MXPA01005098A - Bioengineered tissue constructs and methods for producing and using them - Google Patents

Abstract

Cultured tissue constructs comprising cultured cells and endogenously produced extracellular matrix components without the requirement of exogenous matrix components or network support or scaffold members. Some tissue constructs of the invention are comprised of multiple cell layers or more than one cell type. The tissue constructs of the invention have morphological features and functions similar to tissues and their strength makes them easily handleable. Preferred cultured tissue constructs of the invention are prepared in defined media, that is, without the addition of chemically undefined components.

Description

BIODISED TISSUE CONSTRUCTIONS AND METHODS FOR PRODUCING AND USING THEM FIELD OF THE INVENTION The invention is in the field of tissue design. This invention is directed to an in vitro method for inducing cells to produce an extracellular matrix. This living extracellular matrix, which has tissue-like properties, can be used for clinical or testing purposes.

BACKGROUND OF THE INVENTION The field of tissue design combines biodesign methods with the principles of the science of life to understand the structure and function relationships in normal and pathological mammalian tissues. The goal of tissue design is the development and ultimate application of biological substitutes to restore, maintain, or improve tissue functions. Thus, through tissue design, it is possible to design and manufacture a biodesigned tissue in a laboratory. Bioengineered tissues may include cells that are usually associated with native mammalian or human tissues and synthetic scaffolds or exogenous matrix scaffolds. The new biodesigned tissues must be functional when they are grafted to a host, and be permanently incorporated into the body of the host or progressively biodesigned by the cells from the recipient host patient. The fabrication of a tissue equivalent without a support member or scaffold leads to scientific challenges to create the new biodesigned tissues.

BRIEF DESCRIPTION OF THE INVENTION The invention is directed to bioengineered tissue constructs of cultured cells and which endogenously produce components of the extracellular matrix without the requirements of exogenous components of the matrix or of maintenance networks or of scaffold members. The invention can thus advantageously be made completely from human cells, and from the components of the human matrix produced by those cells, for example, when bioengineered tissue constructs are designed for use in humans. The invention is also directed to methods for producing tissue constructions by stimulating cells in culture, such as fibroblasts, to produce components of the extracellular matrix without the addition of either exogenous matrix components, maintenance networks or scaffold members. The invention is also directed to methods for producing tissue constructions by stimulating cells in culture, such as fibroblasts, to produce components of the extracellular matrix in a defined medium system and / or without the use of undefined components or biological components not derived from human, such as bovine serum or organ extracts. In addition, this tissue construct can be made by a series of seeds of different cell types to produce a cultured tissue construct that mimics the cellular composition and tissue structures of native tissues. Additionally, tissue construction is produced and self-assembled by cultured cells without the need for support scaffolds or the addition of exogenous components of the extracellular matrix. The strength characteristics of the tissue constructions make them manageable so that they are easily removed and separated from the culture apparatus in which they are formed and transplanted directly without the need of any support or carrier in clinical or assay applications. The tissue constructions of the invention are useful for clinical purposes such grafts to a patient with tissue or organic defects, such as skin ulcers or wounds or for in vitro tissue tests or animal grafts such as for safety testing or validation of pharmaceutical products. , cosmetics, and chemicals.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph describing the increase in collagen concentration as determined by the hydroxyproline assay as compared to the cell number in a dermal construct derived from human neonatal foreskin cells described in example 1. Figure 2 is a microphotograph (20x objective) of a fixed section, embedded in paraffin, stained with hematoxylin and eosin of a cell-matrix construction formed from cultured fibroblasts of human dermis in a chemically defined medium at 21 days. The porous membrane appears as a thin translucent band between the construction. Figure 3 shows transmission electron microscopy images of two amplifications of a cell-matrix construct formed from cultured fibroblasts of human dermis in a chemically defined medium at 21 days. Figure 3A is a 7600X amplification showing the endogenous matrix including the alignment of the collagen fibers between the fibroblasts. Figure 3B is a 19000X amplification of fully formed endogenous collagen fibers demonstrating fibrillary arrangement and packaging. Figure 4 is a photomicrograph (20x objective) of a fixed section, embedded in paraffin, stained with hematoxylin and eosin of a cultured skin construct formed in chemically defined medium in the absence of exogenous components of the matrix comprising a cell-matrix construction formed from cultured fibroblasts of human dermis in a chemically defined medium with a differentiated, multilayer epidermis formed from human keratinocytes cultured in chemically defined medium.

DETAILED DESCRIPTION OF THE INVENTION Hitherto, the currently designed living tissue constructs are not fully assembled cells and must adhere to either the addition or incorporation of exogenous component of the shade or synthetic members for structure or maintenance, or both. The biodesigned tissue constructions described herein exhibit many of the native characteristics of the tissue from which these cells are derived. The tissue constructions thus produced can be used for grafting to a patient for in vitro testing. A preferred embodiment is a cell-matrix construct comprising a first cell type and an endogenously produced extracellular matrix wherein the first cell type is capable of synthesizing and secreting an extracellular matrix to produce cell-matrix construction. Another preferred embodiment is a bilayer construct comprising a first cellular type and an extracellular matrix endogenously produced and a layer of cells of a second type disposed on it or within the cell-matrix construction formed by the first cell type. A more preferred embodiment is a cell-matrix construct comprising fibroblasts, such as those derived from the dermis, to form a cultured dermis construct. Another more preferred embodiment is a cell-matrix construct comprising fibroblasts, such as those derived from dermis, to form a dermis construct cultured with a layer of keratinocytes grown thereon to form an epidemic layer to result in a cultured skin construct. in bilayer. The cultured skin constructions of the invention express many physical, morphological, and biochemical characteristics of the native skin. In an even more preferred embodiment, the cell-matrix construct is a tissue construct that is similar to the dermal layers of the skin, a human dermal construct that is formed in a defined system comprising human-derived cells that do not use components. chemically undefined during its cultivation. In the most preferred mode. The tissue constructs of the invention are manufactured in a chemically defined system comprising cells derived from human but not undefined chemical components or non-human biological components or cells. A preferred embodiment of the invention comprises a structural layer of at least one type of extracellular matrix producing cells and endogenously produced extracellular matrix components, more simply referred to as "matrix", where the matrix is completely synthesized by the cell and assembled by culturing the cells. This layer is referred to herein as a "cell-matrix construction" or a "cell-matrix layer" because the cells secrete and contain themselves in and around their matrix. Cultured tissue constructs do not require, therefore, do not include exogenous components of the matrix, that is, components of the matrix not produced by culturing cells but introduced by other means. In a more preferred embodiment, it is shown that the cell-matrix construction produced by human dermal fibroblasts has a predominant concentration of collagen similar to that of the native skin. As evidenced by electron microscopy, the matrix is fibrous in nature comprising collagen that exhibits the band pattern of 67 nm in four planes, as well as the organization of fibril packing and fibril aces similar to the native collagen. The delayed reduction of SDS-PAGE has detected the presence of collagen type I and type III in these constructions, the types of predominant collagen were found in native human skin. Using standard immunohistochemistry (IHC) techniques, the dermal cell-matrix construct stains positively for decorin, a proteoglycan dermatan sulfate known to associate with collagen fibrils and is thought to regulate fibril diameter in vivo. Decorina can also be visualized in the TEM construction. The tissue produced is also stained positively for tenascin, a matrix glycoprotein extracellular found, for example, in mesenchyme or in tissues under repair. Very similarly to the tissue under repair in vivo, the tissue produced in culture has been shown to increase its ratio of collagen type I to type III as the matrix is formed. Although one does not wish to be bound by theory, it is believed that the cells fill the open spaces between them quickly with a loose matrix analogous to the granulation tissue comprised primarily of collagen type III and fibronectin, and then remodel their loose matrix with a denser matrix comprising mainly type I collagen. The produced cell-matrix has been shown to contain glycosaminoglycans (GAG), such as hyaluronic acid (HA); fibronectin; proteoglycans aside from decorin such as biglucan and versican; and, a profile of sulfated glycosaminoglycans such as di-hyaluronic acid, di-chondroitin-O-sulfate; di-chondroitin-4-sulfate; di-chondroitin-6-sulfate; di-chondroitin-4,6-sulfate; di-chondroitin-4-sulfate-UA-2S; and di-chondroitin-6-sulfate-UA-2S. These structural or biochemical characteristics are exhibited by them as the construction takes place in culture and are distinctly evident when the construction approaches its final form. The presence of these components in the cell-matrix construction from fully formed dermal culture indicates that the construction has structural and biochemical characteristics that approach that of the normal dermis. Although the aforementioned list is a list of the biochemical and structural characteristics a cell-matrix construction formed from dermal fibroblasts should recognize that cultured cell-matrix constructs formed from other types of fibroblasts will produce many of these characteristics and other phenotypic characteristics for the type of tissue from which they originate. In some cases, fibroblasts can be induced to express non-phenotypic components either by chemical exposure or contact, physical stress, or by transgenic means. Another preferred embodiment of the invention is a cell-matrix layer having a second layer of cells disposed thereon. The second cell layer is grown on the cell-matrix layer to form a bilayer bio-engineered tissue construct. In a more preferred embodiment, the cells of the second layer are of epithelial origin. In the most preferred embodiment, the second layer comprises human cultured keratinocytes which together with a first cell-matrix layer, a cell-matrix construct formed from dermal fibroblasts and endogenous matrix to form a dermis layer, comprise a living, skin. When fully formed, the epidermal layer is a multilayer, stratified, well-differentiated keratinocyte layer that exhibits a basal layer, a suprabasal layer, a granular layer and a stratum corneum. The skin construct has a well-developed basement membrane present in the dermis-epidermis junction as exhibited by transmission electron microscopy (TEM). The basement membrane appears thicker around hemidesmosomes, marked by anchoring fibrils that comprise collagen type VII, and visualized by TEM. Anchoring fibers they can be seen existing from the basement membrane and trapping the collagen fibers in the dermal layer. These anchoring fibers, as well as the other components of the basement membrane, are secreted by keratinocytes. It is also known that while keratinocytes are able to secrete components of the basement membrane on their own, a recognizable basement membrane will not form in the absence of fibroblasts. Immunohistochemical staining of the skin constructs of the present invention have also shown that laminin, a basal membrane protein is present. In a preferred method of the invention for forming a cell-matrix construction, a first cell type, a cell type that produces extracellular matrix, is sown on a substrate, cultivated, and induced to synthesize and secrete an extracellular matrix organized around them to form a cell-matrix construction. In another preferred method of the invention, a surface of the cell-matrix construction is seeded with cells of a second cell type and cultured to form a tissue bilayer construct. In a more preferred method, a full-thickness tissue construct having similar characteristics to native human skin is formed by cultured fibroblasts, such as human dermal fibroblasts, under conditions sufficient to induce matrix synthesis to form a cell-matrix of dermal cells and matrix, a dermal layer, on which epithelial, human cells, such as keratinocytes, are they sow and cultivate under sufficient conditions to form a fully differentiated stratified epidermal layer. Therefore, a method for obtaining the tissue constructs of the present invention comprises: (a) cultivating at least one extracellular matrix producing cell type in the absence of exogenous extracellular matrix components on a structural support member; and, (b) stimulating the cells of step (a) to sesize, secrete, and organize extracellular matrix components to form a tissue construct comprised of cells and matrix sesized by those cells; wherein steps (a) and (b) can be done simultaneously or consecutively. To form a bilayer tissue construct comprising a cell-matrix construction and a second cellular layer thereon, the method further comprises the step of: (c) culturing cells of a second type on a surface of the formed tissue construct to produce a tissue construction in two layers. An extracellular matrix producing cell type for use in the invention can be any cell type capable of producing and secreting extracellular matrix components and organizing the extracellular matrix components to form a cell-matrix construction. More than one cell type producing extracellular matrix can be grown to form a cell-matrix construction. Cells of different cell types or tissue origins can be cultured together as a mixture to produce component complementary and structures similar to those found in native tissues. For example, the extracellular matrix producing cell type may have other cell types blended therewith to produce an extracellular amount that is not normally produced by the first cell type. Alternatively, the extracellular matrix producing cell type can also be mixed with other cell types that form specialized tissue structures in the tissue but do not contribute substantially to the complete formation of the matrix aspect of the cell-matrix construct, such as in certain skin constructions. of the invention. Although any extracellular matrix producing cell type can be used in accordance with this invention, preferred cell types for use in this invention are derived from mesenchyme. The most preferred cell types are fibroblasts, stromal cells, and other cells that maintain the connective tissue, more preferably human dermis fibroblasts found in human dermis for the production of a human dermal construct. The fibroblastic cells produce a number of extracellular matrix proteins, mainly collagen. There are several types of collagen produced by fibroblasts, however, collagen type I is the most prevalent in vivo. Strains of human fibroblast cells can be derived from a number of sources, including, but not limited to, the foreskin of the neonate, dermis, tendon, lung, umbilical cord, cartilage, urethra, corneal stroma, oral mucosa or intestine. Human cells may include but are not necessarily limited to fibroblasts, but may include: smooth muscle cells, chondrocytes and other connective tissue cells of mesenchymal origin. It is preferred, but not required, that the origin of the matrix producing cell used in the production of a tissue construct be derived from a tissue type that resembles or mimics after employing the culture methods of the invention. For example, in the embodiment wherein a skin construct is produced the preferred matrix producing cell is a fibroblast, preferably of dermal origin. In another preferred embodiment, fibroblasts isolated by microdissection can be used from the dermal papilla of the hair follicles to produce the matrix alone or in association with other fibroblasts. In the modality where a corneal construction occurs, the matrix-producing cell is derived from the corneal stroma. Cell donors may vary in development and age. The cells can be derived from donor tissues of embryos, neonates, or of elderly individuals including adults. Embryonic progenitor cells such as mesenchymal stem cells can be used in the invention and induced to differentiate to develop into the desired tissue. Although human cells are preferred for the use of the invention, the cells to be used in the method are not limited to cells from human sources. Cells from other mammalian species include, but are not limited to, equine, canine, porcine, bovine and ovine sources, or rodent species such as mice or rats that may be used. In addition, cells that are spontaneously, Chemically or virally transfected or recombinant cells or genetically engineered cells can also be used in this invention. For those embodiments that incorporate more than one cell type, chimeric mixtures of normal cells can be employed from two or more sources; mixtures of normal and genetically modified or transfected cells; or mixtures of cells from two or more species or tissue sources. Recombinant or genetically engineered cells can be used in production in cell-matrix construction to create a tissue construct that acts as a graft for drug administration to a patient in need of increased levels of natural cellular products or treatment with a therapeutic cells can produce and administer to the patient via the cellular products of the recombinant graft, growth factors, hormones, peptides or proteins, for a continuous amount of time or as needed when biologically, chemically, or thermally signaling due to the conditions present in the patient. The expression of the gene product either in the long term or in the short term is desired, depending on the indication of the use of the cultured tissue construct. Long-term expression is desired when the cultured tissue construct is implanted to deliver therapeutic products to a patient for an extended period of time. Conversely, short-term expression is desired in the cases where the cultured tissue construct is grafted to a patient who has a wound in where the cells of the cultured tissue construct will promote normal or near-normal healing or reduce the scarification of the wound site. Once the wound has healed, the gene products from the cultured tissue construct are no longer needed or are no longer desired on the site. The cells can also be genetically engineered to express proteins or different types of extracellular matrix components that are either "normal" but that are expressed at high levels or can be modified to make a grafting apparatus comprising extracellular matrix and living cells that is therapeutically advantageous to improve healing, facilitating or directing neovascularization, or minimizing the formation of scars or keloids. These procedures are generally known in the art, and are described in Sambrook et al, Molecular Cloninq, A Laboratorv Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), incorporated herein by reference. All cell types mentioned above are included within the definition of a "matrix producing cell" as used in this invention. The predominantly predominant extracellular matrix component produced by fibroblasts is fibrillar collagen, particularly type I collagen. Fibrillar collagen is a key component in the cell-matrix structure; however, this invention is not limited to matrices comprised of only this protein or type of protein. For example, other collagen, both fibrillar and non-fibrillar collagen from the collagen family such as collagen types II, III, IV, V, VI, VII, IX, X, XI, Xll, XIII, XIV, XV, XVI XVII, XVIII, XIX, can be produced by the use of the appropriate cell type. Similarly, other matrix proteins that can be produced and deposited using the current method include, but are not limited to, elastin; proteoglycans, such as decorin, or biglucan; or glycoproteins such as tenascin; vitronectin; fibronectin; laminin; thrombospondin I and glycosaminoglycan (GAG) such as hyaluronic acid (HA). The matrix producing cell is cultured in a container suitable for animal cell culture or tissue culture, such as culture dishes, bottles, or rotating bottles, which allow the formation of a three dimensional, tissue-like structure. Suitable cell culture surfaces on which the cells can grow can be of any biologically compatible material to which the cells can adhere and provide an anchoring means for the cell-matrix construction to be formed. The materials such as glass; stainless steel; polymers, including polycarbonate, polystyrene, polyvinylchloride, polyvinylidene, polydimethylsiloxane, fluoropolymers, and propylene fluorinated ethylene; and silicone substrates, including fused silica, polysilicon, or silicone crystals can be used as cell growth surfaces. The cell growth surface material may be chemically treated or modified, electrostatically, or coated with biological elements such as poly-1-lysine or peptides. An example of a coat peptide is the RGD peptide.

