MXPA97003321A - In vitro fabric and equivalen organ models - Google Patents

Abstract

The present invention relates to an organ equivalent of the part of the cornea of the eye, made by the use of tissue culture systems. The method to construct the cornea equivalent results in a structure analogous to the cornea of the eye in vivo. The equivalent of cornea is an in vitro model on the eye, which can be used for transplantation or implantation in vivo or to select compounds in vitro. This invention is also directed to the use of endothelial cells in other tissues and organo equivalents to promote the development of the basement membrane.

Description

IN VITRO TISSUE AND MODELS OF EQUIVALENT ORGANS Csampo of the Invention This invention is in the field of tissue culture systems and is directed to an equivalent organ of the cornea of the eye: an equivalent cornea model. The tissue culture method for constructing the equivalent cornea model results in an analogous construction to the cornea of the eye in vivo. The cornea equivalent is an in vi tro model of the eye that can be used for in vivo transplantation or implantation or to mask in vi tro compounds. This invention is also directed to the use of endothelial cells in other tissues and equivalent organs to promote the development of the basement membrane. BACKGROUND OF THE INVENTION Tissue culture techniques are being successfully used to develop tissue and organ equivalents. The basis of these techniques includes collagen matrix structures, which are capable of being remodeled into tissues and functional organs by employing the correct combination of living cells, nutrients and culture conditions. Tissue equivalents have been described extensively in many patents including U.S. Patent Nos. 4,485,096; 4,485,097; 4,539,716; 4,546,500; 4,604,346; and 4,837,379, all of which are incorporated herein by reference. A successful application of the tissue equivalent is the equivalent of living skin, which has a morphology similar to real human skin. The living skin equivalent is composed of two layers: the upper portion is made of differentiated and stratified human epidermal keratinocytes that cover a thicker lower layer of human dermal fibroblasts in a collagen matrix. Bell et al., "Recipes for Reconstitutin Skin", (Recipes for Reconstituting the Skin), J. of Biochemical Engineering, 113: 113-119 (1991). Studies have been done on the culture of corneal epithelial and endothelial cells. Xie et al., "A simplified technique for the short-term tissue culture of rabbit corneal cells", (A simplified technique for short-term tissue culture of rabbit cornea cells), In Vitro Celular & Developmental Biology, 25: 20-22 (1989) and Simmons et al., Corneal Epithelial ound Closure in Tissue Culture: An in vi tro Model of Ocular Irritancy "(Cornea Epithelial Wound Closure in Tissue Culture: An Irritation Model Ocular in vi tro), Toxicology and Applied Pharmacology, 88: 13-23 (1987) The development of an organ equivalent in vi tro of the cornea of the eye is of particular interest for use in toxicity tests in vi tro to serve as non-animal, accurate and economic predictive models of potential in vivo eye and dermal irritation for many types of products and raw materials SUMMARY OF THE INVENTION This invention is directed to an equivalent organ of the cornea of the eye. cornea according to this invention involves the generation, by tissue culture, of the three distinct cell layers in the cornea: the outer layer, a stratified squamous epithelium, the middle layer, fibers of collagen; and the inner layer, a simple squamous epithelium, also called the cornea endothelium. The method to construct the equivalent of cornea results in a structure analogous to the cornea of the eye in vi vo. This invention is based, in part, on the discovery that the inclusion of an endothelial layer is required, not only for the transparency of the cornea in vivo, but also for a morphology, expression of biochemical and physiological markers, cell diffusion, subjection epithelial to the matrix, and uniformity of epithelial coverage in vivo, improved. The endothelium promotes the development of the basement membrane in the cornea equivalent. The results on the influence of the endothelium in obtaining a higher level of epithelial differentiation in vi tro were unexpected.

Based on this discovery, it was found that the use of the endothelium in other tissue and organ equivalents also promotes the development of the basement membrane. In this manner, this invention is also directed to the use of endothelial cells in those constructions equivalent to tissue and to organs utilizing collagen or epithelial cells. DESCRIPTION OF THE FIGURES Figures 1A, IB, 1C, ID, 1E and 1F are immunofluorescent photomicrographs of cornea equivalents showing the distribution of laminin in cornea equivalents with an endothelial cell layer from various species and sources. The cornea constructions after 7 days at the moisture interface exhibit a small amount of laminin deposited in the area of the basement membrane. The HUVEC cultures (figure C, Mag. = 375X) and SEC 006A (figure 1E, Mag. = 375X) showed a continuous distribution of laminin. The DVEC (figure IB, Mag. = 375X), the SCE2 (figure ID, Mag. = 375X) and the SEC 023VC (figure 1F, Mag. = 375X) showed an almost continuous distribution. ECB (figure 1A, Mag. = 375X) showed a distribution similar to that previously obtained in moisture cultures lacking an endothelial cell layer. Figures 2A, 2B, 2C, 2D, 2E, 2F and 2G are immunofluorescent photomicrographs of cornea equivalents showing the distribution of ZO1 protein in cornea equivalents with an endothelial cell layer from various species and sources. The positive MCE (Figure 2F, Mag. = 375X) and DVEC (Figure 2A, Mag. = 375X) controls showed an evident localization in the uppermost layers of the epithelium. Cultures of SCE2 (Figure 2C, Mag. = 375X) and SEC 023VC (Figure 2D, Mag. = 375X) also showed a limited distribution, although cultures of HUVEC (Figure 2B, Mag. = 375X), BCE (Figure 2E , Mag. = 375X) and SEC 006A (figure 2G, Mag. = 375X) showed a widely distributed staining indicating the lack of specialized localization of the ZO1 protein. Figures 3A, 3B and 3C are a