WO2020183047A1

WO2020183047A1 - Decellularised sclerocorneal limbus

Info

Publication number: WO2020183047A1
Application number: PCT/ES2020/070168
Authority: WO
Inventors: Manuel CARO MAGDALENO; Miguel Alaminos Mingorance
Original assignee: Universidad De Granada; Servicio Andaluz De Salud
Priority date: 2019-03-08
Filing date: 2020-03-09
Publication date: 2020-09-17

Abstract

The invention describes a biomimetic limbus obtained from decellularised sclerocorneal limbus, artifical fabric and composition comprising said limbus, in addition to its use in the preparation of a drug for treating limbal insufficiency or as a cell factor and/or drug release system.

Description

LIMBO SCLEROCORNEAL DESCELLULES CURLY

FIELD OF THE INVENTION

The present invention is within the field of medical treatments, regenerative medicine and tissue engineering, and more specifically within the field of treating patients using stem cells and biomaterials.

BACKGROUND OF THE INVENTION

In unilateral total limbal insufficiencies (IL), in which the adelph eye has been shown to be healthy, the optimal option is the limbar autograft, provided the patient is willing to allow surgery to remove part of the sclerocorneal limbus from its only healthy eye, which is rare. On the other hand, in the case of bilateral total ILs, a histocompatible living donor (DVH) allogeneic transplant is possible, normally a relative of the patient, which will be the choice, seeking the greatest possible adjustment for histocompatibility. Likewise, in many cases it is difficult to find histocompatible relatives who allow such surgery, never without risks. As a last option, due to a higher failure rate, the cadaver donor allogeneic (DC) (Miri et al., 2010. Ophthalmology 117 (6): 1207-13). One of the advantages of the autograft and the histocompatible donor is freshness, since the time from when the limbal explants are extracted until they are transplanted is minimal. In the cadaveric donor, they will spend a minimum of 24-48 hours -with luck- before being transplanted. Although it should be noted that the main advantage of these is the better histocompatibility between donor and recipient, being perfect in the case of autograft and worse in DVH, all the worse depending on the level of equality of the histocompatibility complexes. In the case of a cadaver donor, in terms of histocompatibility, it will be the worst option of the three mentioned. On the other hand, in the case of autograft, the problem of possible infectious infections is avoided.

As we have mentioned, if the limbic insufficiency is bilateral, due to the impossibility of carrying out the transplantation of autologous cells, an allogeneic cadaver donor transplant is used (Tan et al., 1996. Ophthalmology 1996; 103 (1): 29 - 36).

One of the advantages is the greater availability that we have of this, since we can take advantage of the limbal rim of keratoplasties and we will have the entire limbus. Donor corneas discarded due to refractive surgery, have leukoma, or any other defect can also be used, since only the impeller and we are going to decellularize it. On the contrary, the main disadvantage is the lack of immunocompatibility and freshness, at least 24-48 h after the extraction, not counting the inadequate manipulation of the blade in said extraction. In the longest series, cadaveric allogeneic transplants do not have a survival greater than 5 years (Miri et al., 2010. Ophthalmology 117 (6): 1207-13).

The risk of rejection and the need to follow a postoperative immunosuppressive treatment -with its side effects- if HLA compatibility is not complete is another of its great disadvantages (Young et al., 1999. Br J Ophthalmol Dec; 83 (12): 1409). Maruyama-Hosoi et al. (Cornea, 25 (2006), pp. 377-382) found rejection reactions in a series of 121 limbus allogeneic transplants in 13% of cases despite systemic immunosuppressive treatment, with generalized rejection in those who received only topical immunosuppression except in one case.

The surgical technique for removing the limbal explant from a cadaveric donor will depend on whether we have the entire globe or only the anterior sclerocorneal cap. The latter is used more frequently in penetrating and lamellar keratoplasties, and therefore, we have it more easily. Using the entire eyeball and fixing it with gauze, it is possible to lick them from the cornea towards the limbus with a Graefe knife. Holland modified this technique from Thoft's keratoepithelioplasty, calling this technique lenticule (Holland EJ. Epithelial transplantation for the management of severe ocular surface disease. In: Transactions of the American Ophthalmological Society 1996. p. 677-743) . Dua et al. (2010; Clin Experiment Ophthalmol [Internet] 2010 Mar [cited 2014 Dec 2]; 38 (2): 104-17) describes another technique using the entire eyeball, maintaining ocular pressure by injecting air with a syringe through the optic nerve, in order to perform a partial trepanation at 200-250 microns and then with a crescent-type knife, dissect towards the limbus and cut radially with Westcott-type scissors (Dua H et al., 2010. Clin Experiment Ophthalmol 38 (2): 104-17). On the other hand, when only the sclerocorneal cap is available, after trepanning the corneal button for keratoplasty, the limbal rim can be dissected in different ways. A first way would be to carry out a lamellar dissection from a cut at a marked depth, with a precalibrated blade, either diamond or steel with a stop, to obtain a lamella of 150-250 microns. After this dissection, we will pull with the help of forceps from one end to the other to perform a mechanical dissection by traction, as we would when cutting a cloth (Dua H et al., 2010. Clin Experiment Ophthalmol 38 (2): 104-17 ). Finally, we can also dissect from the cornea to the sclera, with the help of an assistant who fixes the tissue, dissecting a lamella with a knife to a depth such as to leave the anterior third of the corneal thickness, dividing each keratolimbar ring into two halves or " crescents ”(crescent). Holland called this last technique corneoscleral crescent extraction (Holland EJ. Epithelial transplantation for the management of severe ocular surface disease. In: Transactions of the American Ophthalmological Society 1996. p. 677-743) (Holland et al., 2003. Ophthalmology 110 (1): 125-30). Then, the dissection with a crescent knife is carried out until the limbus passes, reaching one millimeter of the sclera, leaving a small conjunctiva flap to preserve the structure of the Vogt palisades. Subsequently, it is cut radially with Westcott scissors to obtain fragments of the desired shape, using two donor eyes for each one of the recipient, since the graft will be placed on the posterior edge of the recipient's limbus, thus increasing the circumference to be covered, so that we will use three halves of two eyes or a complete ring plus a wedge of the second eye.

It would therefore be necessary to find a substitute that presents a similar anatomy, composition and structure, that does not cause immunogenicity problems or causes rejection despite immunosuppressive treatment, and if possible, that does not require immunosuppressive treatment.

BRIEF DESCRIPTION OF THE INVENTION

A first aspect of the invention refers to a decellularized human sclerocorneal limbus, preferably obtained by a method that comprises washing the leaflet with at least one decellularizing agent.

Decellularizing agents can be physical, chemical, or enzymatic.

In a preferred embodiment of this aspect of the invention, the decellulating agent is a nonionic surfactant.