Although tissue constructions of the construct can be grown on a solid cell growth surface, a cell growth surface with pores communicating both the upper and lower surface of the membrane is preferred to allow bilateral contact of the medium with the construction of tissue in development or for contact only from the bottom of the crop. The bilateral contacts allow the medium to contact both the upper and lower surface of the construction under development for a maximum exposure of the surface area to the nutrients contained in the medium. The medium can also be contacted only with the lower part of the tissue construction grown in formation so that the upper surface can be exposed to air, such as the development of a skin culture construct. The preferred culture vessel is one which uses a carrier insert, a culture member treated with a waterproof such as a porous membrane which is suspended in the culture vessel containing the medium. Typically, the membrane is secured to one end of a tubular member or frame that is inserted into and interfacing with the base, such as petri dishes or culture boxes that can be covered with a mesh. The culture vessels incorporate a carrier insert with a porous membrane known in the art and which is preferred to carry out the invention and is described in a number of United States patents in the field, some of which are commercially available, including for example: 5,766,937, 5,466,602, 5,366,893, 5,358,871, ,215,920, 5,026,649, 4,871,674, 4,608,342, the description of which is incorporated herein. When these types of culture vessels are used, the tissue construction is produced on a surface of the membrane, preferably the upper part, the surface facing towards the upper part and the culture is brought into contact by the cellular medium on both surfaces in the upper and lower. The pores in the growth surface allow the passage of the culture medium to supply nutrients to the lower part of the culture through the membrane, thus allowing the cells to be fed bilaterally or only from the lower part. A preferred pore size is one that is small enough that it does not allow cell growth through the membrane, but large enough to allow the free passage of nutrients contained in the culture medium to the lower surface of the construction cell-matrix, such as by capillary action. Preferred pore sizes are about less than 3 microns but have a range between about 0 microns to about 3 microns, more preferably between about 0.2 microns to about 1 microns and more preferably about 0.4 microns to about 0.6 pore size micras that are used. In the case of human dermal fibroblasts, the most preferred material is polycarbonate having a pore size between about 0.4 to about 0.6 microns. The maximum pore size depends not only on the size of the cell but also on the ability of the cell to alter its shape and pass through the membrane. It is important that the tissue-like construction adhere to the surface but do not incorporate or wrap the substrate so that it is removed from it by detachment with minimal force. The size and shape of the tissue construction formed is dictated by the size of the surface of the vessel or the membrane on which it grows. The substrates may be round or angular or of angular shapes with rounded ends, or of irregular shapes. The substrates can also be flat or contoured as a mold to produce a shaped construction to interface with a wound or mimic the physical structure of the native tissue. To have larger surface areas of the growth substrate, more cells are seeded to the surface and a larger volume of medium is needed to sufficiently bathe and nourish the cell. When the proprietary construction is finally formed, either in a single-layer cell-matrix construction or in a bilayer construction, it is removed by detachment from the membrane substrate before being grafted to a patient. The cultured tissue constructions of the invention do not adhere to synthetic or bioabsorbable members, such as a mesh member for the formation of the tissue construct. The mesh members are organized as a fabric, a weave, or a felt material. In systems where a mesh member is employed, the cells are grown on a mesh member and grown on either side and within the interstices of the mesh to develop and incorporate the mesh into the cultured tissue construct. The final construction formed by methods that incoforan such as a mesh are adhered to the physical support and the volume form, examples of the tissue culture constructions that abide by synthetic mesh members are found in U.S. Patents. No. 5,580,781, 5,443,950, 5,266,480, 5,032,508, 4,963,489 or Naughton, et al. The system for the production of the cell-matrix layer can either be static or can use a perfusion medium for the culture medium. In the static system, the culture medium is still relatively motionless in contrast to the perfusion system where the medium is in motion. The perfusion of the medium affects the viability of the cells and increases the development of the matrix layer. Perfusion means include, but are limited to: using a magnetic bar or motorized propeller in the adjacent culture dish (below) or adjacent to the carrier substrate containing the culture membrane to agitate the medium; pumping means in or through the culture box or chamber, gentle agitation of the culture box on a rotary shaker or platform; or spin, if it occurs in a bottle to rotate. Other perfusion media can be determined by one skilled in the art for use in the methods of the invention. Formulations of the culture medium suitable for use in the present invention are selected based on the cell types to be cultured and on the tissue structure to be produced. The culture medium that is used and the specific culture conditions need to promote cell growth, matrix synthesis, and viability that will depend on the type of cell to be grown.