series of electron transmission micrographs showing the effects of various strains of endothelial cells in the formation of basal lamina in three-layer cornea constructions. Figure 3A is an electron transmission micrograph showing the formation of basal lamina in a three-layer construct made by the use of transformed mouse cornea endothelial cells (MCE). After seven days in the moisture interface, the basal lamina was observed in the stromatic-epithelial junction with numerous hemidesmosomes (asterisk), anchoring filaments (small arrowheads), a well-defined dense lamina (large arrowheads) and Anchoring fibrils (small arrow). Barium = 0.2μm. Figure 3B is an electron transmission micrograph showing the formation of the basal lamina in a three-layer construct made by the use of non-transformed sheep cornea endothelial cells (SCE2). After seven days at the moisture interface, a basal lamina was observed at the stromal-epithelial junction with hemidesmosomes (asterisk), anchoring filaments (small arrow heads) and a dense lamina (large arrowheads). Barium = 0.2μm. Figure 3C is an electron transmission micrograph showing the irregular basal lamina (unequal) in a three-layer construct made by the use of non-transformed sheep arterial endothelial cells (SEC 006A). After seven days at the moisture interface, a basal lamina consisting of hemidesmosomes (asterisk) and a dense lamina (arrowheads) was observed at the stromal-epithelial junction. Barium = 0.2μm. Figures 4A and 4B are immunofluorescent photomicrographs of the keratin 3 distribution under different environmental conditions. Keratin 3 is a specific marker for corneal epithelial cells and normally occurs in all the suprabasal cell layers of the cornea limbus and the cells of the central cornea. (Schermer, A., Galvin, S., and Sun, TT, "Expression regarding the differentiation of a major 64K cornea keratin in vivo and in culture suggests the limbal location of corneal epithelial stem cells" ( Differentiation-related expression of a major 64K corneal Keretin in vivo and in culture suggests limbal location of cornea epithelal sten cells), J. Cell Biology 103: 49 (1986) .The photomicrographs illustrate cornea constructions submerged 14 days (Figure 4A , Submerged, Mag. = 375X) and 7 days after the air extraction of the moisture (Figure 4B, Air Extraction of Moisture, Mag. = 375X) showing that the dyeing for keratin 3 (labeled by the use of the antibody AE5) is strong and occurs in all suprabasal layers as in the limbus of normal cornea in vivo Figures 5A and 5B are immunofluorescent photomicrographs of the distribution of alpha6-integrin under ambient or submerged air extraction conditions. Moisture: Alpha6-integrin is a marker for hemidesmosomes in the cornea epithelium. The photomicrographs illustrate cornea constructions submerged 14 days (Figure 5A, Submerged, Mag. = 375X) and 7 days after air extraction of moisture (Figure 5B, Air Extraction of Moisture, Mag. = 375X) showing the Location of alpha6-integrin as a continuous band along the epithelial cell-matrix interface that suggests the polarization of basal epithelial cells. Figures 6A and 6B are immunofluorescent photomicrographs of corneal equivalents showing the distribution of collagen VII type in cornea equivalents under different environmental conditions. Photomicrographs illustrate cornea constructions submerged 14 days (Figure 6B, Submerged, Mag. = 375X) and 7 days after air extraction of moisture (Figure 6A Moisture Air Extraction, MAG = 375x), showing the Collagen constructs of type VII collagen in a continuous band in the stromatic-epithelial junction. Figures 7A and 7B are immunofluorescent photomicrographs of corneal equivalents showing the distribution of Laminin in cornea equivalents under different environmental conditions. Photomicrographs illustrate cornea constructions submerged 14 days (Figure 7A, Submerged, Mag. = 375X) and 7 days after air extraction of moisture (Figure 7B, Air Extraction of Moisture, Mag. = 375X) showing the display of a continuous band of laminin deposited in the basement membrane zone. Figures 8A and 8B are immunofluorescent photomicrographs showing the distribution of enolase under different environmental conditions. Enolase is a marker for the proliferating cell population in the cornea epithelium. It usually occurs in basal cells of the limbal region. (Zieske, JD, Bukusoglu, G., Yankauckas, MA, Characterization of a potential marker of corneal epithelial sten cells "(Characterization of a potential marker of corneal epithelial stem cells), Invest. Opthal ol. Vis. Sci. : 143-152 (1992)). The suprabasal layers of the epithelium stain enolase positively when the culture is immersed 14 days (Figure 8A, Submerged, Mag. = 375X) or 7 days after air extraction of moisture. (Figure 8B, Air Extraction of Moisture, Mag. = 375X) to approximate more closely what is observed in vi vo. Figures 9A and 9B are a series of electron transmission micrographs showing the ultrastructural specialization in three layer constructions in a submerged state. Figure 9A is an electron transmission micrograph showing vermiform ridges on the epithelial surface of the submerged culture at 13 days (equivalent to 6 days after air extraction of moisture). The layer of apical cells expresses vermiform ridges along the anterior surface (arrowheads). Barium = 0.5μm. Figure 9B is an electron transmission micrograph showing the formation of basal lamina in three layer constructions in a submerged state at 13 days. A basal lamina was observed in the stromal-epithelial junction with numerous hemidesmosomes (asterisk), anchoring filaments (small arrowheads), a well-defined dense lamina (large arrow heads) and anchoring fibrils (arrow). Barium = O.lμm. Figures 10A and 10B are a series of electron transmission micrographs showing the production of basal lamina in equivalent skin constructions with an endothelial layer (Figure 10A) and without an endothelial layer (Figure 10B). Figure 10A is an electron transmission micrograph showing the presence of a basal lamina in the epithelial stromal junction with hemidesmosomes (asterisk) and a dense lamina (large arrow head) of an equivalent skin construction with an endothelial layer 7 days after air extraction.