In another preferred embodiment, the decellularizing agent is selected from sonication, UV rays, NaCl, Triton X-100, Benzalkonium Chloride (BAK), Igepal, or SDS. More preferably it is about 0.1% SDS.

A second aspect of the invention relates to a recellularized sclerocorneal limbus comprising the decellularized human sclerocorneal limbus according to the first aspect of the invention. Preferably the cells that are used to recellularize are selected from the list consisting of: corneal stromal cells (keratocytes), connective tissue cells (fibroblasts), mesenchymal stem cells, or any combination thereof.

A third aspect of the invention relates to an artificial tissue comprising the decellularized human sclerocorneal limbus according to the first aspect of the invention, or the recellularized sclerocorneal limbus according to the second aspect of the invention. A fourth aspect of the invention refers to a composition comprising the decellularized human sclerocorneal limbus according to the first aspect of the invention, the recellularized sclerocorneal limbus according to the second aspect of the invention, or the artificial tissue according to the third aspect of the invention.

A fifth aspect of the invention relates to the decellularized human sclerocorneal limbus according to the first aspect of the invention, the recellularized sclerocorneal limbus according to the second aspect of the invention, the artificial tissue according to the third aspect of the invention, or the composition according to the fourth aspect aspect of the invention, for use as a medicine.

A sixth aspect of the invention relates to the decellularized human sclerocorneal limbus according to the first aspect of the invention, the recellularized sclerocorneal limbus according to the second aspect of the invention, the artificial tissue according to the third aspect of the invention, or the composition according to the fourth aspect of the invention, to increase, restore or partially or totally replace the functional activity of a diseased or damaged tissue or organ.

A seventh aspect of the invention relates to the decellularized human sclerocorneal limbus according to the first aspect of the invention, the recellularized sclerocorneal limbus according to the second aspect of the invention, the artificial tissue according to the third aspect of the invention, or the composition according to the fourth aspect aspect of the invention, for the treatment of partial or total unilateral or bilateral limbar insufficiency.

An eighth aspect of the invention relates to the decellularized human sclerocorneal limbus according to the first aspect of the invention, the recellularized sclerocorneal limbus according to the second aspect of the invention, the artificial tissue according to the third aspect of the invention, or the composition according to the fourth aspect aspect of the invention, for the treatment of any epitheliopathy, of a corneal ocular disease that does or does not involve corneal neovascularization.

A ninth aspect of the invention relates to the use of decellularized human sclerocorneal limbus according to the first aspect of the invention, the recellularized sclerocorneal limbus according to the second aspect of the invention, the artificial tissue according to the third aspect of the invention, or the composition according to the fourth aspect of the invention, as a release system for cellular factors and / or drugs.

A tenth aspect of the invention refers to a kit or device comprising the elements necessary to obtain the decellularized human sclerocorneal limbus according to the first aspect of the invention, the recellularized sclerocorneal limbus according to the second aspect of the invention, the artificial tissue according to the third aspect of the invention, or the composition according to the fourth aspect of the invention

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. (A) Original limbus. (B) Decellularized limbus. The figures show the limbus fragment carved in its "original" state prior to decellularization (A) and subsequently decellularized (B). In the latter, it can be seen that there are no cell nuclei stained with DAPI (fluorescent dye that specifically binds to DNA)

Figure 2. Recellularized blade with WH. Decellularized fragments (LimboScaffoId ©) and recellularized with mesenchymal cells of the umbilical cord (WH) Wharton jelly are observed. It is observed how the cells are marked with DAPI mainly in the superficial zone, although some cells appear inside the stroma of the limbus.

Figure 3. Recellularized limbus with FOM. Decellularized fragments (LimboScaffoId ©) and recellularized with human oral mucosa fibroblasts (FOM) are observed. It is observed how the cells are marked with DAPI mainly in the superficial zone, although some cells appear inside the stroma of the limbus.

DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention have developed an artificial limbus, which comprises a scaffold and mesenchymal cells, where the scaffold or support has an anatomy, composition and structure identical to the original, since it is decellularized libus, but does not present immunogenicity to not presenting donor cells. For the first time, a method is described to obtain a totally decellularized human limbus, which represents a great advance in avoiding possible rejections. The decellularized limbus serves as a natural niche for stem cells and other cell types, and can be obtained from both cadavers and donors undergoing keratoplasty. In addition, it allows its use as a delivery system for cellular factors or drugs.

DECELULARIZED LIMBO OF THE INVENTION

As the sclerocorneal limbus is a surplus tissue from both penetrating and lamellar keratoplasties and complete limbs are available, great material is available to use as a scaffold without the need to generate it through tissue engineering. Therefore, a first aspect of the invention relates to a decellularized human sclerocorneal limbus, preferably obtained by a method that comprises washing the leaflet with at least one decellularizing agent.

Decellularizing agents are known in the state of the art, and can be, among others, physical, chemical or enzymatic agents.

Sodium dodecyl sulfate (SDS or NaDS) is an anionic surfactant compound with amphiphilic properties required for all detergents.

Even more preferably, the decellularization method is an in vitro method that comprises: a) cutting the sclerocorneal cap into 4 semicircular fragments of similar lengths, b) trimming the clear cornea until leaving only 1 mm of cornea and 2 mm of sclera, c ) fix each fragment on a support, preferably a pad, d) delaminate and dissect the anterior 1/3 of the corneo-scleral stroma, e) wash the sclero-corneal limbus resulting from step (e) in physiological serum or phosphate buffer (PBS ), f) immerse in a decellularizing agent solution, preferably in SDS, and even more preferably in 0.1% SDS in water or in PBS, g) incubate at room temperature for 12 to 48 hours with shaking (between 100 and 300 rpm), renewing this solution every 12 hours. h) wash with PBS for 24-48 hours at room temperature while stirring, renewing the PBS every 12 hours.

Fixation of the fragments on the pad of step (c) can be done, among other means and without limitation, by using sterile surgical cyanoacrylate. Other alternatives may be the use of needles. The pad can be anything that is sterile, a Petri dish, or a microtome holder. The delamination of step (d) results in sheets of approximately, but not limited to, between 50 and 350 microns, more preferably between 80 and 300 microns, even more preferably between 100 and 200 microns, and in a particular embodiment the thickness of the sheets is approximately 150 microns, preferably 150-250 microns. It can be carried out, for example, using a 2.75 mm triangular knife with cutting edges that is inserted in the middle of the semicircular fragment trying to go to ½ or 1/3 corneal and then dissect to the left and right

In another preferred embodiment of this aspect of the invention, the sclerocorneal limbus is obtained from a cadaveric donor. In another preferred embodiment, the sclerocorneal limbus can be obtained from leftover tissue from keratoplasties, whether penetrating or lamellar. In another preferred embodiment of this aspect of the invention, said limbus has a depth of between 50-300 pm, more preferably between 100-250 pm, and even more preferably approximately 200 pm.