In some cases, such as in the manufacture of bioengineered bilayer skin constructions of the present invention, the composition of the medium varies with each stage of manufacture since different supplementation is necessary for different purposes. In a preferred method, the cell-matrix layer is formed under defined conditions, that is, it is grown in chemically defined medium. In another preferred method, a tissue construct comprises a cell-matrix layer provided with a second layer of cells disposed thereon wherein both cell types are grown in a defined culture medium system. Alternatively, the tissue construct comprises a cell-matrix layer manufactured under defined medium conditions and a second layer formed thereon under undefined conditions of the medium. In contrast, the tissue construct comprises a cell-matrix layer that can be manufactured under undefined medium conditions and the second layer formed on it under defined medium conditions. The use of chemically defined culture medium is preferred, medium free of extracts from animal organs or undefined animal tissues is preferred, for example, serum, pituitary extract, hypothalamic extract, placenta extract or embryonic extract or proteins and secreted factors by cells fed. In a more preferred embodiment, the medium is free of undefined components and biologically defined components derived from non-human sources. Although the addition of undefined components is not preferred, they can be used in accordance with the methods described at any point in the culture in order to manufacture tissue constructions successfully. When the invention is carried out using human selection cells using chemically defined components not derived from non-human sources, the resulting tissue construct is a defined human tissue construct. Synthetic functional equivalents may also be added to the chemically defined medium supplemented within the scope of the definition of chemically defined for use in the most preferred manufacturing method. Generally, an expert in the cell culture technique will be able to determine suitable natural human equivalents, human recombinants or synthetic equivalents to commonly know the animal components to supplement the culture medium of the invention without conducting research or experimentation. The advantages of using such construction in the clinic are that the concern for contamination and infection by adventitious animals or viruses that cross species is diminished. In the test scenario, the advantages of a chemically defined construction are that when tested, there is no opportunity for the results to be confused due to the presence of undefined components. The culture medium comprises base nutrients usually supplemented additionally with other components. The skilled artisan can determine the appropriate nutrient base in the animal cell culture technique with reasonable expectation to successfully produce a tissue construction of the invention. Many commercially available nutritional sources are used in the practice of the present invention. These include commercially available nutrient sources that supply inorganic salts, and energy sources, amino acids, and B vitamins such as Dulbecco's Modified Eagle Medium (DMEM); minimum essential medium (MEM); M199; RPMI 1640; Dulbecco medium modified by Iscove (EDMEM). Essential minimal medium (MEM) and M199 require additional supplementation with phospholipid precursors and non-essential amino acids. Commercially available vitamin-rich mixtures that supplement the additional amino acids, nucleic acids, with enzymatic factors, phospholipid precursors, and inorganic salts include Ham F-12, Ham F-10, NCTC 109, and NCTC 135. Although in various concentrations, all Basal media provides a basic source of nutrients for cells in the form of glucose, amino acids, vitamins and inorganic ions, along with other basic components of the medium. The most preferred base medium of the invention comprises a nutrient base of either calcium-free or low-calcium-modified Dulbecco Eagle medium (DMEM), or, alternatively, DMEM and Ham F-12 between a 3 to 1 ratio to a ratio 1 to 3, respectively. The base medium is supplemented with components such as amino acids, growth factors and hormones. The culture medium defined for the cultivation of the cells of the invention is described in U.S. Patent No. 5,712,163 to Patenteau and in the PCT International Publication No. WO 95/31473, the description of which is incorporated herein by reference. reference. Other means are known in the art such as those described in Ham and McKeehan, Methods in Enzymology, 58: 44-93 (1979), or for other appropriate chemically defined media, in Bottenstein et al., Methods in Enzymology, 58:94 -109 (1979). In the preferred embodiment, the base medium is supplemented with the following components known to those skilled in animal cell culture: insulin, transferrin, triiodothyronine (T3), and either or both ethanolamines and o-phosphoryl-ethanolamine, wherein the concentrations and substitutions of supplements can be determined by expert technicians. Insulin is a polypeptide hormone that promotes the taking of glucose and amino acids to provide long-term benefits over multiple passages. Insulin or insulin-like growth factor (IGF) supplementation is necessary for long-term cultures since there will be an eventual decrease in the ability of cells to take glucose and amino acids and possible degradation of the cell genotype. The insulin can be derived either from an animal, for example bovine, human sources, or by recombinant means such as recombinant human insulin. Therefore, a human insulin could be qualified as a chemically defined compound not derived from a non-human biological source. Insulin supplementation is recommended for cell culture and is supplied to the medium at a wide range of concentrations. A preferred concentration range is between about OJ μg / ml to about 500 μg / ml, more preferably about 5 μg / ml to about 400 μg / ml, and more preferably about 375 μg / ml. Appropriate concentrations for supplementation of insulin-like growth factor, such as IGF-1 or IGF-2, can be readily determined by one skilled in the art for cell types chosen for culture. Transferrin is in the middle for the regulation of iron transport. Iron is an essential trace element found in serum. Since the iron can be toxic to the cells in their free form, in the serum it is supplemented to the cells attached to the transferrin at a concentration range of preferably between about 0.05 to about 50 μg / ml. More preferably at about 5 μg / ml. Triiodothyronine (T3) is a basic component and is the active form of thyroid hormone that is included in the medium to maintain the rates of cellular metabolism. Triiodothyronine is supplemented to the medium at a concentration range between about 0 to about 400 pM, more preferably between about 2 to about 200 pM and more preferably to about 20 pM. One or both ethanolamine and o-phosphoryl-ethanolamine, which are phospholipids, are added whose function is an important precursor in the inositol pathway and fatty acid metabolism. Lipid supplementation normally found in serum is necessary in a serum-free medium. Ethanolamine and o-phosphoryl-ethanolamine are supplied to the medium at a concentration range between about 10"6 to about 10 'M, more preferably about 1 x 10" 4M. During the duration of the culture, the base medium is further supplemented with other components to induce synthesis or differentiation or to improve cell growth such as hydrocortisone, selenium, and L-glutamine. Hydrocortisone has been shown in keratinocyte cultures that promotes the keratinocyte phenotype and therefore improves the differentiated characteristics such as the content of involucrin and keratinocyte transglutaminase (Rubin et al., J. Cell Physiol., 138: 208-214 (1986)). Therefore, hydrocortisopa is a desirable additive at times where these characteristics are beneficial such as in the formation of keratinocyte sheet grafts or in skin constructions. The hydrocortisone may be provided at a concentration range of about 0.01 μl / ml to about 4.0 μl / ml, more preferably between about 0.4 μl / ml to 16 μl / ml. Selenium is added to the serum-free medium to supplement the trace elements of selenium normally provided by the serum. Selenium can be provided at a concentration range of about 10"9M to about 10" 7M; more preferably around 5.3 x 10 ^ M. The amino acid L-glutamine occurs in some nutrient bases and can be added in cases where there are no or insufficient amounts present. L-glutamine can also be provided in stable form such as that sold under the trademark, GlutaMAX- ™ (Gibco BRL, Grand Island, NY). GlutaMAX-1 ™ is the stable dipeptide form of L-alanine-L-glutamine and can be used interchangeably with L-glutamine and is provided in equimolar concentrations as a substitute for L-glutamine. The dipeptide provides stability to L-glutamine for degradation in storage time and during incubation that can lead to a lack of certainty in the effective concentration of L-glutamine in the medium. Typically, the base medium is supplemented with preferably between about 1 mM to about 6 mM, more preferably between about 2 mM to about 5 mM, and more preferably 4 mM L-glutamine or GlutaMAZ-1 ™ Growth factors such as epidermal growth factor (EGF) can also be added to the medium to aid in the establishment of the cultures during the increase in cell number and seeding. EGF can be used in the native form or in the recombinant form. Human, native or recombinant forms of EGF are preferred for use in the medium when a skin equivalent is made that does not contain non-human biological components. EGF is an optional component and can be provided at a concentration between about 1 to 15 ng / mL, more preferably between about 5 to 10 ng / mL. The medium described above is typically prepared as set forth below. However, it should be understood that the components of the present invention can be prepared and assembled using conventional methodology compatible with their physical properties. Is It is well known in the art to substitute certain components with an appropriate analogue or functional equivalent that acts as an agent for the purposes of availability and economy and achieve a similar result. Naturally occurring growth factors can be substituted with recombinant or synthetic growth factors that have similar qualities and that result when used in the development of the invention. The means according to the present invention are sterile. The sterile components are purchased sterile or rendered sterile by conventional methods, such as filtration, before preparation. Appropriate aseptic procedures were used through the following examples. DMEM and F-12 were initially combined and the individual components were added to complete the medium. Storage solutions for all components can be stored at -20 ° C, with the exception of the source of nutrients that can be stored at 4 ° C. all storage solutions are prepared at the final 500X concentrations listed above. A storage solution of insulin, transferrin and triiodothyronine (all from Sigma) is prepared as follows: triiodothyronine is initially dissolved in absolute ethanol in 1 N hydrochloric acid (HCl) at a ratio of 2: 1. The insulin is dissolved in dilute HCl (approximately OJ N) and the transferrin is dissolved in water. The three are mixed and diluted in water at a concentration of 500X. Ethanolamine and o-phosphoryl-ethanolamine are dissolved in water at a concentration of 500X and sterilized by filtration. Progesterone is dissolved in absolute ethanol and Dissolve with water. Hydrocortisone is dissolved in absolute ethanol and diluted in phosphate pH regulator (PBS). Selenium is dissolved in water at a concentration of 500X and sterilized by filtration. EGF is purchased sterile and dissolved in PBS. Adenine is difficult to dissolve but can be dissolved by any number of methods known to those skilled in the art. Serum albumin can be added to certain components in order to stabilize them in solution and is currently derived from human sources. For example, human serum albumin (HSA) or bovine serum albumin (BSA) can be added for prolonged storage to maintain the activity of progesterone and EGF storage solutions. The medium can be used either immediately after preparation or stored at 4 ° C. If stored, EGF should not be added until the time of use. In order to form the cell-matrix layer by the culture of the matrix-producing cells, the medium is supplemented with additional agents that promote matrix synthesis and deposition by the cells. These supplemental agents are compatible with the cells, are defined to a high degree of purity and are free of contaminants. The medium used to produce the cell-matrix layer is called "means for matrix production". To prepare the medium for matrix production, the base medium is supplemented with an ascorbate derivative such as sodium ascorbate, ascorbic acid, or one or more chemically stable derivatives such as Magnesium salt phosphate L-n-hydrated ascorbic acid. Ascorbate is added to promote proline hydroxylation and procollagen secretion, a soluble precursor for deposited collagen molecules. Ascorbate has also been shown to be an important cofactor for the post-translational processing of other enzymes as well as an overregulator for type I and type III collagen synthesis. Although one does not wish to stick to a theory, supplementation of the medium with amino acids involved in protein synthesis preserves cellular energy by not requiring the cell to produce the amino acids themselves. The addition of proline and glycine is preferred since they, as well as the hydroxylated form of proline, hydroxyproline, are basic amino acids that make up the structure of collagen. Although not required, the medium for matrix production is optionally supplemented with a neutral polymer. The cell-matrix constructions of the invention can be produced without a neutral polymer, but again although one does not wish to be bound by a theory, their presence in the matrix production medium can make the processing and deposition of collagen more consistently between the samples. A preferred neutral polymer is polyethylene glycol (PEG), which has been shown to promote in vitro processing of soluble procollagen precursors produced by cells grown for collagen matrix deposition. PEG grade tissue culture is preferred in the medium of the invention witthe range of from about 1000 to about 4000 MW (molecular weight), more preferably between about 3400 to around 3700 PM. Preferred PEG concentrations are for use in the method which may be at concentrations of about 5% p / v or less, preferably around 0.01% p / v around 0.5% p / v, more preferably between about 0.025% w / v about 0.2% w / v, more preferably around 0.05% w / v. Other crop-grade neutral polymers such as dextran, preferably dextran T-40, or polyvinylpyrrolidone (PVP), preferably in the range of 30,000-40,000 PM, may also be used at concentrations of about 5% w / w or less, preferably about 0.01% w / v about 0.5% w / v, more preferably between about 0.025% w / w about 0.2% w / v, more preferably about 0.05% w / v. Other cell culture grade agents and agents compatible with cells that improve collagen processing and deposition should be evaluated by the skilled artisan in the culture of mammalian cells. When the cell-producing cells are confluent, and the culture medium is supplemented with components that assist in the synthesis of matrix, secretion, or organization, it is said that the cells are stimulated to form a tissue construct comprising cells and matrix synthesized by those cells. Therefore, a formulation of the preferred matrix production medium comprises: a 3: 1 base mixture of Dulbecco's modified Eagle medium (DMEM) (high glucose formulation without L-glutamine) and medium Hams F-12 supplemented with either 4 mM L-glutamine or its equivalent, 5 ng / ml epidermal growth factor, 0.4 μg / ml hydrocortisone, 1 x 10_4 M ethanolamine, or 1 x 10 O ^ M phosphoryl-ethanolamine, 5 μg / ml insulin, 5 μg / ml transferrin, 20 pM triiodothyronine, 6.78 ng / ml selenium, 50 ng / ml L-ascorbic acid, 0.2 μg / ml L-proline, and OJ μg / ml glycine. To the production medium, other pharmacological agents can be added to the culture to alter the nature, amount, or type of secreted extracellular matrix. These agents may include polypeptide growth factors, transcription factors or inorganic salts for upregulating collagen transcription. Examples of polypeptide growth factors include transforming growth factor-beta 1 (TGF-β1) and tissue plasminogen activator (TPA), both of which are known to upregulate collagen synthesis. Raghow et al., Journal of Clinical Investigation, 79: 1285-1288 (1987); Pardes et al., Journal of Investigative Dermatology, 100: 549 (1993). An example of an inorganic salt that stimulates the production of collagen is cerium. Shivakumar et al., Journal of Molecular and Cellular Cardiology 24: 775-780 (1992). The cultures are maintained in an incubator to ensure sufficient environmental conditions to control the temperature, humidity, and gas mixture for cell culture. Preferred conditions are between about 34 ° C to about 38 ° C, more preferably 37 ± 1 ° C with an atmosphere between about 5-10 ± 1% CO2 and a relative humidity (Hr) between about 80-90 %.