Barium = O.lμm. Figure 10B is an electron transmission micrograph showing rudimentary basal lamina (asterisk) structures located in invaginations in the epithelial stromal junction of an equivalent skin construct 14 days after air extraction without an endothelial layer. Barium = O.lμm. Figure 11 shows a schematic representation of the tissue culture model. Figures 12A and 12B show a human eye diagram. Figure 12A is a diagram of the eye cut in a meridional plane that passes through the equator of the eye horizontally, dividing the eye into an upper half and a lower half. Figure 12B is a section through the human cornea, showing the five layers. (Diagrams of Functional Histology, Borysenko et al., Little Brown, publishers, pages 216-217, 1979). DESCRIPTION DETAIL OF THE INVENTION The outermost layer of the eye is the fibrous tunic, composed of dense avascular connective tissue. The fibrous tunic has two different regions: the sclera and the cornea. The sclera, the "white" of the eye, forms the posterior portion of the fibrous tunic. The sixth anterior of the fibrous tunic is modified to form the transparent cornea. (Figure 12A). The cornea is a multilayer tissue consisting of an outer epithelial layer, a preceding basal membrane, an extracellular collagen matrix (called a stroma), a second basal membrane (called the Descemet membrane), and a stratified internal endothelium of a single cell. The epithelium of the cornea is five to seven layers of cells, with a single layer of columnar basal cells, two to three cell layers of wing cells, and two to three cell layers of flattened surface cells. At the edge of the cornea there is a transition zone (the limbus), which is the interface between the cornea and the conjunctival epithelium. The external stratified squamous epithelium joins with the ocular conjunctiva at the sclera-cornea junction. A simple squamous epithelium, also called the cornea endothelium, delineates the inner side of the cornea. The middle layer of the cornea is clear, the result of the regular arrangement of its collagen fibers. (Figure 12B). Basal membranes are specialized extracellular matrices that function to support cells, and appear to be involved in molecular selection as well as in regulation of cell attachment, development, and differentiation. These functions involve the binding of cells to the basement membrane through a group of binding proteins that include the integrin family. However, the mechanisms involved in the assembly of a basement membrane are not known. It has been found that the assembly of the basement membrane regulates the differentiation of the epithelium and the formation of the basement membrane in the tissue in vi tro and the organ equivalents. It has also been found that the culture environment influences the development and epithelial differentiation of the cornea. 1. Construction of a Cornea In Vltro Model The construction of the cornea equivalent according to this invention involves tissue culture and generation of the three distinct cell layers in the cornea: the outer layer, a stratified squamous epithelium; the middle layer, collagen fibers; and the inner layer, the cornea endothelium. The method to construct the cornea equivalent results in a structure analogous to the cornea of the eye in vivo. The following description of the preferred embodiment of the cornea equivalent should be understood as illustrative, not limiting. Modifications can be made to the cells and culture parameters and are still within the scope of the invention. For ease of description, reference is made to Figures HA to 11D, which show a tissue culture model. In figure HA, the endothelial cells are seeded in a culture graft and grow to confluence. In Figure 11B, a layer of collagen containing stromal fibroblasts overlaps the confluent endothelial cell layer and is allowed to contract, immersed in the medium. In Figure 11C, a suspension of corneal epithelial cells is seeded in the central area of the contracted reticulum and grown submerged until the epithelial coverage of the raised central area is almost complete. In Figure 11D, the culture is placed in a wet air-liquid interface. In the first stage of construction of the cornea in vi tro model, the endothelial cells are seeded into membranes of a cell culture graft. These endothelial cells will form the inner layer, or basal layer, of the cornea equivalent. The walls of the cell culture graft may consist of polystyrene, polycarbonate, resin, polypropylene (or other biocompatible plastic) with a porous membrane base of polycarbonate or other porous membrane compatible with the culture such as membranes made of collagen, cellulose, fiber of glass or nylon attached to the bottom in which the cells can be grown. The porosity of the membrane can vary from 0.2 μm to 10 μm, 3 μm being preferable. The graft is either suspended or supported in the culture cuvette to allow the culture medium to access the lower side of the culture. A layer of acellular collagen is melted in the cell culture membrane and allowed to gel at room temperature.

The amount of molten acellular layer will depend on the cell culture membrane used, but will typically be from 1 mL to about 5 L. In the preferred method, a K-RESIN® culture graft with a porous polycarbonate membrane base is used. of 3 μm of approximately 2 cm2 of area. A 1 mL acellular layer is melted on the polycarbonate membrane and allowed to gel. The acellular collagen layer comprises 686 μg of bovine tendon collagen extracted by acid in 0.05% acetic acid, 8.1% of Essential Eagle's Medium Minimum 10X, 4 mM of 1-glutamine, 50 μg / ml of gentamicin, 1. 8 mg / mL of sodium bicarbonate and 10% Dulbecco's Modified Eagle Medium (DMEM) containing 10% newborn calf serum (NBCS). Once this has gelled, endothelial cells of 3 x 104 are seeded (6.7 x 103 / cm2) in the gel. The endothelial layer is then immersed in DMEM containing 10% NBCS, 4 mM 1-glutamine, and 50 μg / ml gentamicin for four days at 37 ° C, 10% C02. Alternatively, the acellular collagen layer can be omitted and the endothelial cells seeded directly into the porous membrane. The use of an acellular layer is preferable when transformed endothelial cells are used in order to inhibit the overgrowth of the non-contact-inhibited cells on the underside of the membrane. Alternatively, the acellular layer can be made of Type IV collagen, laminin or a hydrogel. The endothelial cells used to form the endothelial layer can be derived from a variety of sources. Untransformed corneal endothelial cells derived from sheep, rabbit and cow have been used. The mouse cornea endothelial cells were transformed with a large SV40 T antigen. (Muragaki, Y., Shiota, C. Inoue, M., Ooshima, A., Olsen, BR, and Ninomiya, Y., "transcripts of the collagen gene alpha-1-VIII encode a short-chain collagen polypeptide and are expressed by various epithelial and mesenchymal endothelial cells in newborn mouse tissues, "Eur. J. Biochem. 