In another preferred embodiment, the decellularized limbus of the invention comprises only the anterior part of the sclerocorneal limbus. In this specification, "anterior part of the sclerocorneal limbus" is understood as the area comprising the anterior third (1/3) of the sclerocorneal cap that includes the sclerocorneal limbus.

Although most conditions were able to eliminate a significant percentage of corneal cells, very few protocols were able to eliminate more than 90% of the cells from the tissues (González-Andrades et ai, 2015. Transí Vis Sci Technol. 2015; 4 ( 2): 13).

These authors found that the efficiency of the most important protocols did not depend on time, suggesting that the type of agent and its concentration are the most important factors related to the efficiency of cell elimination. Although it is an important factor for the decreaseiularisation of other types of tissues, the presence of the SCL barrier in entire corneas can even decreaseiularisation for the longer incubation times. Therefore, the deceleration of whole corneas may not be time dependent, with the exception of certain detergents that are known to have a strong deceleration power, such as SDS. As expected, higher concentrations of SDS were associated with improved deceiularization efficiencies, with a positive correlation with agent concentration.

The term "sclerocorneal limbus" refers to the area that corresponds to the transition between the sclera and the cornea. It is especially relevant because its blood vessels supply the cornea. The main importance of the sclerocorneal limbus is that it is where the limbal stem cells are in charge of re-epithelializing the cornea in constant desquamation. The entire corneal epithelium is replaced every 7 days. The area where it is collected the aqueous humor is behind everything and is called the iridocorneal angle. Another important function is that it is the area where the lymphatic vessels are in addition to the blood vessels and, of course, where the sensory nerves penetrate that collect the sensory information from the cornea and protect it from possible attacks, forcing the mechanism of blinking and phenomenon Bell, protector of the main lens or diopter of the eye). From the outside inwards, it is composed of: conjunctiva, Tenon's capsule, episcleral lamina, stroma of the sclerocorneal limbus, aqueous humor drainage system, and Schlemm's duct. On the other hand, it is characterized in that the stroma presents a transitional tissue between the cornea and the sclera, and its collagen fibers become disorganized as in the sclera.

The term "decellularization" refers to a process for the isolation of the extracellular matrix of a tissue from the cells that inhabit it, leaving a scaffold or scaffold of the original tissue. That is, a natural biomaterial is created that supports cell growth and differentiation and tissue growth. They are obtained through combinations of physical, chemical and enzymatic treatments, ensuring that the structure is not lost and they are suitable for the tissue where they are used. There are mainly two decellularization processes: by perfusion and by immersion.

In another specific embodiment of this aspect of the invention, the scaffold contains growth factors or other agents.

RECELLULARIZED SCLEROCORNEAL LIMBO OF THE INVENTION

A second aspect of the invention relates to recellularized sclerocorneal limbus, hereinafter "recellularized sclerocorneal limbus of the invention", which comprises the decellularized human sclerocorneal limbus and, in addition, at least one cell. Preferably the cells that are used to re-cellularize are human cells, more preferably the cells are selected from the list consisting of: corneal stromal cells (keratocytes), connective tissue cells (fibroblasts), stem cells, or any of their combinations.

In another preferred embodiment of this aspect of the invention, the stem cells are selected from the list consisting of mesenchymal stem cells, limbal cells of the eye, hemotopoietic stem cells, non-human embryonic stem cells, induced pluripotent stem cells, adult stem cells, mesothelial cells, umbilical cord blood stem cells, or any combination thereof. Preferably the stem cells are umbilical cord mesenchymal cells and / or limbal cells.

The term "stem cell" refers to a cell with clonogenic, self-renewing and differentiating capacity in multiple cell lines. In particular, cells Stem essences have the ability to proliferate extensively and form colonies of fibroblast cells. As used in the present invention, the term "stem cell" refers to a pluripotent or multipotent cell, capable of generating one or more differentiated cell types, and which also possesses the ability to self-regenerate, that is, to produce more stem cells. The “totipotent stem cells” can give rise to both embryonic components (such as the three embryonic layers, the germ line and the tissues that will give rise to the yolk sac), as well as extra-embryonic components (such as the placenta). That is, they can form all cell types and give rise to a complete organism. The "pluripotent stem cells" can form any cell type corresponding to the three embryonic lineages (endoderm, ectoderm and mesoderm), as well as the germ and the yolk sac. They can, therefore, form cell lines, but a complete organism cannot be formed from them. "Multipotent stem cells" are those that can only generate cells from the same layer or embryonic lineage of origin. The bone marrow is home to at least two distinct stem cell populations: mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs). In the context of the present invention, stem cells are selected from the group comprising mesenchymal stem cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, adult stem cells, or combinations thereof. In a particular embodiment, the stem cells are stem cells from a mammal, preferably human.

In another preferred embodiment of this aspect of the invention, the pluripotent stem cells have been obtained by a method that does not involve the destruction of human embryos.

For their part, mesenchymal cells have a regenerative and immunomodulatory effect, and therefore, an anti-inflammatory effect (immune pathologies), they also have a “homing” effect (they go where there is damage), plasticity (conversion to the cellular phenotype influenced by the humoral environment where they are placed), are not potentially tumorous and have little or no immunogenicity.

Therefore, in a particular embodiment, the stem cells are mesenchymal stem cells, preferably human mesenchymal stem cells. Even more preferably they are human mesenchymal cells derived from adipose tissue. In another even more preferred embodiment, they are human mesenchymal cells from the umbilical cord.

The term "adult stem cell" refers to that stem cell that is isolated from a tissue or organ of an animal in a growth state subsequent to the embryonic state. Preferably, the stem cells of the invention are isolated in a postnatal state. They are preferably isolated from a mammal, and more preferably from a human, including neonates, juveniles, adolescents, and adults. Adult stem cells can be isolated from a wide variety of tissues and organs, such as bone marrow (mesenchymal stem cells, multipotent adult progenitor cells, and hematopoietic stem cells), adipose tissue, cartilage, epidermis, hair follicle, skeletal muscle, heart muscle, intestine , liver, neuronal.

The term "embryonic stem cell" or "ESC" are cells derived from the inner cell mass of embryos in the blastocyst stage, with the capacity for self-renewal and differentiation in all types of adult cells. Embryonic stem cells are able to proliferate indefinitely in vitro, maintaining an undifferentiated state and a normal karyotype through prolonged culture. They also have the ability to differentiate into cells of the three embryonic germ layers (mesoderm, endoderm and ectoderm; (Itskovitz-Eldor, et al., Mol. Med. 6: 88-95, 2000) and germ lineage. Embryonic stem cells represent a powerful system model for investigating the mechanisms underlying pluripotent cell biology and differentiation in the early embryo, as well as providing opportunities for genetic manipulation. Embryonic stem cells have been isolated from ICM from multiple species blastocyst stage embryos (Bhattacharya, et al., BMC Dev. Biol. 5:22, 2005), including mice (Solter and Knowles, Proc. Nati. Acad.