In the preferred embodiment, the cell-matrix construction is a dermal construct formed of dermal fibroblasts and their secreted matrix. Preferably, human dermal fibroblasts are used, derived as primary cells from dermis or more preferably from serial or subcultured passages from established storage cells or banks that have been selected against viral and bacterial contamination and tested for purity. . The cells are cultured under sufficient conditions in growth medium to cause them to proliferate at an appropriate number to seed the cells in the culture substrate upon which a cell-matrix construction is formed. Alternatively, cells from frozen storage cells can be seeded directly into the culture substrate. Once a sufficient number of cells have been obtained, the cells are harvested and seeded on a suitable culture surface and grown under appropriate growth conditions to form a confluent sheet of cells. In the preferred embodiment, the cells are seeded onto a porous membrane that is submerged to allow contact of the medium from the bottom of the culture through the pores and directly toward the top. Preferably, the cells are resuspended either in a base culture medium or in growth medium and are seeded onto the cell culture surface at a density between about 1 x 10 5 cells / cm 2 to about 6.6 x 10 5 cells / cm 2, more preferably between about 3 x 105 cells / cm2 to about 6.6 x 105 cells / cm 2 and more preferably at about 6.6 x 10 5 cells / cm 2 (cells per square centimeter of surface area). The cultures are grown in growth medium to establish the culture and are cultivated until they are between 80% to 100% confluence at which time they are induced chemically by medium changes for the matrix production medium in order to upregulate the synthesis and secretion of the extracellular matrix. In an alternative method, the cells are sown directly in production medium to eliminate the need to change from the base medium to the production medium but it is a method that requires high densities of seeding. During culture, the fibroblasts organize the secreted matrix molecules to form a tissue-like two-dimensional structure but do not exhibit significant contractile forces to cause the formation of the cell-matrix construction to contract and separate itself from the substrate of culture. The change of the medium is done every two to three days with fresh matrix production medium and over time, the secreted matrix increases in thickness and in organization. The time needed to create a cell-matrix construction depends on the capacity of the initial seed density, the cell type, the age of the cell line, and the ability of the cell line to synthesize and secrete the matrix. The fully formed constructions of the invention have large thicknesses due to the fibrous matrix produced and organized by the cells; there are no cell cultures ordinarily confluent or excessively confluent where the cells can lose adherence to each other. Fibrous qualities give constructions cohesive properties similar to tissues unlike ordinary crops because they resist physical damage, such as separation or cracking, with routine management in clinical settings. In the manufacture of a cultured dermal construct, the cells will form a matrix organized around it on the surface of the cell culture preferably of at least about 30 microns in thickness or more, more preferably between about 60 to about 120 microns in thickness around the surface of the membrane; however, the thickness that has been obtained in excess of 120 microns is suitable for use in assay or clinical applications where said larger thicknesses are necessary. In a more preferred method, a cellular epithelial layer is applied to a surface, preferably at the top, upwardly facing the surface of the cell-matrix construction. To the cell-matrix construction, the epithelial cells can be sown and cultivated to form a multilayer tissue construct. In the most preferred method, keratinocytes derived from the skin are grown on cell construction to form a skin construct. In other preferred embodiments, corneal epithelial cells, also referred to as corneal keratinocytes, can be seeded onto the cell-matrix construct to form a corneal construct. Epithelial cells from the oral mucosa can grow on the cell-matrix construction to form a oral mucosa construction. The epithelial cells from the esophagus can be planted on the cell-matrix construction to form an esophageal tissue construct. The uroepithelial cells from the urogenital tract can be planted on the cell-matrix construction to form a uroepithelium construct. Other cells of epithelial origin can be selected to form a tissue construct from which these cells are derived. Methods for providing epidermal cells to a dermal substrate, and methods for their cultivation, including the induction of differentiation and comification to form a differentiated keratinocyte layer, are known in the art and are described in US Pat. No. 5,712,163 to Parenteau, et al. and in the patent of E.U.A. No. 5,536,656 to Kemp, et al, the contents of which are incorporated herein by reference. Typically to carry out the epidermalization of the cell-matrix construction, the keratinocytes are seeded into the cell-matrix constructs and grown on them until the layer is about one to three cells in thickness. The keratinocytes are then induced to differentiate so that they form a multilayer epidermis and are then induced to cornify to form a stratum corneum. In the method for forming a differentiated epidermal layer, the cultured keratinocytes are taken from the storage cells and the cell number is expanded. When a necessary number of cells has been obtained, they are released from the culture substrate, they are resuspended, counted, diluted and then sown on the surface of the cell-matrix construction at a density between about 4.5 x 103 cells / cm2 at about 5.0 x 105 cell / cm2, more preferably between about 1.0 x 104 cell / cm2 at about 1.0 x 10 5 cells / cm 2 and more preferably at about 4.5 x 10 4 cells / cm 2. The constructions are then incubated for about 60 to about 90 minutes at 37 + 1 ° C, 10% CO2 to allow the keratinocytes to anchor. After incubation the constructions are immersed in epidermalization medium. After a sufficiently long time in culture, the keratinocytes proliferate and spread to form a confluent monolayer through the cell-matrix construction. Once confluent, the formulation of the cell medium is changed to differentiation medium to induce cell differentiation. When a multilayer epithelium has been formed, the cornification medium is then used and the culture is brought to the air-liquid interface. For the differentiation and cornification of the keratinocytes, the cells are exposed to a liquid air interface with low or dry moisture. A dry or low humidity interface can be characterized as trying to double the low levels of skin moisture. Over time, keratinocytes will express most or all of the keratins and other characteristics found in the native skin when exposed to these conditions.

As mentioned above, the system for the production of a cell-matrix construction can be used in the formation of a corneal construction. Corneal epithelial cells can be derived from a variety of mammalian sources. The preferred epithelial cell is a rabbit or human corneal epithelial cell (corneal keratinocyte) but any mammalian corneal keratinocyte can be used. Other epithelial keratinocytes such as those derived from the sclera (outer white opaque portion) of the eye or epidermis may be substituted, but corneal keratinocytes are preferable. In the method for forming a corneal construction, the medium is removed from the culture insert (containing the cell-matrix construction) and its surroundings. Rabbit normal corneal epithelial cells are expanded via subculture, trypsinized to remove them from the culture substrate, suspended in culture medium, and seeded at the top of the membrane at a density of between about 7.2 x 104 at about 1.4 x 105 cell / cm2. The constructs are then incubated without medium for about 4 hours at 37 + 1 ° C, 10% C02 to allow the epithelial cells to anchor. After incubation, the constructions are immersed in corneal maintenance medium (CMM) (Johnson et al, 1992). The epithelial cells are cultured until the cell-matrix construction is covered with epithelial cells. The integrity of the epithelial covering can be evaluated by a variety of methods, for illustration by staining the culture with a solution of nyl blue sulfate (1: 10,000 in phosphate salt buffer). Once the cell-matrix construction is covered, after approximately 7 days, the constructions are transferred aseptically to new culture devices with sufficient corneal maintenance medium (CMM) to achieve a level of fluid just at the surface of the construction to maintain a moisture interface without submerging the epithelial layer. These constructions are incubated at 37 + 1 ° C, 10% CO2, and humidity greater than 60%, with the CMM, making medium changes, as necessary, typically three times a week.

For differentiation, but not for the cornification of the epithelial cell layer, as is necessary in the production of a corneal construct, the surface of the epithelial cell is exposed to a wet-liquid air interface. Methods for providing a wet-liquid air type are described in the US patent. No. 5,374,515 of Parenteau. As used herein, the term "moisture interface" is meant 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 moisture and humidity in the culture environment is not critical, but it must be sufficiently moist and humid to prevent the formation of cornified cells. A wetted interface can be characterized as trying to duplicate similar humectant levels of the human eye.

In an alternative preferred embodiment, the seeding of a second matrix producing cell on a first cell-matrix construction formed to obtain a cell-like construction can be carried out. thicker matrix or a cell-matrix construction in bilayer. The second planting can be carried out with the same cell type or with a strain or with a different cell type or with a strain, depending on the desired result. The second seeding is carried out under the same conditions using the methods and means of matrix production used in the production of the first layer. One result for carrying out secondary seeding with a cell of a different type is to have a matrix formed with profiles of different matrix components or a densely packed matrix to affect healing when the construct is grafted to a patient. The first cell sown produces a matrix analogous to the reticular layer of the dermis, a densely packed type I collagen layer and constituent components of the extracellular matrix. The second cell sown would produce a matrix similar to the papillary layer of the dermis characterized by laxative collagen fibers and extracellular matrix. Another result is that the second cell type can produce a therapeutic substance that could affect healing, such as improvement of graft taking or graft integration or minimization or prevention of scarring.

In another preferred embodiment, populations of mixed cells of two or more cell types can be cultured together during the formation of a cell-matrix construct which provides that at least one of the cell types used is capable of synthesizing extracellular matrix. The second cell type may be one that is needed to carry out other Tissue functions or to develop particular structural characteristics of tissue construction. For example, in the production of a skin construct, dermal papilla cells or epithelial cells from adnexa that can be cultured with the matrix producing cells to allow the formation of epithelial appendages or their components. Epidermal appendages such as structures of sweat or sebaceous glands or components of hair follicle structures or components that can be formed when they are cultured together with the matrix-producing cells. The epithelial cells can be derived from the structures appendages of the glands and the hair follicle located in the deep dermis, such as by microdissection, and include eccrine cells, myoepithelial cells, cells of the secretory glands, and cells of the stems of the cells. hair follicles. Other cell types that are normally found in the skin that also constitute skin may be added such as melanocytes, Langerhans cells and Merkel cells. Similarly, vascular endothelial cells can be co-cultured to produce rudimentary components for new vascular formation. Adipocytes can also be cultured with matrix producing cells to form a construct used for reconstructive surgery. As an alternative way to administer this second cell type, cells can be seeded locally as a point or in an array of any number of cell sites on or within a cell-tissue matrix in formation or fully formed to localize the development of these structures . For Seeding the cells within the cell-matrix construction, the cells can be injected between the lower and upper surface, inside the cell-matrix, so that the cells grow, form specialized structures and carry out their specialized functions. To produce a three-layered tissue construct, a first seed of cells comprising a cell-producing type of matrix or a cell type that does not produce matrix is seeded onto the growing substrate for a sufficient time to produce a cell-matrix construction or a cellular layer. Once the initial cell-matrix construction or cell layer is formed, a second seed of cells comprising a cell-producing type of matrix is seeded onto the surface of the first cell-matrix or cell layer construction and grown for a short time conditions sufficient to form a second cell-matrix construction on the first construction. On the second cell-matrix construction, a third seed of a third cell type is sown and cultivated under conditions sufficient to produce the third layer. As an example, to produce a three-layered corneal construct, the cell of the first cell type may be comprised of the original endothelium, such as corneal endothelial cells; the second cell type may comprise cells of collective tissue origin, such as corneal keratinocytes, and the third cell type may comprise cells of epithelial origin, such as corneal epithelial cells. As another example of a three-layer skin construct, the first-seeded cell may be of vascular origin for providing components for vascularization, the cells of the second seed can comprise dermal fibroblasts to form a cell-matrix construct to serve as a dermal construct, and the cells of the third seed can be epidermal keratinocytes to form an epidermal layer. The tissue constructions of the invention can be stored at cryogenic temperatures when vitrification or cryopreservation methods are employed. Vitrification methods of tissue constructions are described in the U.S. patent. No. 5, 518,878 and methods for cryopreservation are described in the U.S. Patents. Nos. 5,689,961 and 5,891, 617 and in the PCT international application WO 96/24018, the description of which is incorporated herein by reference. The skin constructions of this invention can be used in tissue testing systems for in vitro toxicology testing. Assay systems incorporating skin constructs for testing purposes are described in the U.S. patent. No. 4,835,102 the description of which is incorporated herein by reference. Because the cell produced in skin building has a similar structure, and, more importantly, an organization similar to skin, it can be a valuable test system as an alternative or replacement for absorption tests, toxicity and in many cases products. effective in tests on live animals or humans. The production of the matrix has been shown to mimic several of the processes exhibited in the production of the matrix as well as the repair of the matrix in vivo. Due to this, the described system can be a valuable tool in the analysis of healing and tissue generation and also for the testing and analysis of chemical stimulants and / or physical tissue repair. The most preferred use for the skin constructions of this invention is for grafting or implantation of a mammalian host to restore or repair the skin due to injury or disease. Indications for grafting a skin construct include but are not limited to reconstructive or plastic surgery, skin wounds, burns, psoriasis, venous and diabetic ulcers, and basal cell carcinoma. The skin constructions of the invention are useful both for protecting tissue wounds as well as serving as a scaffold for host tissue growth. It is believed that the level of tissue organization produced in this invention can serve to facilitate and possibly accelerate healing actions. The cell matrix constructs of the invention have cohesive properties. "Cohesive" as used herein means that it is capable of maintaining physical unitary integrity and tissue-like handling properties. The physical properties that mainly give the cohesive properties to the construction of the invention are thickness and structure in fibrous matrix. The fibrous extracellular matrix is formed from collagen synthesized by cells and other components of the matrix, mainly fibrillar collagen aggregated into fibrils and bundles of fibrils, and which gives the construction its thickness. The cell-matrix constructions of the invention are manageable, this is, they can be manually separated from their growing substrate, without carrier support or specialized tools, and applied to patients or a testing device. Damage such as ripping or narrowing can be avoided from ordinary manipulation in the clinic without detriment to the structure or function. When applied to a patient, they can be secured in one place with sutures or staples. To graft the skin construction of the present invention to a patient, the graft area is prepared according to standard practice. For indications of burns, sites with burn wounds to be grafted are prepared for the graft so that the area of burned skin is completely removed. The removed beds will appear clean and clinically uninfected before grafting. For thick partial depth wounds due to a surgical incision, the pre-operative area is shaved if necessary, cleaned with an antimicrobial, the skin rinsed antiseptically and rinsed with normal saline. Local anesthesia usually consists of intradermal administration of lidocaine or epinephrine or both. Once the anesthesia has been performed, a dermatome is used to remove the skin to an appropriate depth, creating a partial thickness depth wound. Hemostasis can be achieved by compression with epinephrine containing lidocaine and by electrocautery. The skin construction is then applied to the wound bed and, if necessary, secured by sutures or stapled to its place, and then bandages are applied appropriately.