207 (3): 895-902 (1992)). Preferred cell types are the transformed mouse cornea endothelial cell line, or normal corneal endothelial cells derived from sheep or rabbit. Most preferred are normal rabbit cornea endothelial cells. Normal rabbit endothelial cells are derived from the enzymatically decoupled cornea endothelium or cornea explants and are grown serially in the MSBM medium (Johnson, E., Meunier, SF, Roy, CJ, and Parenteau, NL, " Serial Cultivation of Normal Human Keratinocytes: A Defined System Studying the Regulation of Growth and Differentiation "(Serial Cultivation of Normal Human Keratinocytes: A Defined System Studying the Regulation of Growth and Differentiation), In Vitro Cell, Dev. Biol. 28A: 429-435 (1992)) modified by the addition of 50 μg / mL heparin and 0.4 μg / mL heparin binding growth factor-1 (MSBME). Transformed endothelial cells are cultured in DMEM-10% NBCS. Endothelial cells from a different origin to the cornea can also be used in this invention. Endothelial cells of non-corneal origin that have also been used in this invention include ovine and canine vascular endothelial cells and human umbilical vein. The endothelial cells can be transformed with a recombinant retrovirus containing the SV40 large T antigen (Muragaki et al., 1992 supra). Transformed cells continue to grow in the cornea equivalent and form lifts in the upper part of the acellular layer due to their lack of contact inhibition. Untransformed cells will form a monolayer that extends below the cellular-collagen, stromal layer. Alternatively, normal endothelial cells can be transfected as above, but with the addition of a recombinant construct expressing a heat sensitive gene. These cells - lí transformed will grow in a continuous culture under reduced temperature. After the establishment of a confluent endothelial cell layer, the temperature can be raised to deactivate the transforming gene, allowing the cells to resume their normal regulation and exhibit contact inhibition, to form an endothelial cell monolayer similar to untransformed cells. Most peptides are sensitive to heat (with the exception of heat shock proteins) so there is a wide choice of peptides that can be turned off by raising the temperature of the culture. Transformation in this manner also facilitates the use of high vacuum to obtain and culture cell types such as human cornea endothelial cells. In the second stage, the collagen is mixed with keratocytes (stromatic fibroblast cells) to achieve a cell-collagen mixture. The cell-collagen mixture contains approximately 100 stromal fibroblast cells per μg of bovine tendon collagen extracted by acid. The fibroblasts contract the gel to form an elevated area (table) of approximately 2.5 cm2. The cell-stromal collagen mixture comprises the middle layer of the cornea equivalent. The types of collagen that can be used are bovine tendon collagen extracted by acid, bovine tendon collagen extracted above, or rat tail collagen. Alternatively, collagen may also consist of a mixture of Type I and III collagens as they are commonly extracted from the dermis or a mixture of Types I, V and VI as extracted from the corneal stroma. Preferably, Type I collagen extracted by purified bovine tendon acid is used for the initial gel. In organotypic construction, stromal fibroblasts will synthesize additional collagen types such as V and VI as well as additional Type I collagen insofar as they modify the collagen matrix during culture. The epithelial cells will contribute with Type IV and VII collagen in the epithelial stromal junction and the endothelial cells will contribute with Type XII collagen and other membrane components of Descemet in the stromatic-endothelial junction. (Muragaki et al., 1992, supra). Any mammalian stromal fibroblast can be used in this cell layer. Any fibroblast of connective tissue such as those derived from the sclera, dermis, tendon, or face can be used. When corneal cells are used, fibroblasts derived from human or rabbit corneal stroma are preferable. The cells are enzymatically dissociated from the stroma of the normal cornea, cultured in 10% NBCS of DMEM and serially passed. The cells incorporated in the construction are preferably used in step four. Once the endothelial cell culture is ready, to prepare the second cell layer, the cell-collagen mixture, the medium is removed from the cell culture grafts containing the confluent endothelial layer (typically 1.7-2.5 x XlO x). 5 cells / graft). The cell-collagen mixture is transferred and brought into contact with the surface of the endothelial cell layer. The cell-collagen mixture contains the same proportions of materials as the acellular layer with the addition of 5 x 104 stromatic fibroblasts / mL of molten mixture. Three L of this mixture are pipetted into each cell culture graft and allowed to gel. The construction is then immersed in NBCS at 10% DMEM and allowed to contract at 37 ° C, at 10% C02 for seven days. These two layers, which will eventually comprise the endothelial layer and the collagen layer of the cornea model, are cultured under conditions, known to those skilled in the art, to form a fused collagen lattice, preferably upon immersion in 10% NBCS. of Dulbecco at 37 ° C, C02 at 10% for seven days, to form a central raised area or a "table", resulting in the contraction of collagen by stromal fibroblasts. Normal rabbit stromal fibroblasts are cultured for seven days, but culture times may be shorter or longer (usually 2-10 days) depending on the species, cell type and number used. NBCS at 10% DMEM is the preferred culture medium but any medium that normally supports the development of fibroblasts can be used. In the third stage, once the condensed collagen reticulum is formed, the corneal epithelial cells are laminated over the raised area of the collagen to form the outer, apical layer of the cornea equivalent. Corneal epithelial cells can be derived from a variety of mammalian sources. The preferred epithelial cell is an epithelial cell of human or rabbit cornea (cornea keratinocyte) but any mammalian corneal epithelial cell can be used. Other epithelial or keratinocyte cells such as those derived from the sclera (outer white opaque portion) of the eye or epidermis may be substituted, but corneal epithelial cells are preferable. The medium is removed from the culture graft (containing the contracted stromal matrix and the endothelial layer) and around it. The normal rabbit corneal epithelial cells, step 4, are passaged by trypsin and seeded at the top of the membrane at a density of 7.2 x 104 - 1.4 x 10 5 cells / cm 2. The constructs are then incubated without medium for four hours at 37 ° C, 10% C02 to allow the epithelial cells to bind. After incubation, the constructions are immersed in a Cornea Maintenance Medium (CMM) (Johnson et al., 1992, supra). The epithelial cells are cultured until the table is covered with epithelial cells. The term epithelial coverage can be ascertained by a variety of methods, as an illustration, by dyeing the culture with a solution of Nile Blue sulfate (1: 10,000 in phosphate-buffered saline). Once the table is covered, after approximately seven days, the constructions are