USA 75: 55655569, 1978.), swine (Chen, et al., Theriogenology 52: 195-212, 1999), and non-human primates (Thomson, et al., Proc. Nati. Acad. USA). USA 92, 7844-7848, 1995).

Methods for obtaining embryonic stem cells are widely known and can be put into practice by the skilled person without the need for excessive experimentation. Primate embryonic cells can be isolated from blastocysts of different primate species (Thomson et al., Proc. Nati. Acad. Sci. USA, 92: 7844). Methods for obtaining human embryonic stem cells without the need to destroy the embryo are also known.

In order to avoid the use of human embryos, it is possible to use non-human transgenic animals as a source of embryonic stem cells. In particular, US5,523,226 describes methods for generating transgenic pigs that can be used as donors for xenotransplantation to humans. WO97 / 12035 describes methods for producing transgenic animals suitable for xenotransplantation. Also, W001 / 88096 describes immunocompatible animal tissues. These immunocompatible animals can be used to generate pluripotent embryonic cells as described in US6,545,199. Likewise, the use of embryonic stem cell lines is possible, which can be of different origin.

The term "mesenchymal stem cell" or "MSC", as used herein, refers to a multipotent stromal cell, originating from the mesodermal germ layer, that can differentiate into a variety of cell types, including osteocytes (bone cells), chondrocytes (cartilage cells), and adipocytes (fat cells). Markers expressed by mesenchymal stem cells include CD105 (SH2), CD73 (SH3 / 4), CD44, CD90 (Thy-1), CD71, and Stro-1 as well as adhesion molecules CD106, CD166, and CD29. Among the negative markers for MSCs (not expressed) are the hematopoietic markers CD45, CD34, CD14, and the costimulatory molecules CD80, CD86 and CD40 as well as the adhesion molecule CD31. MSCs can be obtained from, without being limited to, bone marrow, adipose tissue (such as subcutaneous adipose tissue), liver, spleen, testes, menstrual blood, amniotic fluid, pancreas, periosteum, synovium, skeletal muscle, dermis, pericytes, trabecular bone, human umbilical cord, lung, dental pulp and peripheral blood. MSCs according to the invention can be obtained from any of the above tissues, such as from bone marrow, subcutaneous adipose tissue or umbilical cord. Most of these methods rely on the ability of MSCs to adhere to plastic, so that while non-adherent cells are removed from culture, attached MSCs can be expanded in culture dishes. MSCs can also be isolated from subcutaneous adipose tissue following a similar procedure, known to those skilled in the art. In a particular embodiment of the invention, the mesenchymal stem cells are obtained from umbilical cord, preferably human umbilical cord.

The terms "pluripotent stem cell" or "pluripotent stem cell" and grammatical equivalents are used interchangeably in the context of the present invention to refer to undifferentiated or poorly differentiated cells, of any species, with the capacity to divide indefinitely without losing their properties and capable of forming any cell of the three embryonic lineages (mesoderm, endoderm, ectoderm) and germ lineage as well as the germ lineage when cultured under certain conditions. The invention contemplates the use of any type of pluripotent stem cell that is capable of generating progeny of any of the three germ layers including cells derived from embryonic tissue, fetal tissue, adult tissue and other sources. Suitable stem cells for use in the present invention include embryonic stem cells, embryonal carcinoma cells, induced stem cells (iPS), and primordial germ cells. Likewise, the invention contemplates the use of stem cells pluripotent cells of any species including, without limitation, human, mouse, rat, bovine, sheep, hamster, pig, and the like cells.

The term "induced pluripotent stem cell" or "iPS", as used in the present invention, refers to cells that are substantially genetically identical to a differentiated somatic cell from which they are derived but that show similar characteristics in terms of differentiation and capacity. proliferative to pluripotent embryonic stem cells. Typically, iPS express on their surface one or more markers selected from the group consisting of SSEA-3, SSEA-4, TRA-I -60, TRA-1-81, TRA-2-49 / 6E and Nanog. Typically, iPS express one or more genes selected from the group of Oct-3/4, Sox2, Nanog, GDF3, REXI, FGF4, ESGI, DPP A2, DPP A4 and hTERT. IPS can be generated using methods described in the state of the art such as the methods described by Takahashi and Yamanaka (Cell, 2006, 126: 663-676), Yamanaka et al., (Nature, 2007, 448: 313-7) , Wernig et al, (Nature, 2007, 448: 318-24), Maherali (Cell Stem Cell, 2007, 1: 55-70); Maherali and Hochedlinger (Cell Stem Cell, 2008, 3: 595-605), Park et al, (Cell, 2008, 134: 1-10); Dimos et al., (Science, 2008, 321: 1218-1221), Blelloch et al. (Cell Stem Cell,

2007, 1: 245-247); Stadtfeld et al., (Science, 2008, 322: 945-949) and Okita et al., (Science,

2008, 322: 949-953). They are cells reprogrammed in vitro from somatic cells terminally differentiated by retroviral transduction of the transcription factors Oct3 / 4, Sox2, Klf4 and c-Myc. Typically, iPS cells are obtained from somatic cells by expressing in said cells the Oct-3/4 and Sox2 proteins, the Oct-3/4, Sox2 and Klf4 proteins, the Oct-3/4 proteins. , Sox2, Klf4 and c-Myc and / or the Oct-4, Sox2, Nanog and LIN28 proteins.

The recellularized sclerocorneal limbus of the invention is obtainable by an in vitro method that comprises the following steps: a) obtaining the decellularized sclerocorneal limbus (scaffold) of the invention, b) culturing an isolated cell or isolated cells in or on the decellularized sclerocorneal limbus (scaffold) resulting from step (a).

In another preferred embodiment of this aspect of the invention, cells can be genetically modified by any conventional method including, for example, but not limited to, by transgenesis processes, deletions or insertions in their genome, etc.

Isolated cells can be cells of autologous, allogeneic or xenogeneic origin. In a preferred embodiment of the invention, said cells are of autologous origin and are isolated of the subject to which they will be administered, thus reducing the possible complications associated with antigenic and / or immunogenic responses to said cells.

In general, there are advantages associated with the use of autologous cells or tissues, or with the intracellular content of autologous cells, including: (a) a significant reduction in the number of infections from donor to recipient by infectious agents, and (b) the absence of the immune rejection effect, therefore, the patient does not have to undergo other treatments, and the effects and problems associated with immunosuppression are prevented.

