or 35.

37. Use of the well according to any of claims 34 or 35, or of the support according to claim 36, for the preparation of the fibrin gel according to any of claims 16 or 17.

38. Use of the fibrin gel according to any one of claims 1 to 5 as a support for the proliferation of CEP.

39. Use according to claim 38, wherein the CEP are stem cells.

40. Use of the fibrin gel according to any of claims 1 to 19 for the preparation of a medicament.

41. Use of the fibrin gel according to claim 40 for the preparation of a medicament for use in the treatment and / or prevention of lesion, degenerative, inflammatory or genetic disease of the ocular surface.

42. Use of the fibrin gel according to claim 41 for the treatment and / or prevention of a limbic insufficiency syndrome.

43. Use of the fibrin gel according to any of claims 41 or 42 for the treatment and / or prevention of corneal blindness.

44. Use of the fibrin gel according to any of claims 1 to 19 for the in vitro evaluation of the behavior of the CEP and / or CES.

45. Use according to claim 44, wherein the CEPs are stem cells and the ESCs are fibroblasts.

46. Pharmaceutical composition comprising the gel according to any one of claims 1 to 19.

47. Pharmaceutical composition according to claim 46, further comprising a pharmaceutically acceptable carrier.

48. Pharmaceutical composition according to any of claims 46 or 47, further comprising another active ingredient.