transferred aseptically to new culture trays with enough corneal maintenance medium (CMM) to achieve a fluid level right up to the construction surface in order to of maintaining a wet interface without submersion of the epithelial layer. The constructions are incubated at 37 ° C, 10% C0, and more than 60% humidity, with the CMM changing medium, as necessary, typically three times a week. As used herein, the term "wet interface" is intended to mean a culture environment that is regulated so that the construction surface is wet, with high humidity, but not dry or submerged. The exact level of juiciness and moisture in the culture environment is not critical, but it must be sufficiently juicy and moist to prevent the formation of cornified cells. A wet interface can be characterized by trying to duplicate humidity levels similar to those of the human eye. Immunocytochemical comparison of incubation of the construct in (1) a true aerial interface (dry) versus (2) submerged incubation versus (3) incubation in a wet (but not submerged) interface showed that the environment depended on the production of an epithelium; which approaches a normal cornea. However, incubation in a dry interface caused the cornea epithelium to suffer from abnormal squamous differentiation (dermal line). These are several alternatives to achieve a wet interface of the epithelial layer and the medium. An alternative method to achieve a wet interface in the epithelial layer uses a liquid / mucin mixture to simulate a tear film. The specialized tear film can be formulated using a physiologically regulated salt solution containing lipid-protein or lipid surfactants and / or mucin, glycosaminoglycans, hyaluronic acid or other substance that maintains moisture. The film drop is placed on the top of the table to maintain a moisture barrier between the epithelium and the atmosphere. The film is typically replaced when the medium is changed. Alternatively one or more of the components of the tear film can be added directly to the medium and braided on the construction surface during cultivation to form the wet surface interface. Alternatively, the maintenance of a wet interface can also be aided by the use of an artificial layer that can attract and maintain moisture on the surface of the crop. This can be achieved by applying a thin layer made of agarose, hydrogel, or alginate. In another alternative, a wet interface can be achieved through the use of a dialysis membrane or polymer, such as contact lens material, a cut slightly larger than that of the table can be used to attract and maintain fluid and prevent moisture loss . 2. Use of the Endothelium in Other Organ Equivalents. The inclusion of an endothelial layer promotes a morphology, expression of biochemical and physiological markers, cellular diffusion, epithelial attachment to the matrix and uniformity of the epithelial coverage in vi tro, improved. The results of the influence of the endothelium in obtaining a higher level of epithelial differentiation in vi tro and the promotion of a basement membrane formation were applied in other culture methods in vi tro of tissue equivalents. Examples of tissue equivalents that can be modified in accordance with this invention include US Series No. 08 / 193,809, incorporated herein by reference, and specifically dermal and skin equivalents. In general, to make a skin equivalent, the past dermal fibroblasts are mixed with type I collagen to form a cellular collagen lattice within a culture graft. This grid then serves as a substrate for epidermal keratinocytes. The keratinocytes grow to confluence and stratify while the culture remains submerged in the culture medium during the first 4 days. The skin equivalent is then grown at the air-liquid interface to allow differentiation of the epidermis to proceed. In preparing tissue or organ equivalents using collagen according to this invention, a first layer of endothelial cells can be cultured, as described above in section one, before melting collagen in the endothelial layer. In a preferred embodiment, the endothelial cell layer is used to modify equivalent models of skin in vi tro, such as those described in US 4,485,096, incorporated herein by reference, to promote epithelial differentiation and basement membrane formation . 3. Uses for the Cornea Equivalent Model. The Draize eye irritation test has served as the standard for evaluating the potential for eye irritation of a product during the last forty-five years. (Draize, JH, Oodard, G., Calvery, HO, "Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes" (Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes), J. Pharmacel, Exp. Ther.82: 377-390 (1994)). A variety of examination models and protocols have been proposed as in vitro grids to assess ocular irritation. (Booman, KA, De Prospo, J., Demetrulias, J., Driedger, A., Griffith, JF, Grochoski, G., Kong, B., McKormick, .C., North-Root, H., Rosen, MG, Sedlak, RI, "In vitro methods for estimating eye irritation of cleaning products, Phase I: Preliminary assessment" (In vitro methods for estimating ocular irritation of cleaning products, Phase I: Preliminary evaluation), J. Toxicel. . &Ocular Toxicol 7: 173-185 (1988)). Cell cultures used in conjunction with objective, quantifiable terminal points to assess cytotoxicity that has shown good correlation with in vi tro data sets. (Bruner, LH, Kain, DJ, Roberts, DA, Parker, RD, "Evaluation of seven in vitro alternatives for ocular safety testing", Evaluation of seven in vitro alternatives for ocular safety examination), Appl. Toxicol. 17: 136-149 (1991)). However, cells in monolayer cultures have inherent limitations as model systems for predicting irritation in complex organs such as the eye. Typically, cells in a monolayer culture are susceptible to irritants in concentrations well below those required to induce in vivo irritation. The test samples must first be solubilized in a cell culture medium before being introduced into the culture system. This can lead to secondary toxicities due to the effects on osmolarity, pH or components of the medium. In addition, facts arising from the dilution of the test sample can mask the toxicity and lead to overestimation of the irritation potential of a sample. The level of epidermal differentiation obtained in a monolayer culture only poorly mimics the degree of differentiation observed in vivo. The protective barrier function of the cornea epithelium, which includes cytoskeletal keratin networks, desmosomes and light junctions, is not known to play an important role in the protection of ocular tissue from chemical attack. (Holly, F.J., "Physical chemistry of the normal and disordered tear film" (Physical chemistry of the normal and disordered tear film), Trans Opthalmol, Soc. U.K. 104: 374-380 (1985)). The proposed organotypic model overcomes some of the inherent limitations of monolayer culture by providing a model system that more accurately simulates the target organ of interest. In addition, the physical configuration of this test cornea allows the topical application of test