When donors are used to obtain stem cells, the immune response can be minimized by matching the haplotypes of the donors to those of the recipients. In a preferred embodiment of the invention, the stem cell population is considered not to trigger an immune response if at least about 70% of the cells in the isolated stem cell population do not trigger an immune response. In another preferred embodiment at least about 80%, at least about 90%, or at least about 95%, 99% or more of the cells of the isolated stem cell population do not elicit an immune response. Preferably the cells of the invention do not trigger an antibody-mediated immune response and / or do not trigger a humoral immune response and / or do not trigger a mixed lymphocyte immune response.

If desired, the isolated cells can be expanded by cloning using methods suitable for the cloning of cell populations. Isolated cells can be cloned at low density (eg, in a Petri dish) and can be isolated from other cells using suitable devices (eg, cloning rings). The clonal population can be expanded in a suitable culture medium.

The term "isolated" indicates that the cell or cell population to which it refers is not within its natural environment. The cell or cell population has been substantially separated from the surrounding tissues. In a preferred embodiment of the invention, the cell or population of cells is substantially detached from surrounding tissue if the sample contains at least about 75%, and at least about 85%, at least about 90%, and at least about 95% of the cells. In other words, the sample is substantially separated from the surrounding tissue if the sample contains less than about 25%, less than about 15%, and less than about 5% of materials other than cells. These percentage values refer to the percentage by weight or the number of cells. The term encompasses cells that have been removed from the organism from which they are derived, and exist in the crop. The term also includes cells that have been removed from the organism from which they originated, and are subsequently reinserted into an organism. The organism containing the reinserted cells may be the same organism from which the cells were removed, or it may be a different organism, that is, a different individual of the same species.

The blade of the invention has cavities to house the cells and that they can easily nest. In addition to the natural cavities that it already has, more microcavities can be made by methods known in the state of the art, such as, but not limited to, using microneedles. Thus, for example, mesenchymal cells can transform into limbal stem cells, which are very sensitive to ultraviolet light, and are normally hidden in crypts within the sclera.

ARTIFICIAL TISSUE OF THE INVENTION

A third aspect of the invention relates to an artificial tissue, hereinafter artificial tissue of the invention, comprising the decellularized human sclerocorneal limbus of the invention, or the recellularized sclerocorneal limbus of the invention.

COMPOSITION OF THE INVENTION

A fourth aspect of the invention refers to a composition, hereinafter composition of the invention, comprising the decellularized human sclerocorneal limbus according to the first aspect of the invention, the recellularized sclerocorneal limbus according to the second aspect of the invention, or the artificial tissue according to the third aspect of the invention.

In a preferred embodiment of this aspect of the invention, the composition is a pharmaceutical composition.

In another preferred embodiment of this aspect of the invention, the composition of the invention further comprises a pharmaceutically acceptable carrier.

In another preferred embodiment of this aspect of the invention, the composition of the invention further comprises another active ingredient.

The term "active ingredient", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" refers to any component that potentially provides pharmacological activity or other different effect in diagnosis, cure, mitigation, treatment or prevention of disease, which affects the structure or function of the human body or the body of other animals. Examples of active ingredients of biological origin include growth factors, hormones and / or cytokines. A variety of therapeutic agents are known in the art and can be identified by their effects. Certain therapeutic agents are capable of regulating cell proliferation and differentiation. For example: nucleotides, chemotherapeutic drugs, hormones, non-specific (which are not antibodies), proteins, oligonucleotides (for example, antisense oligonucleotides that bind to a target nucleic acid sequence (for example, mRNA sequence), peptides, and peptidomimetics.

The pharmaceutical composition of the present invention can be used in a method of treatment in an isolated manner or in conjunction with other pharmaceutical compounds.

The pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the adjuvants and vehicles known to those skilled in the art and commonly used in the preparation of therapeutic compositions.

The term "pharmaceutically acceptable excipient" refers to the fact that it must be approved by a regulatory agency of the federal government or a national government or one that appears in the United States Pharmacopoeia or the European Pharmacopoeia, or some other generally recognized pharmacopoeia. for use in animals and humans. The term "vehicle" refers to a diluent, excipient, vehicle or adjuvant with which the cell or cells of step (b) are to be administered; Obviously, such a vehicle must be compatible with the cells. Illustrative, non-limiting examples include any physiologically compatible vehicle, for example isotonic solutions (eg, sterile saline (0.9% NaCl), phosphate-buffered saline (PBS), Ringer's lactate solution, etc.) , optionally supplemented with serum, preferably with autologous serum; culture media (eg, DMEM, RPMI, McCoy, etc.); or, preferably, a solid, semisolid material, gelatinous or viscous support medium, such as collagen, collagen-glycosamine-glycan, fibrin, polyvinyl chloride, poly-amino acids, such as polylysine, or polyornithine, hydrogels, agarose, silicone dextran sulfate.

The pharmaceutical composition of the invention may, if desired, also contain when necessary additives to increase and / or control the desired therapeutic effect of the cells, for example, buffer agents, surface active agents, preservatives, etc. The pharmaceutically acceptable carrier may comprise a cell culture medium that supports the viability of the cells. The medium will generally be serum-free in order to avoid eliciting an immune response in the recipient. The vehicle will generally be buffered and / or pyrogen-free. Furthermore, for the stabilization of the cell suspension, it is possible to add metal chelating agents. The stability of cells in the medium Liquid of the pharmaceutical composition of the invention can be improved by adding additional substances, such as, for example, aspartic acid, glutamic acid, etc. Pharmaceutically acceptable substances that can be used in the pharmaceutical composition of the invention are generally known to a person skilled in the art and are commonly used in the production of cellular compositions. Examples of suitable pharmaceutical carriers are described in "Remington Pharmaceutical Sciences" by EW Martin. Additional information on such vehicles can be found in any pharmaceutical technology manual (ie, Galenic pharmacy).

The pharmaceutical composition of the invention is administered in a suitable dosage form. For this, the pharmaceutical composition of the invention will be formulated according to the chosen administration form. The formulation will be adapted to the form of administration. In a specific embodiment, the pharmaceutical composition is prepared in a liquid, solid or semi-solid dosage form, for example, in suspension form, in order to be administered by implantation, injection or infusion to the subject in need of treatment.

The administration of the pharmaceutical composition of the invention to the subject in need will be carried out using conventional means. In all cases, the pharmaceutical composition of the invention will be administered using equipment, apparatus and devices suitable for the administration of cellular compositions and known to a person skilled in the art. In another preferred embodiment, direct administration of the pharmaceutical composition of the invention to the site where the benefit is intended to occur may be advantageous. In this method, direct administration of the pharmaceutical composition of the invention to the desired organ or tissue can be achieved by direct administration (for example, by injection, etc.) to the external surface of the affected organ or tissue by insertion of a suitable device, eg, a suitable cannula, by infusion (including reverse flow mechanisms) or by other means described in this patent or known in the art.