The skin constructions of the present invention can also be networked before grafting to a patient. The network formation improves the conformation of the skin construction towards the wound bed and provides a means to drain the exudate from the wound under the graft. The term "network forming" is defined as a mechanical method by which a fabric is punched through long cuts to form a network-like arrangement. The network formation is preferably obtained by the use of a conventional skin network former (ZIMMER®; BIOPLASTY®). One can also manually pierce a tissue with a scalpel or with a needle. The netted skin can expand by stretching the skin so that the tears open and then it is applied to the wound bed. The expanded mesh fabric provides a wound area with maximum coverage. Alternatively, the mesh skin can be applied without expansion, simply as a sheet with a non-expanded hole arrangement. The mesh skin construction can be applied alone or with the patient's own skin from another area of the body. The tissue constructions may also have perforations or fenestration and pores provided by other means. Fenestrations can be applied manually using a laser, punch, scalpel, needle or pin. The skin construction of the invention can be applied to wounds other than surgical wounds or burned areas. Other wounds such as venous ulcers, diabetic ulcers, decubitus ulcers may experience a healing benefit through the application of skin construction described. Other congenital skin diseases such as epidermolysis bulosa may also benefit. The following examples are provided to better express the practice of the present invention and should not be construed in any way that limits the scope of the present invention. Those skilled in the art will recognize that various modifications can be made to the methods described herein while not departing from the spirit and scope of the present invention.

EXAMPLES EXAMPLE 1 Formation of a collagen matrix by human neonatal foreskin fibroblasts Human neonatal foreskin fibroblasts (originating from Organogenesis, Inc. Canton, MA) were seeded at 5 x 10 5 cells / 162 cm 2 in tissue culture treated bottles (Costar Corp., Cambridge, MA, cat # 3150) and grown in the midst of growth. The growth medium consists of: Dulbecco's Modified Eagle Medium (DMEM) (high glucose formulation, without L-glutamine, Bio Whittaker, Walkersville, MD) supplemented with 10% newborn calf serum (NBCS) (HyClone Laboratories, Inc., Logan, Utah) and 4mM L-glutamine (Bio Whittaker, Walkersville, MD). The Cells were maintained in an incubator at 37 ± 1 ° C with a CO2 atmosphere at 10 ± 1%. The medium was replaced with freshly prepared medium every two to three days. After 8 days in culture, the cells had grown to confluence, that is, the cells had formed a monolayer packaged along the bottom of the tissue culture flask, and the medium was aspirated from the culture flask. To rinse the monolayer, sterile saline phosphate buffer was added by filtering to the bottom of each culture flask and then aspirated from the flasks. The cells were released from the flask by adding 5 mL trypsin-glutamine versne (Bio Whittaker, Waikersville, MD) to each vial and gently rotating it to ensure complete coverage of the monolayer. The cultures were returned to the incubator. As soon as the cells were released 5 ml of SBTI (Soy trypsin inhibitor) was added to each vial and mixed with the suspension to stop the action of trypsin-versen. The cell suspension was removed from the flasks and also divided between sterile, conical centrifuge tubes. The cells were collected by centrifugation at approximately 800-1000 x g for 5 minutes. The cells were resuspended using fresh medium at a concentration of 3.0 x 10 6 cells / ml, and seeded on treated grafts for tissue culture with a pore size of 0.4 microns, with a diameter of 24 mm (TRANSWELL®, from Corning Costar) , in a 6-well device at a density of 3.0 x 106 cells / inserts (6.6 x 105 cells / cm2). Cells were maintained in an incubator at 37 ± 1 ° C with an atmosphere of CO2 aM ± 1% and were fed fresh production medium every 2 to 3 days for 21 days. The production medium comprises: a 3: 1 base mixture of DMEM medium and Hams F-12 (Quality Biologics Gaithersburg, MD), 4 mM, GlutaMAX-1 ™ (Gibco BRL, Grand Island, NY) and additives for a resulting concentration of: 5 ng / ml human recombinant epidermal growth factor (Upstate Biotechnology Lake Placid, NY), 2% newborn calf serum (Hyclone, Logan, Utah), 0.4 ug / ml hydrocortisone (Sigma St. Louis, MO) ), ethanolamine 1 x 10 ~ 4 M (Fluka, Ronkonkoma, NY ACS grade), o-phosphoryl-ethanolamine 1 x 10"4 M (Sigma, St. Louis,), 5 μg / ml insulin (Sigma, St. Louis, MO), 5 μg / ml transferrin (Sigma, St. Louis, MO), 20 pM triiodothyronine (Sigma, St. Louis, MO), and 6.78 ng / ml selenium (Sigma Aldrich Fine Chemicals Co., Milwaukee , Wl), 50 ng / ml L-ascorbic acid (WAKO Chemicals USA Inc. # 013-12061), 0.2 μg / ml L-proline (Sigma, St. Louis, MO), 0 J μg / ml glycine ( Sigma, St. Louis, MO) and 0.05% polyethylene glycol (PEG) 3400-3700 PM (cell culture grade) ( Sigma, St. Louis, MO). Samples for histological analysis were taken on days 7, 14 and 21 and fixed in formalin, then embedded in paraffin. The formalin-fixed samples were embedded in paraffin and sections of 5 microns were stained with hematoxylin-eosin (H &E) according to procedures known in the art. Using the stained sections, with (H & E) thickness measurements were made on ten microscopic fields taken at random using a 10X eye piece mounted with a 10MM / 100 micron grid.

The results for the two different cellular strains of human thermal fibroblasts are summarized in Table 1, which shows the thickness of the cell-matrix construction and its development.

Samples were also submitted for analysis of collagen concentration on days 7, 14 and 21. Collagen content was estimated using a colorimetric assay for hydroxyproline content known in the art (Woessner, 1961). At the same times the number of cells was also determined. Table 2 summarizes the collagen concentration and Table 3 is a summary of cell data from the cell-matrix constructs produced from two different cell strains (B156 and N119) using the procedure described above.

Samples of human cells derived from the dermal matrix on days 7, 14, and 21 were analyzed by slow reduction in SDS-PAGE to determine the collagen composition relevant to the alpha type I and type III collagen bands in the samples. The biochemical characteristics of the dermal matrix were determined using immunohistochemical methods. The identification of fibronectin was carried out on the sections fixed in paraffin using the stytavidin-biotin system Zymed Histostain (Zymed Laboratories Inc., South San Francisco, C.A.). The presence of tenascin was determined by staining with the anti-tenascin primary antibody (Dako, Carpintheria, CA) followed by anti-mouse horseradish peroxidase-labeled antibody (Calbiochem) as a secondary antibody. Samples were visualized by applying diaminobenzidine (Sigma St. Louis, MO) and counterstained with nuclear fast red. The glycosaminoglycan (GAG) quantification was carried out in the samples of day 21 using the method previously described (Farndale, 1986). The tests show the presence of 0.44 grams of GAG per cm2 in a sample of human cells derived from the dermal matrix taken 21 days after sowing.

EXAMPLE 2 Total thickness of the skin construction Using a dermal construct formed using the method described in Example 1, the epidermal keratinocytes of the foreskin of the normal human neonate (originating from Organogenesi, Inc. Canton, MA) were placed on the cell-matrix construction to form the epidermal layer of the construction of skin. The medium was removed aseptically from the culture insert and its surroundings. Normal keratinocytes of human epidermis were taken from passage 4 of cells stored in a freezing culture to confluence. The cells were then released from the culture dishes using trypsin-versen, pooled, centrifuged to form a cell pellet, resuspended in epidermalization medium, counted and plated on a membrane at a density of 4.5 x 104 cells. / cm2. The constructs were incubated for 90 minutes at 37 ± 1 ° C, 10% C02 to allow the keratinocytes to anchor. After incubation, the constructions were immersed in epidermalization medium. The epidermalization medium is comprised of: a 3: 1 mixture base of Dulbecco's Modified Eagle Medium (DMEM) (Bio Whittaker, Waikersville, MD) (high glucose formulation, no L-glutamine (Bio Whittaker, Waikersville, MD) and Hams F-12 medium (Quality Biologics Gaithersburg, MD), supplemented with 0.4 μg / ml hydrocortisone (Sigma St.