samples in vehicles (eg, petrolatum and mineral oil) that approximate the exposure mode in vi. Researchers have used both animal models and cells grown in an effort to approximate the human condition. However, there is a wide separation in direct applicability. Animals may be too different in their physiological response to damage and analyzes using a traditional cell culture may be too simplified for direct correlation with possible human responses in vi vo. Although these methods are necessary and useful, the use of human organotypic constructs helps eliminate the discrepancy between the human and animal response, and links the separation between the cultured cells and the complex organism. Cell-cell interactions and the response to damage or pharmacological agents can be easily examined in an organotypic, controlled environment. The organotypic culture method can also be used to form human graft tissue either as an adjunct to conventional transplantation or as a substitute. The use of cultured corneal endothelial cells has already been shown to be beneficial as a replacement for inadequate or often damaged endothelium of graft material. (Insler, M.S., López, J.G., "Transplantation of vultures human neonatal corneal endothelium" (Cultured human neonatal cornea endothelial transplantation), Curr. Eye Res. 5 (12): 967-72 (1986)). The use of cultured cornea epithelium has also shown some benefit in the promotion of wound closure. (Roat, M.I., Thoft, R.A., "Ocular Surfece Epithelial Transplantation", Opthalmol Int. Clin. 28 (2): 169-174 (1988)). The organotypic cornea construction comprising an endothelium, stroma and epithelium could be used for the closure of eye injuries and for the repair of the total thickness of the cornea. Although it is not transparent in vi tro, it is expected that the endothelial cells provided by the construct will regulate fluid transport to the corneal stroma and will further stimulate the fibroblasts to continue to organize the matrix and produce the appropriate collagens and glycosaminoglycans needed to the clarity of cornea. The equivalent of cornea in vi tro can be constructed with more or less extracellular matrix or stroma to facilitate remodeling. The closure of the wound would be maintained by the presence of well-attached corneal epithelium, thereby limiting hyperproliferation and intimidation of the stromal matrix. The invention is further illustrated by the following examples, which should not be taken as limiting in any way. EXAMPLES Example 1: Comparison of Normal Endothelial Cells of Various Origins. Purpose: This study was carried out to determine whether non-transformed endothelial cells of cornea and non-cornea origin can effect epithelial differentiation and basement membrane formation. Materials and Methods: Transformed mouse cornea endothelial cells (MCE) described in S. E. U. N. 07 / 974,740 and compared with the following normal strains: a. Corneal endothelial cells, ovine (SCE2), b. Arterial, ovine endothelial cells (SEC 006A), c. Ovine endothelial cells (SEC 023VC), d. Vena cava, canine endothelial cells (DVCEC), e. Human umbilical vein endothelial cells (HUVEC), and f. Endothelial cells of the cornea, bovine (BCE 15960). As a negative control, the constructs were prepared without endothelial cells. The Cornea Maintenance Medium (CMM) consisted of a 3: 1 mixture of Medium: Ham's F-12 modified with Dulbecco's, calcium-free with 1.1 μM of hydrocortisone, 5 μg / mL of insulin, 5 μg / mL of transferrin, 20 pM triiodothyronine, 10"4M ethanolamine, 10 ~ 4M o-phosphoryl-ethanolamine, 1 mM strontium chloride, 50 μg / mL gentamicin, 4 M 1-glutamine, 90 μM adenine, 3 x 10"6M selenium, 1.8 mM calcium chloride and 0.3% NBCS. Endothelial cells were taken from frozen deposits and cultured in one step before being used in corneal constructions. The endothelial cells were seeded at 3.0 x 10 4 cells per transcrisol on a 1 mg / ml acellular layer of type I collagen gel and developed to confluence for 7 days while they were immersed. The HUVEC, SEC 023VC, BCE 15960, and SEC 006A were cultured in Ham's F-12 + 10% NBCS, 25 μg / mL Heparin, 50 μg / mL ECGS and 50 μL / mL gentamicin. MCEC, DVEC, SCE2, and HEC were grown in DMEM + 10% NBCS and 50 μg / mL gentamicin while they were incubated at 37 ° C in an atmosphere of 10% C02. Rabbit cornea keratocytes at a density of 5.0 x 10 5 cells / mL in 3 mL of 1 mg / mL of Type I collagen were spread over the top of the endothelial layer. The collagen containing the gelled cells and the medium (DMEM - 10% NBCS and 50 μg / mL gentamicin) were added to the outside of the transcrisol. The constructs were incubated at 37 ° C with 10% C02 for the next seven days while the lattice lattice of collagen containing the fibroblasts was contracted by the corneal fibroblasts. The collagen reticles have contracted away from the sides of the transcrisol to form a table. The medium was removed from the crucible and 50 μL of rabbit cornea epithelial cells in suspension (3.6 x 10 6 cells / mL) were seeded in the center of the table, in 1.8 x 105 cells / table in CMM. 12 mL of CMM was added again, submerging the entire construction. Seven days after post-epithelialization, the rabbit corneal epithelial cells proliferated to completely cover the table. The epithelial coverage was monitored by dyeing with Nile Blue. The dyeing was done by the addition of 8 mL of 1; 10,000 Nile Blue Sulfate in phosphate buffered saline (PBS) for 30 minutes. Adherence was examined when attempting to peel off the stained epithelium from the collagen reticulum with forceps. The resistance to barking indicated a positive effect of the endothelial cell layer. When the epithelial coverage was completed, the constructs were transferred to a new tray containing two cotton pads and sufficient medium, 11 mL, (9 mL in feeds hereafter since the cotton pads together hold 2 mL) to cover only the surface of the crop and braid on the surface of the construction. The completion of this stage provided a wet apical surface to avoid abnormal squamous differentiation. The cultures were maintained in the incubator at 37 ° C in 10% C02 and medium changes were made every 2-3 days during the rest of the culture period. Results: General observations: the Nile Blue sulfate staining was complete, although the coverage varied in all conditions. Qualitative barking examination indicated adherence in cultures of MCE (positive control) and BCE, slight adhesion in SEC 023VC and SCE2 and no adherence in HUVEC, DVEC, SEC 006A and no culture of endothelium 7 days after -epithelialization. Immunocytochemistry: Immunocytochemistry using indirect fluorescence was carried out using an anti-laminin antibody to detect the deposition of basement membrane components, and anti-ZO1 antibody to detect the distribution of protein associated with light attachment. Distribution of Laminin: in previous studies described in the U.S.S.N. 07 / 974,740, small amounts of laminin were located in spots along the basement membrane zone in air-displacing cultures of moisture, which lack endothelial cells.