The pharmaceutical composition of the invention can be stored until the time of its application by conventional methods known to a person skilled in the art. For short-term storage (less than 6 hours), the pharmaceutical composition of the invention can be stored at or below room temperature in a sealed container, supplemented or not with a nutrient solution. Medium-term storage (less than 48 hours) is preferably carried out between 2-8 ° C, and the pharmaceutical composition of the invention also includes an isosmotic buffered solution in a container made or lined with a material that prevents the cell adhesion. Longer term storage is preferably effected by means of cryopreservation and proper storage under conditions that promote the retention of cell function.

In a specific embodiment, the pharmaceutical composition of the invention can be used in combination therapy. Said additional drugs can be part of the same pharmaceutical composition or they can, alternatively, be supplied in the form of a separate composition for simultaneous or successive (sequential in time) administration with respect to the administration of the pharmaceutical composition of the invention.

MEDICAL USES OF THE INVENTION

An eighth aspect of the invention relates to the decellularized human sclerocorneal limbus according to the first aspect of the invention, the recellularized sclerocorneal limbus according to the second aspect of the invention, the artificial tissue according to the third aspect of the invention, or the composition according to the fourth aspect aspect of the invention, for the treatment of any disease of the ocular surface, preferably limbar insufficiency, corneal epitheliopathy, and other pathologies of the anterior pole in combination with other active principles, such as glaucoma, uveitis, infections, as well as other ocular pathologies, among them those that produce neovascularization. A ninth aspect of the invention relates to the use of decellularized human sclerocorneal limbus according to the first aspect of the invention, the recellularized sclerocorneal limbus according to the second aspect of the invention, the artificial tissue according to the third aspect of the invention, or the composition according to the fourth aspect of the invention, as a release system for cellular factors and / or drugs

A tenth aspect of the invention relates to the decellularized human sclerocorneal limbus according to the first aspect of the invention, the recellularized sclerocorneal limbus according to the second aspect of the invention, the artificial tissue according to the third aspect of the invention, or the composition according to the fourth aspect aspect of the invention, for the release of cells or drugs.

As discussed above, the limbus of the invention can be used to house stem cells that can differentiate into limbal cells or other cell lines. In addition, it can contain cells genetically modified to produce useful therapeutic compounds, which can act as active ingredients. It can also function as a "depot", either directly, housing therapeutic compounds of interest, or employing nanoparticles or other mechanisms to contain and release them.

By way of example, not limiting, useful therapeutic agents according to the invention include the following therapeutic categories: analgesics, such as non-steroidal anti-inflammatory drugs, opiate agonists and salicylates; anti-infective agents, such as anthelmintics, antianaerobics, antibiotics, aminoglycoside antibiotics, antifungal antibiotics, cephalosporins, macrolide antibiotics, various beta-lactam antibiotics, penicillins, quinolone antibiotics, sulfonamide antibiotics, tetracycline antibiotics, antimycobacterials, antimycobacterials, antimycobacterials antimalarials, antiviral agents, antiretroviral agents, scabicides, anti-inflammatory agents, anti-inflammatory corticosteroids, antipruritic / local topical anesthetics, anti-infectives, topical antifungal agents, anti-infective topical antivirals, acidic inhibitors, electrolytic agents, alkaline agents, and renal agents of carbonic anhydrase, diuretics, loop diuretics, osmotic diuretics, potassium-sparing diuretics, thiazide diuretics, electrolyte supplements, and uricosuric agents, in zymes, such as pancreatic enzymes and thrombolytic enzymes, gastrointestinal agents, such as antidiarrheals, antiemetics, gastrointestinal gastrointestinal anti-inflammatory agents, anti-inflammatory agents salicylate, anti-gastric ulcer antacids, acid pump inhibitor agents, antiulcer agents, mucosa gastric antiulcer H2 blocking agents, antiulcer agents, cholelitholytic agents, digestants, emetics, laxatives and stool softeners and prokinetic agents, general anesthetics such as halogenated inhalation anesthetics inhalation anesthetics, intravenous anesthetics, barbiturates, benzodiazepines intravenous anesthetics, intravenous anesthetics of opiates and intravenous anesthetics agonists, hormones and hormonal modifiers, such as abortifacients, adrenal corticosteroids, and androgens, anti-renal agents, antobiol agents, agents such as immunoglobulins, immunosuppressants, toxoids, and vaccines; local anesthetics, such as amide from local anesthetics and ester-type local anesthetics, musculoskeletal agents, such as anti-gout, anti-inflammatory agents, anti-inflammatory agents, corticosteroids, anti-inflammatory agents, gold compounds, immunosuppressive agents, anti-inflammatory agents , non-steroidal anti-inflammatory drugs (NSAIDs), salicylate anti-inflammatory agents, minerals and vitamins, such as vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, and vitamin K.

In a particular embodiment, useful therapeutic agents according to the previous categories include: (1) analgesics in general, such as lidocaine or its derivatives, and analgesic non-steroidal anti-inflammatory drugs (NSAIDs), including diclofenac, ibuprofen, ketoprofen, naproxen and, ( 2) opioid agonist pain relievers, such as codeine, fentanyl, hydromorphone, and morphine, (3) salicylate pain relievers, such as aspirin (ASA), (4) antihistamine H1 blockers, such as terfenadine, clemastine, and (5) anti-infective agents, such as mupirocin; (6) anti-infective antianaerobics, such as chloramphenicol and clindamycin; (7) antifungal anti-infective antibiotics, such as amphotericin B, clotrimazole, fluconazole, and ketoconazole; (8) against macrolide antibiotics - infectious, such as azithromycin and erythromycin; (9) various anti-infective beta-lactams, such as aztreonam and imipenem; (10) anti-infective penicillin antibiotics, such as nafcillin, oxacillin, penicillin G, penicillin V and; (11) quinolone anti-infective antibiotics, such as ciprofloxacin and norfloxacin; (12) anti-infective tetracycline antibiotic, such as doxycycline, minocycline, and tetracycline; (13) anti-infective antimycobacterial antituberculosis drugs, such as isoniazid (INH), rifampicin and; (14) anti-infective antiprotozoals, such as atovaquone and dapsone; (15) antimalarial antiprotozoal anti-infectives, such as chloroquine and pyrimethamine; (16) anti-infective antiretrovirals, such as ritonavir and zidovudine; (17) anti-antiviral-infectious agents, such as acyclovir, ganciclovir, interferon alfa, and rimantadine; (18) topical anti-infective antifungals, such as amphotericin B, clotrimazole, miconazole, nystatin and; (19) anti-infective topical antivirals, such as acyclovir; (20) electrolytic and kidney agents, such as lactulose; (21) loop diuretics, such as furosemide; (22) potassium-sparing diuretics, such as triamterene; (23) thiazide diuretics, such as hydrochlorothiazide (HCTZ), (24) uricosuric agents, such as probenecid; (25) enzymes, such as RNase and DNase; (26) antiemetics, such as prochlorperazine; (27) salicylate gastrointestinal inflammatory antiants, such as sulfasalazine; (28) gastric acid pump anti-ulcer inhibitor agents, such as omeprazole; (29) H2 blockers, anti-ulcer agents, such as cimetidine, famotidine, nizatidine, and ranitidine; (30) digestives, such as pancrelipase; (31) prokinetic agents, such as erythromycin (32; ester) local anesthetics, such as benzocaine and procaine; (33) Musculoskeletal corticosteroid anti-inflammatory agents, such as beclomethasone, betamethasone, cortisone, dexamethasone, hydrocortisone, and prednisone; (34) musculoskeletal immunosuppressive anti-inflammatory drugs, such as azathioprine, cyclophosphamide, and methotrexate; (35) non-steroidal anti-inflammatory musculoskeletal drugs (NSAIDs), such as diclofenac, ibuprofen, ketoprofen, ketorlac, and naproxen; (36) minerals, such as iron, calcium, and magnesium; (37) Vitamin B compounds, such as cyanocobalamin (vitamin B12) and niacin (vitamin B3); (38) vitamin C compounds, such as ascorbic acid, and (39) vitamin D compounds, such as calcitriol.