Louis, MO), ethanolamine 1 x 10"4 M (Fluka, Ronkonkoma, NY), o-phosphoryl-ethanolamine 1 x 10" 4 M (Sigma, St. Louis, MO), 5 μg / ml insulin (Sigma, St. Louis, MO), 5 μg / ml transferrin (Sigma, St. Louis, MO), 20 pM triiodothyronine (Sigma, St. Louis, MO), 6.78 ng / ml selenium (Aldrich), 24.4 μg / ml of adenine (Sigma Aldrich Fine Chemicals Company, Milwaukee, Wl), 4 mM L-glutamine (Bío Whittaker, Walkersvílle, MD), 0.3% chelated newborn calf serum (Hyclone, Logan, Utah), 0.628 ng / ml progesterone (Amersham Ariington Heights, IL), 50 μg / ml sodium L-ascorbate salt (Sigma Aldrich Fine Chemicals Company, Milwaukee, Wl), 10 ng / ml epidermal growth factor (Life Technologies Inc., MD) , 50 μg / ml gentamicin sulfate (Amersham, Arington Heights, IL). The constructs were cultured in the epidermalization medium for two days at 37 ± 1 ° C, 10% C02. After two days the construction was submerged in a medium composed of; a 3: 1 mixture of Dulbecco's Modified Eagle Medium (DMEM) formulation with high glucose, without L-glutamine, Bio Whittaker, Waikersville, MD), Hams F-12 medium (Quality Biologics, Gaithersburg, MD), supplemented with 0.4 μg / ml of hydrocortisone (Sigma, St. Louis, MO), ethanolamine 1 X 10"4 M (Fluka, Ronkonkoma, NY), o-phosphoryl-ethanolamine 1 x 10" 4 M (Sigma, St. Louis, MO), 5 μg / ml insulin (Sigma, St. Louis, MO), 5 μg / ml transferrin (Sigma, St. Louis, MO), 20 pM triiodothyronine (Sigma, St. Louis, MO), and 6.78 ng / ml of selenium (Sigma Aldrich Fine Chemicals Company, Miiwaukee, Wl), 24.4 μg / ml adenine (Sigma Aldrich Fine Chemicals Company), 4 nM L-glutamine (BioWhittaker, Waikersville, MD), 0.3% chelated newborn calf serum (BioWhittaker, Waikersville, MD), 0.628 ng / ml progesterone (Amersham, Ariington Heights, IL), 50 μg / ml of sodium ascorbate, 265 μg / ml of calcium chloride (Mallinckrodt, Chesterfield, MO), and 560 μg / ml of gentamicin sulfate (Amersham, Ariington Heights, IL). Again the construction was incubated at 37 ± 1 ° C, 10% C02 for 2 days. After 2 days the vehicle containing the construction was aseptically transferred to new culture devices with a sufficient amount of cornification medium, 9 ml, to achieve a level of fluid just to the surface of the carrier membrane to maintain a dry interface for allow the stratification of the epithelial layer. The constructs were incubated at 37 ± 1 ° C, 10% C02, and at low humidity, in medium with medium changes every 2-3 days for 7 days. This means is composed of; a 1: 1 mixture of Dulbecco's modified Eagle's medium (DMEM) (high glucose formulation, without L-glutamine BioWhittaker, Waikersville, MD), Hams F-12 medium (Quality Biologics, Gaithersburg, MD), supplemented with 0.4 μg / ml of hydrocortisone (Sigma, St. Louis, MO), ethanolamine 1 × 10 4 M (Fluka, Ronkonkoma, NY), o-phosphoryl-ethanolamine 1 × 10 4 M (Sigma, St. Louis, MO), 5 μg / ml insulin (Sigma, St. Louis, MO), 5 μg / ml transferrin (Sigma, St. Louis, MO), 20 pM triiodothyronine (Sigma, St. Louis, MO), 6.78 ng / ml selenium (Aldrich), 24.4 μg / ml adenine (Sigma Aldrich Fine Chemicals Company), 4 mM L-glutamine (BioWhittaker, Waikersville, MD), 2% newborn calf serum (BioWhittaker, Waikersville, MD) 50 μg / ml sodium ascorbate, and 50 μg / ml gentamicin sulfate (Amersham, Ariington Heights, IL) After 7 days the construction was fed for 10 more days, with changes every 2-3 days, with a maintenance medium. maintenance was composed of: a 1: 1 mixture of Dulbecco's Modified Eagle Medium (DMEM) (high glucose formulation, without L-glutamine BioWhittaker, Waikersville, MD), Hams F-12 medium (Quality Biologics, Gaithersburg, MD) , supplemented with 0.4 μg / ml hydrocortisone (Sigma, St. Louis, MO), 1 × 104 M ethanolamide (Fluka, Ronkonkoma, NY), or 1 × 104 M phosphoryl-ethanolamine (Sigma, St. Louis, MO) , 5 μg / ml insulin (Sigma, St. Louis, MO), 5 μg / ml transferrin (Sigma, St. Louis, MO), 20 pM triiodothyronine (Sigma, St. Louis, MO), 6.78 ng / ml of selenium (Aldrich), 24.4 μg / ml of adenine (Sigma Aldrich Fine Chemicals Company), 4 mM L-glutamine (BioWhittaker, Waikersville, MD), 1% newborn calf serum (BioWhittaker, Waikersville, MD) 50 μg / ml sodium ascorbate, and 50 μg / ml gentamicin sulfate (Amersham, Ariington Heights, IL). The final samples were subjected to a procedure for hematoxylin and eosin as described in Example 1 to determine the appearance of thickness under light microscopy. The resulting construction consisted of a lower (dermal) layer consisting of fibroblasts surrounded by matrix having characteristics described in Example 1, and which was completely covered by a multilayer, stratified and well-differentiated keratinocyte that exhibited a basal layer, a layer suprabasal, and a granular layer and a stratum corneo similar to that of the skin in situ. The construction The skin has a well-developed basement membrane present in the dermal-epidermal junction as exhibited by transmission electron microscopy (TEM). The basement membrane appears thicker around the hemidesmosomes, marked by anchoring fibrils comprising collagen type VII, as visualized by TEM. As expected, these anchoring fibrils can be easily seen existing from the basement membrane and encompassing the collagen fibrils. The presence of laminin, a basal membrane glycoprotein, was shown using the avidin biotin immunoenzymatic technique previously described (Guesdon, 1979).

EXAMPLE 3 In vitro formation of a collagen matrix by fibroblasts of the human neonatal foreskin in chemically defined medium The human neonatal foreskin fibroblasts were expanded using the procedure described in Example 1. The cells were resuspended at a concentration of 3 x 10 6 cells / nm, and seeded on membrane inserts treated for tissue culture with a pore size 0.4 microns, and 24 mm in diameter in a six well box at a density of 3.0 x 106 cell / TW (6.6 x 105 cell / cm2). These cells were maintained as in Example 1 with fresh calf serum omitted from the culture medium. More specifically the medium contained: a 3: 1 mixture of DMEM, Hams F-12 (Quality Biologics, Gaithersburg, MD), GlutaMax 4 mM (Gibco BRL, Grand Island, NY) (Gibco BRL, Grand Island, NY) and additives: 5 ng / ml human recombinant epidermal growth factor (Upstate Biotechnology, Lake Placid, NY), 0.4 μg / ml hydrocortisone (Sigma, St. Louis, MO), ethanolamine 1 × 104 M, (Fluka, Ronkonkoma, NY cat. # 02400 ACS grade) o-phosphoryl-ethanolamine 1 × 10"4M (Sigma, St. Louis, MO), 5 μg / ml insulin (Sigma, St. Louis, MO), 5 μg / ml transferrin (Sigma, St. Louis, MO), 20 pM triiodothyronine (Sigma, St. Louis, MO), and 6.78 ng / ml of selenium (Sigma Aldrich Fine Chemicals Company, Milwaukee, Wl), 50 ng / ml L-ascorbic acid (WAKO Chemicals USA, Inc.), 0.2 μg / ml L-proline (Sigma, St. Louis, MO), 0J μg / ml glycine (Sigma, St. Louis, MO) and 0.05% polyethylene glycol (PEG) (Sigma, St. Louis, MO) Samples were checked on days 7, 14 and 21 for the concentration of collagen and the cell number using the described procedures.The results are summarized in tables 4 (cell number lar) and 5 (collagen). The samples were also fixed in formalin and processed for staining with hematoxylin and eosin for light microscopy analysis as described in example 1. The histological evaluation showed that the constructs grown in defined medium were similar to those grown in the presence of newborn calf serum at 2%. The samples were also stained positively for fibronectin, using a procedure described in Example 1.

TABLE 4 Collagen (μg / cm2) TABLE 5 Cells (cell / cm2) In addition to the endogenously produced fibrillar collagen, decorin and glycosaminoglycans were also present in cell / matrix construction.

EXAMPLE 4 Total thickness of the skin construction formed using chemically defined medium Using a 25-day dermal construct formed by human dermal fibroblasts under chemically defined conditions similar to the method described in Example 3, the normal epidermal keratinocytes of the human neonatal foreskin were seeded on top of a cell / matrix construct to form the epidermal layer of the leather construction. The medium was removed aseptically from the culture insert and its surroundings. Normal human epidermal keratinocytes were used in passage 4 from cells in frozen subculture storage until confluence. The cells were released from the culture dishes using trypsin-versen, pooled, centrifuged to form a cell pellet, resuspended in epidermalization medium, counted and seeded on the upper part of the membrane at a density of 4.5 x 104 cells / cm2. The constructs were incubated for 90 minutes at 37 ± 1 ° C, 10% C02 to allow the keratinocytes to anchor. After incubation, the constructions were immersed in epidermalization medium. The epidermalization medium is composed of: a 3: 1 base mixture of Dulbecco's Modified Eagle Medium (DMEM) (which does not contain glucose or calcium, BioWhittaker, Waikersville, MD), and Hams F-12 (Quality Biologics, Gaithersburg, MD), supplemented with 0.4 μg / ml hydrocortisone (Sigma, St. Louis, MO), 1 x 104 M ethanolamide (Fluka, Ronkonkoma, NY), 1 x 104 M o-phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 μg / ml insulin (Sigma, St. Louis, MO), 5 μg / ml transferrin (Sigma, St. Louis, MO), 20 pM triiodothyronine (Sigma, St. Louis, MO), 6.78 ng / ml selenium (Aldrich), 24.4 μg / ml adenine (Sigma Aldrich Fine Chemicals Company), 4 mM L-glutamine (BioWhittaker, Waikersville, MD), 2% newborn calf serum (BioWhittaker, Waikersville, MD) 50 μg / ml sodium ascorbate, and 50 μg / ml ml of gentamicin sulfate (Amersham, Arington Heights, IL) The constructs were cultured in the epidermalization medium for 2 days at 37 ± 1 ° C, C02 at 10 ± 1%, after 2 days the medium was changed with fresh medium compound as described above, and returned to the incubator maintained at 37 ± 1 ° C, C02 at 10 ± 1% for 2 days.After 2 days, the vehicle containing the construction was transferred aseptically to new devices s of culture with sufficient medium to achieve a level of fluid just to the surfaces of the carrier membrane to maintain the development of the construction in the air-liquid interface. The contact of the air with the upper surface of the epidermal layer in formation allows the stratification of the epidermal layer. The constructs were incubated at 37 ± 1 ° C, C02 at 10 ± 1%, and at low humidity, in medium with changes of medium every 2-3 days for 7 days. This medium contained a 1: 1 mixture of Dulbecco's Modified Eagle Medium (DMEM) (which does not contain glucose or calcium, BioWhittaker, Waikersville, MD), Hams F-12 medium (Quality Biologics, Gaithersburg, MD), supplemented with 0.4 μg / ml hydrocortisone (Sigma, St. Louis, MO), 1 x 104 M etalonamide (Fluka, Ronkonkoma, NY), o-phosphoryl-ethanolamine 1 x 104 M (Sigma, St. Louis, MO), 5 μg / ml insulin (Sigma, St. Louis, MO), 5 μg / ml transferrin (Sigma, St. Louis, MO) , 20 pM triiodothyronine (Sigma, St. Louis, MO), 6.78 ng / ml (Sigma Aldrich Fine Chemicals Company), 24.4 μg / ml adenine (Sigma Fine Chemicals Company), 4 mM L-glutamine (BioWhittaker, Waikersville, MD), calcium chloride (Mallinckrodt, Chesterfield, MO), 16 μM linoleic acid (Sigma, St. Louis, MO), 1 μM tocopherol acetate (Sigma, St. Louis, MO), 1.25 mM serine (Sigma, St Louis, MO), 0.64 mM choline chloride (Sigma, St. Louis, MO) and 50 μg / ml gentamicin sulfate (Amersham, Ariington Heights, IL). The cultures were fed every 2-3 days, for 14 days. The samples, in triplicate, were subjected 10, 12, and 14 days after the construction was elevated to the air-liquid interface for hematoxylin and eosin processing as described in Example 1 to determine the appearance of thickness under light microscopy. light. The resulting construction consisted of a lower (dermal) layer consisting of fibroblasts surrounded by matrix having characteristics as described in Example 3, and which were overcoated by a layer of stratified and differentiated keratinocytes.

EXAMPLE 5 In Vitro Formation of a Collagen Matrix by Human Agile Tendon Fibroblasts Cell-matrix constructs were formed using the same method described in Example 1 by replacing the fibroblasts of the human neonatal foreskin with human Achilles tendon fibroblasts (HATF). Following 21 days in the production medium, the samples were also subjected to H &E staining and thickness determination using the procedure described in example 1. The resulting construction was visualized as a cell-matrix tissue-like construction with a thickness of 75.00 ± 27.58 microns (n = 2). Endogenously produced fibrillar collagen, decorin and glycinoglycan were also present in the construct.