The addition of MCE greatly improved the laminin deposition resulting in a continuous, strongly stained distribution along the stromal-epithelial junction. The cultures of HUVEC and SEC 006A showed a continuous distribution of laminin. The DVEC, SCE2, and SEC 023VC showed an almost continuous distribution. The ECB showed a distribution similar to that previously obtained in wet cultures that lack an endothelial cell layer. (See figures 1A to 1F). ZOl distribution: ZOl is a protein associated with light junctions in the cornea epithelium. The normal distribution should be limited to different areas in the upper layers of the cornea epithelium. The positive MCE and DVEC controls showed an evident location in the uppermost layers of the epithelium. Cultures of SCE2 and SEC 023VC also showed a limited distribution, although cultures of HUVEC, BCE and SEC 006A showed a widely distributed staining indicating the lack of specialized localization of the ZOl protein. (See figures 2A to 2G). Formation of the Basal Membrane: The assembly of the basement membrane was examined by electron microscopy. (See figures 3A to 3C). The cultures that contain MCE form recognizable basement membrane and well formed hemidesmosomes. Hemidesmosomes and associated basement membrane structures were detected in cultures containing SEC 006A and SCE2. Conclusions: The technical difficulties associated with endothelial cell culture and the survival of the various sources possibly objected to the total expression of all the characteristics of a given endothelial cell strain and led to variability in the results. However, in each case, one or more of the endothelial cell strains experienced imitated characteristics, achieved through the use of the transformed MCE cornea cell line. Since there were always more ECM cells present in these cultures due to their rapid growth and lack of contact inhibition, the differences were attributed to the concentration effects rather than the inherent inability of the various endothelial strains to produce an effect on the epithelium. Of the strains examined, the cells of SCE2 and SEC 006A showed the greatest ability to influence the basement membrane. Epithelial differentiation seemed more affected by SCE2 and DVEC. The refinement of the culture process in order to adapt to the particular requirements to adapt the growth and survival of normal endothelial cells will lead to results similar to those obtained with the mouse line transformed into those strains that show a positive effect. The effects of the endothelium on endothelial differentiation and basal lamina formation is not specific for specific species nor is it limited to endothelial cells of cornea origin. Example 2: Comparison of Wet and Submerged Tissues Containing Endothelial Cells Purpose: This study was carried out to determine if a wet interface is necessary for the total expression of the differentiated characteristics and the formation of basal lamina. Materials and Methods: The cultures were prepared according to the previous example using MCEC in the endothelial layer. At the time of air extraction of moisture, a portion of the crops remained submerged in the culture medium. Both groups were maintained for one and two additional weeks. The cornea constructions were examined 14 days after immersion and 7 days after air extraction of the moisture by histology, immunocytochemistry and electron microscopy. Results: The comparison of the two groups showed no differences in histology or immunocytochemical distribution of keratin 3 (Figures 4A and 4B), alpha-6-integrin (Figures 5A and 5B), Type VII collagen (Figures 6A and 6B), laminin ( Figures 7A and 7B) or alpha-enolase (Figures 8A and 8B). Electron microscopy showed no ultrastructural differences between wet and submerged cultures: the submerged cultures had superficial vermiform ridge specializations (Figure 9A) and exhibited a basement membrane (Figure 9B) identical to those observed in standard moisture cultures containing endothelium. Conclusion: A specialized environment is not necessary to achieve total epithelial differentiation in the presence of endothelial cells. Example 3: Examination of the Endothelial Cell Effect on Skin Constructions. Purpose: This study was carried out to determine if the presence of endothelial cells could assemble the basement membrane in other epithelial cell types. Materials and Methods: Human epidermal keratinocytes and dermal fibroblasts were substituted for corneal epithelial and stromal cells, respectively, in the preparation of the skin construction. The endothelial cells were taken from frozen deposits (the transformed mouse cornea endothelial cells (MCE) of Example 1) and cultured in one step before being used in the construction. Endothelial cells were seeded at 3.0 x 104 cells per transcrisol on a 1 mg / ml acellular layer of Type I collagen gel and grown to confluence for 7 days while immersed in 10% NBCS of DMEM and 50 μg / mL of gentamicin while incubated at 37 ° C in a 10% C02 atmosphere. Human dermal fibroblasts (HDF), at a density of 2.5 x 10 5 cells / mL in 3 mL of Type I collagen 1 mg / mL, were placed on top of the endothelial layer. The collagen containing the gelled cells and the medium (DMEM-10% NBCS and 50 μg / mL gentamicin) was added to the outside of the transcrisol. The constructs were incubated at 37 ° C with 10% C02 for the next 7 days while the collagen lattice containing the fibroblasts was contracted by the corneal fibroblasts. After 6 days, the collagen reticules have contracted away from the sides of the transcrisol to form a table. The medium was removed from the crucible and a suspension of 50 μL of human epidermal cells (3.33 x 106 cells / mL) was seeded in the center of the table in MSBM (3: 1 glucose-free DMEM, calcium free: F-12 of Ham supplemented with 4 mM L-glutamine, 1.1 μM hydrocortisone, 5 μg / mL insulin, 5 μg / mL triiodothyronine, 