In another preferred embodiment, the therapeutic agent can be a growth factor or other molecule that affects cell differentiation and / or proliferation, such as, but not limited to, platelet derivative (PDGF), transforming (TGF), insulin (IGF), hepatic (HGF), epidermal (EGF), vascular endothelium (VEGF), fibroblast growth factor (FGF), or any combination thereof. Growth factors that induce end-state differentiation are well known in the art, and can be selected from any of those factors that have been shown to induce a final state of differentiation. Growth factors for use in the methods described herein may, in certain embodiments, be variants or fragments of a naturally occurring growth factor. For example, a variant can be generated by making conservative amino acid changes and testing the resulting variant in one of the functional assays described above or another functional assay known in the art. Conservative amino acid substitutions refer to the interchangeability of residues that have similar side chains. For example, a group of amino acids that have aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl in their side chains is serine and threonine; A group of amino acids that have amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine and tryptophan; a group of amino acids that have basic side chains is lysine, arginine, and histidine, and a group of amino acids that have sulfur-containing side chains is cysteine and methionine. Preferred amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. As those skilled in the art will appreciate, polypeptide growth factor variants or fragments can be generated using standard techniques, such as mutagenesis, including creation of discrete point mutation (s), or by truncation. For example, the mutation can result in variants that retain substantially the same, or simply a subset, of the biological activity of a polypeptide growth factor from which it was derived.

In another preferred embodiment, the therapeutic agent can be an anti-growth factor agent. More preferably, it could be an anti-VEGF that can be used for the treatment of various ocular pathologies, such as age-related ocular degeneration (DEMAE), diabetic retinopathy, and other ocular pathologies where the efficacy of medications has been demonstrated. anti-VEGF. Anti-VEGF drugs are known in the state of the art, such as, for example, but not limited to, antibodies such as bevacizumab, ranibizumab, lapatinib, sunitinib, sorafenib, axitinib, and pazopanib, or any of their combinations.

Finally, another aspect of the invention refers to the use of the artificial tissue of the invention for the decellularized human sclerocorneal limbus according to the first aspect of the invention, the recellularized sclerocorneal limbus according to the second aspect of the invention, the artificial tissue according to the third aspect of the invention, or the composition according to the fourth aspect of the invention, for the evaluation of a pharmacological, biological and / or chemical product.

Another aspect of the invention refers to a scaffold obtainable by means of a method that comprises: a) obtaining the scaffold of the invention and b) obtaining another scaffold by means of a 3D printing taking the scaffold of the invention as a mold.

3D printing can be developed by any of the methods known in the state of the art, for example, but not limited to, by injection printing, modeling by flux deposition, photopolymerization, printing with ice, etc.

In a preferred embodiment of this aspect of the invention, a component is added to decrease brittleness, for example a polysaccharide. Examples of polysaccharides that may be employed are, but are not limited to, agar-agar, agarose, alginate, chitosan, or carrageenan, or any combination of the foregoing.

Another aspect of the invention refers to the use of the printed element as described in the previous aspect of the invention in the manufacture of a medicine ... The term "drug", as used in this specification, refers to any substance used for the prevention, diagnosis, improvement, alleviation, treatment or cure of diseases in man and animals.

The compounds and compositions of the present invention can be used together with other drugs in combination therapies. The other drugs can be part of the same composition or a different composition, for administration at the same time or at different times.

Throughout the description and claims the word "comprise" 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 characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

EXAMPLE OF THE INVENTION

one_. Carving of the sclerocorneal cap fragments

The sclerocorneal cap is cut into 4 fragments of similar lengths and the clear cornea is trimmed until leaving only 1 mm and 2 mm of sclera. If there is still conjunctiva, it is removed to leave the sclera bare. Each fragment is fixed either with needles or with sterile surgical cyanoacrylate glue on the padded base containing the 10-0 nylon sutures. Once fixed, with a 2.75 mm triangular knife with cutting edges, the thickness of the fragment is incised in its middle third and the anterior 1/3 of the corneo-scleral stroma is delaminated, helping to make this cut with an Adson forceps supported by both sides of the fragment as an isosceles triangle in which two of the upper sides would be the forceps and the base the corneo-sclera fragment. Dissection is continued with a “crescent” type knife or with the lateral edges of the 2.75 mm knife, sliding the knife sideways, until a semicircular segment of the sclera and cornea approximately 1/3 of anterior thickness is finished dissecting.

Subsequently, the anterior corneal corner is sutured with nylon so that the laboratory handling is correct. The transport is carried out in the same medium as the sclerocorneal caps, such as Optisol®. 2. Decellularization of human sclero-corneal limbs.

First, all cells and cellular debris from human sclero-corneal limbs from multiorgan donors or limbal biopsies are removed using a method previously described for the cornea by González-Andrades et al. 2015 and by Oliveira et al. 2013 (Píos ONE 8 (6); e66538) for small intestine based on the anionic surfactant sodium dodecyl sulfate (SDS). To do this, the previously carved sclerocorneal limbs are washed in physiological serum or phosphate buffer (PBS) and immersed in a 0.1% SDS solution in water or in PBS, incubating in this solution at room temperature for 12 to 48 hours with agitation (from 100 to 300 rpm), renewing this solution every 12 hours. After that, they are washed abundantly with PBS for 24-48 hours at room temperature with stirring, renewing the PBS every 12 hours. A sample of the sclero-corneal limbs thus decellularized is fixed with neutral formalin and processed for histological analysis and confirmation of the efficiency of the decellularization process.