EXAMPLE 6 In vitro formation of a collagen matrix by transfected human neonatal foreskin fibroblasts The transfected human dermal fibroblasts were produced using the following procedure. One vial of the viral producers of platelet-derived growth factor JCRP-43 (PDGF) (Morgan, J, et al.) Was thawed, and the cells were seeded at 2 × 10 6 cells / 162 cm2 per vial (Corning Costar, Cambridge, MA). These flasks were fed with growth medium, kept in an incubator at 37 ± 1 ° C with an atmosphere of C02 at 10 ± 1%. The growth medium consists of: Dulbecco's Modified Eagle Medium (DMEM) (high glucose formulation, without L-glutamine BioWhittaker, Waikersville, MD), Hams F-12 medium (Quality Biologics, Gaithersburg, MD), supplemented calf serum newborn at 10% (HyClone Laboratories, Inc., Logan, Utah) and 4 mM L-glutamine (BioWhittaker, Waikersville, MD). On the same day, a vial of human neonatal foreskin fibroblasts (HDFB156) that were also thawed and seeded at 1.5x106 cells / 162 cm2 per bottle (Corning Costar, Cambridge, MA). After three days the JCRP PDGF-43 viral producers were fed fresh growth medium. HDFB156 was fed with growth medium plus 8 μg / ml polybrene (Sigma, St. Louis, MO). The next day the HDFB156 'cells were infected as follows. The medium spent from the JCRIP PDGF-43 wine producers was collected and filtered through a 0.45 micron filter. 8μg / ml polybrene was added to the spent spent medium. The spent medium was placed on HDF. In the following two days the HDF were fed fresh growth medium. The day before HDF will be passed from p5 to p6 a density of 2.5x106 cells / 162 cm2 was seeded per bottle (Corning Costar, Cambridge, MA). The cells were passed as follows; the spent medium was sucked. The bottles were rinsed with a phosphate-buffered saline to remove any residual newborn calf serum. The cells were released from of the bottle by adding 5 ml trypsin-versen to each bottle and moving gently to ensure full coverage of the monolayer. The cultures were returned to the incubator. As soon as the cells were released, 5 ml of SBTI (soybean trypsin inhibitor) was added to each vial and mixed with the suspension to stop the action of trypsin-versen. The cell / trypsin / SBTI suspension was removed from the flasks and equally divided between sterile, conical centrifuge tubes. The cells were collected by centrifugation at approximately 800-1000 x g for five minutes. The cells were resuspended in the growth medium to be seeded at the density listed above. After two days the cells were fed fresh growth medium. The next day the cells were harvested as established above, and were diluted to a density of 1.5x10 6 cells / ml in growth medium containing 10% newborn calf serum (NBCS) with 10% dimethylsulfoxide (DMSO) ( Sigma, St. Louis, MO). The cells were frozen 1 ml / cryovial at about -80 ° C. Production of the collagen matrix for this example uses the same procedure as in Examples 1 and 3, replacing human neonatal foreskin fibroblasts with transformed human neonatal foreskin fibroblasts to produce high levels of platelet-derived growth factor (PDGF) as described above. Samples were taken for staining with H & E as described above at day 18 postsembrated. Samples were also stained using the avidin-biotin methods for the presence of fibronectin listed in Example 10. Samples were taken at 18 days post-sampled for H & E staining as described in example 1, and exhibited a similar thickness appearance to the cell-matrix described in example 1, with a measured thickness of 123.6 microns (N-1). The PDGF output of the transfected cells in the cell-matrix construction was measured as 100 ng / ml by ELISA for the duration of the culture (18 days) although the PDGF control output was not detected.

EXAMPLE 7 Use of the dermal construction as a graft material The cell-matrix constructs prepared according to the methods in Example 1 using human dermal fibroblasts derived from neonatal foreskin were grafted onto wounds with complete incisions created on nude nude mice. The mice were grafted according to the methods described by Parenteau, et al. (1996), the description of which is incorporated here. The grafts were examined at 14, 28 and 56 days for signs of adherence to the wound bed, evidence of wound contraction, areas of graft loss, and presence of vascularization (color). The graft areas were photographed while they were intact on the mouse. A number of mice were sacrificed at each time, and the graft areas and their surroundings were removed together with a small part of murine skin of at least panniculus carnosus. The joints between the graft and the skin of the murine were preserved in each sample. The explanted tissue samples were fixed in 10% formalin in phosphate buffer and fixed in methanol. The samples fixed in formalin were processed for staining with H &E according to the procedure described in example 1. The grafts were able to integrate with the skin of the mouse, with a minimal contraction observed. Within 14 days of grafting, the epidermis of the mouse completely migrated over the graft. Using the samples stained with H &E, the blood vessels were obvious within the graft at 14 days, and through the experiment. By gross observation and by samples stained with H &E, it was determined that the graft persisted and remained healthy containing live cells, without gross abnormalities of the matrix, etc., throughout the experiment.

EXAMPLE 8 Use of the maximum thickness of the skin construction as a skin graft The bilayer skin constructs were prepared as described in Example 2 using human dermal fibroblasts derived from the dermal layer of the neonatal foreskin and human keratinocytes derived from the epidermal layer of a different neonatal foreskin. The skin constructions were able to be manually separated from the membrane, operated without support vehicles, and placed on the site of the graft. The bilayer skin constructs were grafted onto wounds with complete incisions created on athymic nude mice according to the methods described by Parenteau, et al. (1996), the description of which is incorporated here. The time points to take the samples were days 7, 14, 28, 56 and 184 days post-graft. The graft areas were photographed while remaining intact on the mice. A number of mice were sacrificed at each time, and the graft areas and surrounding areas were removed along with a small part that surrounded the mouse skin to at least panniculus carnosus. The junctions between the graft and the mouse skin were preserved in each sample. The explanted tissue samples were fixed in 10% formalin in phosphate buffer and fixed in methanol. The samples fixed in formalin were processed for staining in H &E according to the procedure described in example 1. The grafts were integrated with the tissue of the host within 7 days by observation of the thickness as well as by histological appearance. Through H & E staining, blood vessels were visualized growing within the graft from the host tissue within 7 days of grafting. The grafts remained healthy and persisted during the experiment, with minimal contraction noted. Using an anti-human involucrin stain the persistence of human epidermal cells was observed for the entire graft period.

EXAMPLE 9 In vitro formation of a matrix by human corneal keratinocytes Human corneal keratinocyte cells (originating in Organogenesis, Inc. Canton, MA) were used in the production of a corneal stromal construct. Confluent cultures of human keratinocytes were released from their culture substrates using trypsin-versenne. When they were released, the soy trypsin inhibitor was used to neutralize trypsin-versen, the cell suspension was centrifuged, the supernatant was discarded and the cells were then resuspended in base medium at a concentration of 3x10 6 cells / ml. The cells were seeded on transfer wells treated for tissue culture with a pore size of 0.4 microns, with a diameter of 24 mm in a six-well device at a density of 3.0 × 10 6 cells // TW (6.6 × 10 5 cells / cm 2) . These crops were kept overnight in the middle of sowing. The seed medium is composed of: a 3: 1 base mixture of Dulbecco's Modified Eagle medium (DMEM) and Hams F-12 medium (Quality Biologics, Gaithersburg, MD cat.), 4 mM GlutaMAX (Gibco BRL, Grand Island, NY) and additives: 5 ng / ml epidermal growth factor (EGF) (Upstate Biotechnology Lake Placid, NY), 0.4 μg / ml hydrocortisone (Sigma, St. Louis, MO), 1 × 104 M ethanolamide (Fluka, Ronkonkoma, NY), 1 x 104 M o-phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 μg / ml insulin (Sigma, St. Louis, MO), 5 μg / ml transferrin (Sigma, St. Louis, MO), 20 pM triiodothyronine (Sigma, St. Louis, MO) and 6.78 ng / ml selenium (Sigma Aldrich Fine Chemicals Company). Following this the crop was fed fresh production medium. The fresh production medium was composed of: a 3: 1 base DMEM mixture, Hams F-12 medium (Quality Biologics, Gaithersburg, MD), 4 mM GlutaMAX (Gibco BRL, Grand Island, NY) and additives: 5 ng / ml of human recombinant epidermal growth factor (Upstate Biotechnology Lake Placid, NY), 2% newborn calf serum (HyClone, Logan, Utah), 0.4 μg / ml hydrocortisone (Sigma, St. Louis, MO), 1 × 104 M ethanolamide (Fluka, Ronkonkoma, NY), or - 1 x 104 M phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 μg / ml insulin (Sigma, St. Louis, MO), 5 μg / ml transferrin (Sigma, St. Louis, MO), triiodothyronine 20 pM (Sigma, St. Louis, MO) and 6.78 ng / ml selenium (Sigma Aldrich Fine Chemicals Company), 50 ng / ml L-ascorbic acid (WAKO mash chemicals company), 0.2 μg / ml L- proline (Sigma, St. Louis, MO), 0 J g / ml glycine (Sigma, St. Louis, MO) and 0.05% polyethylene glycol (PEG) (Sigma, St. Louis, MO, cell culture grade). Cells were maintained in an incubator at 37 ± 1 ° C with an atmosphere of 10% C02 ± 1% and were fed fresh production medium every 2-3 days for 20 days (for a total of 21 days in culture) . After 21 days in culture, the keratinocytes had deposited a matrix layer of about 40 microns in thickness, as measured by the method described in example 1. The endogenously produced fibrillar collagen, decorin and glycosaminoglycan were also present in the construction cell-matrix.

EXAMPLE 10 In vitro formation of a collagen matrix by human neonatal prepuce fibroblasts seeded in production medium The human neonatal foreskin fibroblasts (originating in Organogenesis, Inc. Canton, MA) were seeded at 1x105 cells / 0.4 micron pore size, in a six-well device treated for tissue culture, 24 mm in diameter. (TRANSWELL®, Costar Corp. Cambridge, MA) and grew up in growth medium. The growth medium consisted of: Dulbecco's Modified Eagle Medium (DMEM) (high glucose formulation, without L-glutamine, BioWhittaker, Waikersville, MD) supplemented with 10% newborn calf serum (HyClone Laboratories, Inc., Logan , Utah) and 4 mM L-glutamine (BioWhittaker, Waikersville, MD). The cells were maintained in an incubator at 37 ± 1 ° C with an atmosphere of C02 at 10 ± 1%. The medium was replaced every two to three days. After 9 days in culture the medium was aspirated from the culture boxes, and replaced with production medium. The cells were kept in an incubator at 37 ± 1 ° C with an atmosphere of C02 at 10 ± 1% and were fed fresh production medium every 2-3 days for 21 days. The production medium was composed of: a DMEM 3: 1 base, Hams F-12 medium (Quality Biologics, Gaithersburg, MD), 4 mM GlutaMAX (Gibco BRL, Grand Island, NY) and additives: 5 ng / ml of Human recombinant epidermal growth factor (Upstate Biotechnology Lake Placid, NY), 2% newborn calf serum (HyClone, Logan, Utah), 0.4 μg / ml hydrocortisone (Sigma, St. Louis, MO), 1 × 104 M ethanolamide (Fluka, Ronkonkoma, NY) , 1 x 104 M o-phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 μg / ml insulin (Sigma, St. Louis, MO), 5 μg / ml transferrin (Sigma, St. Louis, MO) ), 20 pM triiodothyronine (Sigma, St. Louis, MO) and 6.78 ng / ml selenium (Sigma Aldrich Fine Chemicals Company), 50 ng / ml L-ascorbic acid (WAKO mash chemicals company), 0.2 μg / ml L-proline (Sigma, St. Louis, MO), OJ μg / ml glycine (Sigma, St. Louis, MO) and 0.05% polyethylene glycol (PEG) (Sigma, St. Louis, MO, cell culture grade). The samples were taken on day 21 and fixed in formalin, then embedded in paraffin. The formalin-fixed samples were embedded in paraffin and sections of 5 microns were stained with hematoxylin-eosin (H & E) according to routine techniques used in the art. Using sections stained with H &E, measurements were made in 10 microscopic fields taken at random using a 10X eye piece (Olympus America Inc., Melville, NY) loaded with a 10 mm / 100 micron reticle (Olympus America Inc ., Melville, NY). Constructs created using this method are similar in structure and biochemical composition to those created in Example 1, and have a measured thickness of 82.00 ± 7.64 microns.