10 ~ 4 M ethanolamine, 10"4 M, o-phosphoryl-ethanolamine, 0.18 mM of adenine, 2 x 10"9M of progesterone, 5.26 x 10" 8M of selenium, 0.3% of bovine serum, 1.8 mM of calcium chloride and 10 ng / mL of epidermal growth factor.) The MSBM medium was added again to the outside of the transcrisol, submerging the entire construct The constructions were returned to the incubator at 36 ° C / 10% C02 Four days after the epidermalization, the human epidermal cells proliferated to completely cover the table. removed from the inside and outside of the chambers, the inner chamber was removed and two cotton pads were placed in the crucible to elevate the construction to the air-liquid interface. The medium, 11 mL of cSBM with calcium (1: 1 of DMEM free of glucose, free of calcium: F-12 of Ham supplemented with 4 mM of L-glutamine, 1.1 μM of hydrocortisone, 5 μg / mL of insulin, 5 μg / mL of triiodothyronine, 10 ~ 4 M of ethanolamine, 10 ~ 4 M or -phosphoryl-ethanolamine, 0.18 mM of adenine, 5.26 x 10-8 M of selenium, 2.0% of bovine serum, 1 ng / mL of growth factor epidermal and 1.8 mM calcium chloride) was added again to the transcrisol containing the construct. The constructions were returned to the incubator at 35.5 ° C / 10% C02.

The constructions were maintained under culture conditions suitable for the culture of a skin equivalent. Four days after extraction by air, the medium was replaced with 9 mL of maintenance SBM with calcium (1: 1 of DMEM free of glucose, free of calcium: F-12 of Ham supplemented with 4 mM of L-glutamine, 1.1 μM hydrocortisone, 5 μg / mL insulin, 5 μg / mL triiodothyronine, 10 ~ 4 M ethanolamine, 10"4 M, o-phosphoryl-ethanolamine, 0.18 mM adenine, 5.26 x 10" 8 M selenium, 1.0% of bovine serum, 1 ng / mL of epidermal growth factor and 1.8 mM of calcium chloride). The cultures were maintained at the air-liquid interface on day 4 after the epidermalization. The cultures were maintained for one week at the air-liquid interface and examined for basal membrane formation by electron microscopy seven days after air extraction. Results: The qualitative tests of descortezamiento at the moment of the extraction by air showed a firm subjection of the epidermis to the dermic substrate. Electron microscopy of post-extraction samples by air on day 7 with an endothelial layer (Figure 10A) revealed a basal membrane formation superior to that observed in the absence of endothelium at 14 days post-extraction by air. Unlike corneal constructions, skin constructs cultured at an air-liquid interface without an endothelium show a normal distribution of differentiation markers and the continuous localization of basal lamina components but show only a rudimentary basal membrane and the formation of hemidesmosome after weeks in culture (Figure 10B). These results are with an air-liquid interface. It is expected from the results of Example 2 that improved differentiation of submerged skin cultures would be achieved in the presence of an endothelium. Conclusion: The presence of the endothelial cell layer improves the assembly of the basement membrane by epidermal keratinocytes. Although the above invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious to one skilled in the art that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (9)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and therefore the property described in the following claims is claimed as property. 1. A tissue equivalent comprising: (a) an inner endothelial cell layer; (b) a layer of medium stromal cell-collagen mixture, wherein said stromal cells are derived from fibroblast cells; and (c) an outer epithelial cell layer. The tissue equivalent according to claim 1, characterized in that said inner endothelial layer is derived from one or more cells selected from the group consisting of arterial endothelial cells, vena cava endothelial cells, and endothelial cells of the human umbilical vein, and one or more cells as described above transformed with a recombinant construct. The tissue equivalent according to claim 1, characterized in that said stromal cells of said cell-stromal collagen mixture layer are fibroblast cells and wherein said collagen is Type I or Type III collagen or a mixture thereof. 4. The tissue equivalent according to claim 1, characterized in that said outer epithelial cell layer are keratinocyte cells. 5. A method for producing a tissue equivalent comprising: (a) culturing endothelial cells to form an inner endothelial cell layer; (b) mixing stromal cells with collagen to achieve a cell-stromal collagen mixture, wherein said stromal cells are derived from fibroblast cells; (c) contacting said inner endothelial cell layer of step (a) with said stromal cell-collagen mixture of step (b), thereby forming a middle layer provided on said inner layer; (d) culturing said inner endothelial cell layer and said middle layer; (e) contacting epithelial cells on said middle layer of step (d); (f) culturing said epithelial cells with said middle layer until said middle layer is covered with an outer layer of epithelial cells; and (g) continuing to cultivate said inner, middle and outer layers to form a skin equivalent. The method according to claim 5, characterized in that said endothelial cells of said inner endothelial cell layer are cultured in said first step by contacting said endothelial cells on a porous membrane attached to the lower part of a cell culture graft. The method according to claim 6, characterized in that before said contact, a layer of acellular collagen is melted on said porous membrane. 8. A method for examining the effect of a test substance comprising: (a) exposing the tissue equivalent of claim 1 to the test substance; and (b) determining the effect of said test substance on said equivalent. 9. A method for transplanting the tissue equivalent of claim 1 into a receiver comprising grafting said tissue equivalent into said receiver.