3. Recellularization with human cells.

Once decellularized, the samples are placed in a sterile Petri dish with the anterior surface (corneal surface) facing upwards and they are recellularized. To do this, we start from a primary culture of corneal stromal cells (keratocytes) or connective tissue (fibroblasts) or human mesenchymal stem cells -MSC- (stem cells from adipose tissue, dental pulp, bone marrow or umbilical cord) previously established. This culture is trypsinized to detach the cells, washed in culture medium, and the cells are deposited on the corneal surface of the sclero-corneal limbus fragments or injected into the stroma using a surgical microneedle. After that, the presence of cells was analyzed by staining each of the samples with DAPI (fluorescent dye that specifically binds to DNA and marks the cell nuclei in blue).

The results show the presence of abundant cells in the original sclerocorneal limbus sample not subjected to decellularization, and a complete disappearance of all cells after the decellularization process. Recellularized samples with Wharton's gelatin mesenchymal stem cells from the human umbilical cord (WH) or with fibroblasts from the human oral mucosa (FOM) showed a layer of cells in the surface and, in some cases, some cells within the decellularized tissue, as shown in the figures.

5

Claims

1.- Decellularized human sclerocorneal limbus.

2. The decellularized human sclerocorneal limbus according to the preceding claim, obtainable by an in vitro decellularization method that comprises the use of a decellularizing agent solution, preferably SDS, preferably SDS at approximately the

0.1%.

3. The decellularized human sclerocorneal limbus according to any of claims 1-2, obtainable by an in vitro decellularization method that comprises: a) cutting the sclerocorneal cap into 4 fragments of similar lengths, b) cutting the clear cornea until leaving only 1 mm and 2 mm of sclera, c) fix each fragment on a support, preferably a pad, d) delaminate the anterior 1/3 of the corneo-scleral stroma e) dissect a semicircular segment of sclera and cornea 1/3 thickness above approximately. f) wash the sclero-corneal limbus resulting from step (e) in physiological serum or phosphate buffer (PBS), g) immerse in a solution of decellularizing agent, preferably SDS, and more preferably 0.1% SDSal in water or in PBS , e) incubate at room temperature for 12 to 48 hours with shaking (between 100 and 300 rpm), renewing this solution every 12 hours. f) wash with PBS for 24-48 hours at room temperature while shaking, renewing the PBS every 12 hours

4 - Recellularized sclerocorneal limbus comprising the decellularized human sclerocorneal limbus according to any of claims 1-3.

5. Recellularized sclerocorneal limbus according to the preceding claim, wherein the cells used to recellularize are human cells.

6. Recellularized sclerocorneal limbus according to any of claims 4-5, wherein the cells used to recellularize are autologous cells.

7. Recellularized sclerocorneal limbus according to any of claims 4-6, wherein the cells used to recellularize are stem cells. where stem cells are selected from the list consisting of mesenchymal stem cells, limbal cells of the eye, hemotopoietic stem cells, non-human embryonic stem cells, induced pluripotent stem cells, adult stem cells, mesothelial cells, umbilical cord blood stem cells , or any combination thereof, preferably the stem cells are umbilical cord mesenchymal cells and / or limbal cells.

8.- Recellularized sclerocorneal limbus according to any of claims 4-7, where the cells used to recellularize are selected from the list consisting of: corneal stromal cells (keratocytes), connective tissue cells (fibroblasts), stem cells mesenchymals, or any of their combinations.

9. Recellularized sclerocorneal limbus according to any of claims 4-8, wherein the cells used to recellularize are mesenchymal cells, and more preferably obtained from the umbilical cord.

10.- Recellularized sclerocorneal limbus according to any of claims 4-9, deposited on the corneal surface of the sclero-corneal limbus fragments or injected into the stroma, preferably using a surgical microneedle

11.- Recellularized sclerocorneal limbus according to any of claims 4-10, where the sclerocorneal limbus is obtained from a cadaveric donor

12. Recellularized sclerocorneal limbus according to any of claims 4-11, having a thickness of between 50-300 pm, more preferably between 100-250 pm, and even more preferably approximately 200.

13. An artificial tissue comprising the decellularized human sclerocorneal limbus according to any of claims 1-3, or the recellularized sclerocorneal limbus according to any of claims 4-12.

14. A composition comprising the decellularized human sclerocorneal limbus according to any of claims 1-3, or the recellularized sclerocorneal limbus according to any of claims 4-12, or the artificial tissue according to claim 13.

15. The composition according to the preceding claim, wherein the composition is a pharmaceutical composition.

16. The composition according to any of claims 9-10, further comprising a pharmaceutically acceptable carrier.

17. The composition according to any of claims 9-11, further comprising another active principle.

18. The decellularized human sclerocorneal limbus according to any of claims 1-3, or the recellularized sclerocorneal limbus according to any of claims 4-12, or the artificial tissue according to claim 13, or the composition according to any of claims 14- 17, for use as a medicine.

19. The decellularized human sclerocorneal limbus according to any of claims 1-3, or the recellularized sclerocorneal limbus according to any of claims 4-12, or the artificial tissue according to claim 13, or the composition according to any of claims 14- 17, to increase, restore or partially or totally replace the functional activity of a diseased or damaged tissue or organ

20. The decellularized human sclerocorneal limbus according to any of claims 1-3, or the recellularized sclerocorneal limbus according to any of claims 4-12, or the artificial tissue according to claim 13, or the composition according to any of claims 14- 17, for the treatment of partial or total unilateral or bilateral limbar insufficiency.

21. The decellularized human sclerocorneal limbus according to any of claims 1-3, or the recellularized sclerocorneal limbus according to any of claims 4-12, or the artificial tissue according to claim 13, or the composition according to any of claims 14- 17, for the treatment of any epitheliopathy, of a corneal ocular disease that does or does not involve corneal neovascularization.

22. Use of the decellularized human sclerocorneal limbus according to any of claims 1-3, or the recellularized sclerocorneal limbus according to any of claims 4-12, or the artificial tissue according to claim 13, or the composition according to any of claims 14 -17, as a release system for cellular factors and / or drugs.

23.- A kit or device comprising the elements necessary to obtain decellularized human sclerocorneal limbus according to any of claims 1-3, or the recellularized sclerocorneal limbus according to any of claims 4-12, or the artificial tissue according to claim 13, or the composition according to any one of claims 14-17.

24. Use of the kit or device according to the preceding claim in a method as described in claims 2-3.