EXAMPLE 11 In vitro formation of a collagen matrix by pig dermal fibroblasts Pig dermal fibroblasts (originating in Organogenesis, Inc. Canton, MA) were seeded at 5x105 cells / 162 cm2 in tissue culture treated bottles (Costar Corp., Cambridge, MA, Cat # 3150) and grown in growth medium as described below. The growth medium consisted of; Dulbecco's Modified Eagle Medium (DMEM) (high glucose formulation, without L-glutamine, BioWhittaker, Waikersville, MD) supplemented with 10% fetal calf serum (HyClone Laboratories, Inc., Logan, Utah) and L-glutamine 4 mM (BioWhittaker, Waikersville, MD). Cells were maintained in an incubator at 37 ± 1 ° C with an atmosphere of 10% ± 1% C02. The medium was replaced every two to three days. After confluence, this is the cells have formed a dense layer at the bottom of the tissue culture bottle, the medium is aspirated from the culture box. To rinse the monolayer, sterile phosphate-buffered saline was added and filtered to the monolayer and then aspirated from the culture dish. The cells were released from the flask by adding 5 ml trypsin-verseno glutamine (Bio Whittaker, Waikersville, MD) to each vial and shaking gently to ensure a general coverage of the monolayer. The cultures were returned to the incubator. As soon as the cells were released, 5 ml of SBTI (soybean trypsin inhibitor) was added. to each bottle and mixed with the cell suspension to stop the action of trypsin-versen. The suspension was removed from the bottles and divided equally between sterile, conical centrifuge tubes. The cells were collected by centrifugation at approximately 800-1000 x g for 5 minutes. The cells were resuspended and diluted to a concentration of 3x10 6 cells / ml, and seeded onto transfer wells treated for tissue culture with a pore size of 0.4 microns, and with a diameter of 24 mm at a density of 3.0x106. cells / TW (6.6x105 cells / cm2). The cells were kept overnight in a seeding medium. The seeding medium consisted of; a 3: 1 base mixture of DMEM, Hams F-12 medium (Quality Biologics, Gaithersburg, MD), 4 mM GlutaMAX (Gibco BRL, Grand Island, NY) and additives: 5 ng / ml human recombinant epidermal growth factor (Upstate Biotechnology Lake Placid, NY), 2% newborn calf serum (HyClone, Logan, Utah), 0.4 μg / ml hydrocortisone (Sigma, St. Louis, MO), 1 × 104 M ethanolamide (Fluka, Ronkonkoma , NY), o-phosphoryl-ethanolamine 1 x 104 M (Sigma, St. Louis, MO), 5 μg / ml insulin (Sigma, St. Louis, MO), 5 μg / ml transferrin (Sigma, St. Louis, MO), 20 pM triiodothyronine (Sigma, St. Louis, MO) and 6.78 ng / ml selenium (Sigma Aldrich Fine Chemicals Company), 50 ng / ml L-ascorbic acid (WAKO mash chemicals company), 0.2 μg / ml L-proline (Sigma, St. Louis, MO) and 0J μg / ml glycine (Sigma, St. Louis, MO). The cells were maintained in an incubator at 37 + 1 ° C with an atmosphere of 10 ± 1% C02 and were fed fresh production medium every 2-3 days for 7 days. He Production medium was composed of: a DMEM base 3: 1 mixture, Hams F-12 medium (Quality Biologics, Gaithersburg, MD), 4 mM GlutaMAX (Gibco BRL, Grand Island, NY) and additives: 5 ng / ml human recombinant epidermal growth factor (Upstate Biotechnology Lake Placid, NY), 2% newborn calf serum (HyClone, Logan, Utah), 0.4 μg / ml hydrocortisone (Sigma, St. Louis, MO), ethanolamide 1 × 104 M (Fluka, Ronkonkoma, NY), 1 x 104 M o-phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 μg / ml insulin (Sigma, St. Louis, MO), 5 μg / ml transferrin (Sigma, St. Louis, MO), 20 pM triiodothyronine (Sigma, St. Louis, MO) and 6.78 ng / ml selenium (Sigma Aldrich Fine Chemicals Company), 50 ng / ml L-ascorbic acid (WAKO puree chemicals company), 0.2 μg / ml L-proline (Sigma, St. Louis, MO), 0 J μg / ml glycine (Sigma, St. Louis, MO) and 0.05% polyethylene glycol (PEG) (Sigma, St. Louis, MO) grade cell culture. After 7 days the medium was replaced with production media without newborn calf serum. This medium was used fresh to feed the cells every 2-3 days for more than 20 days, for a total of 28 days in culture. Samples were taken on day 21 and fixed in formalin, then embedded in paraffin. The formalin-fixed samples were embedded in paraffin and 5-micron sections were defined with hematoxylin-eosin (H & E) according to the usual techniques used in the art. Using sections stained with H & E measurements were made in ten microscopic fields taken at random using an eye piece (Olympus America Inc., Melville, NY) loaded with a 10mm / 100 grating micrometer (Olympus America Inc., Melville, NY). The sample exhibited a structure composed of cells and matrix with a measured thickness of 71.20 ± 9.57. In addition to the endogenously produced fibrillar collagen, decorin and glycosaminoglycan were also present in the cell-matrix construction.

EXAMPLE 12 In vitro formation of a bilayer skin construct containing dermal papilla cells A cell-matrix was made according to the method in Example 1 using human neonatal foreskin fibroblasts as an initial cell type producing matrix. The cell-matrix was locally seeded with spots of dental papilla cells as a second cell population that in turn was seeded with keratinocytes with a third cell population, to form a continuum of epidermal layer on the cell-matrix and the cell of the dermal papilla. First, a cell-matrix construct was formed using human dermal fibroblasts (HDF) derived from neonatal foreskin. HDF were grown upon seeding in treated flasks for tissue culture at 5 x 10 5 cells / 162 cm 2 (Costar Corp. Cambridge, MA) in growth medium consisting of: Dulbecco's Modified Eagle Medium (DMEM) (high glucose formulation, without L-glutamine, BioWhittaker, Waikersville, MD) supplemented with 10% newborn calf serum (NBCS) (HyClone Laboratories, Inc., Logan, Utah) and 4 mM L-glutamine (Bio Whittaker, Waikersville, MD). When they were confluent, the HDFs were released from the culture box using trypsin-versen and resuspended using fresh medium at a concentration of 3.0 x 10 6 cells / ml, and were seeded into 0.4 micron tissue culture inserts. pore size, 24 mm in diameter (TRANSWELL®, Corning Costar) in a six-well device at a density of 3.0 x 106 cells / insert (6.6 x 10 5 cells / cm 2). The HDF cultures were maintained at an incubator 37 ± 1 ° C with a CO2 atmosphere at 10 ± 1% and were fed with fresh production medium every 2 to 3 days for 23 days according to the method described in example 1. After the cell-matrix construction had been formed, it was seeded with dermal papilla points as the second cell population. The dermal papilla cell are a discrete population of specialized fibroblasts surrounded by the hair bulb of the hair follicles to play a central role in hair growth. The dermal papilla can be isolated by microdissection of the hair follicle and in vitro culture using the method previously described by Messenger, A.G., The Culture of Dermal Papilla Cells from Human Hair, Follicles Follicles, Br. J. Dermatol. 110: 685-9 (1984), the method that is incorporated herein. When a culture of dermal papilla cells reaches confluence, they form aggregates that can be re-seeded on culture flasks to form new aggregates. The dermal papilla is isolated from a skin biopsy obtained from a 4 week old pig. Cells from the dermal papilla (PDP) were serially cultured in DMEM containing 20% NBCS up to passage 8. After three weeks of culture, the PDP cells reformed the dermal papilla-like structures, or aggregates, and each one had a diameter of approximately 90 to 210 microns. The aggregates were removed from the culture dish by vigorous pipetting of the medium against them, and then plated on the human collagen matrix at a density of 200 aggregates per cm 2. The aggregates were grown submerged for an additional 15 days in DMEM with 20% NBCS with spent medium exchanged for fresh medium every 2-3 days. The cell-matrix cultures containing the dermal papilla cell on these were seeded with keratinocytes and cultured to form a continuous epidermal layer on the cell-matrix and the dermal papilla. Two different constructions were made: the first with human keratinocytes, the second with pig keratinocytes. Normal epidermal keratinocytes were isolated from human neonatal prepuce, or from pig keratinocytes (HEP), using the external growth of explants to establish primary cultures. These cells were then cultured and expanded to passage 3 from the pig strain, or to passage 4 from the human strain. After about 5 to 6 days in culture, cells were released from the culture dishes using trypsin-versen, pooled, centrifuged to form a cell pellet, resuspended in epidermalization medium, counted and sown on top of the membrane at a density of 4.55 x 104 cells / cm2 for HEP cells, or 1.6 x 10 5 cells / cm2 for PEP cells. The epidermalized cultures were cultured for 12 days as previously described in example 2. The final samples were subjected to hematoxylin and eosin processing for light microscopy. The resulting skin constructs exhibited the basic morphological similar to the skin: a dermal layer consisting of fibroblasts surrounded by endogenously produced matrix, including endogenously produced collagen fibers, decorin and glycosaminoglycan, in localized areas of the dermal papilla cell and a Continuous stratified layer of keratinocytes through the cell-matrix construction and the dermal papilla. In both tissue constructions superimposed with either human or pig keratinocytes, the dermal papilla maintained a packed structure that induced the small undulations of the overlying epithelium. Differentiated epithelial cells often present closely to the cells of the dental papilla.

EXAMPLE 13 Measurement of hyaluronic acid by ELISA intercalated Hyaluronic acid (HA) was measured in the constructs formed by normal fibroblasts in medium containing serum and chemically defined medium according to methods 1 and 3, respectively.

The cell-matrix constructions were formed on circular carriers with a diameter of 75 mm that were incorporated into the porous membranes (TRANSWELL®, Corning Costar). Extracts from the cell-matrix constructs were prepared by adding 10 mL of ammonium acetate buffer and 0.5 mg / mL of proteinase K to a test tube containing a cell-matrix construct. The mixture was incubated at 60 ° C overnight. After completing the digestion, the mixture was centrifuged and the supernatant extract was transferred to a separate tube for the hyaluronic acid assay. A 96-well box was covered with 50 μL of 20 μg / mL of HA binding protein in an 0JM NaHCO 3 solution and stored overnight at 4 ° C. The box was then washed three times with 0.85% NaCl containing 0.05% Tween 20. To each well was then added 250 μL of blocking solution (sodium phosphate buffer, 10 mmol, pH = 7.4 containing 3% BSA and 0.9% NaCl, PBS + 3% BSA) and the box was incubated at RT for 2 h. The box was then washed three times with 0.85% NaCl containing 0.05% Tween 20. Then 50 μL of a standard solution of HA and the extracts were added to the box from the experimental conditions, including various solutions of these conditions. The box was incubated at room temperature (around 20 ° C) for 2 hours. The box was then washed three times with 0.85% NaCl containing 0.05% Tween 20 and 50 μL of biotinylated HA (1: 2000 dilution) was added to each well and then incubated for 2 hours at room temperature. The box was washed three times with NaCI at 0.85% containing 0.05% Tween 20 and then 50μL of HRP-avidin D (1: 3000 dilution) is added to each well. The box was then incubated for 45 minutes at room temperature. The box was washed three times with 0.85% NaCl containing 0.05% Tween 20. At each well, 10 μL of ortho-phenylenediamine substrate solution was added. The box was incubated at 37 ° C for 10 minutes. The reaction was stopped by the addition of 50 μL of IM HCl. Finally, using a box reader the absorbance was read at 492 nm and recorded. The absorbance measurements were averaged and converted to quantitative measures. The circular cell-matrix constructions (diameter 75 mm) formed in a medium containing serum were determined as each containing about 200 μg of hyaluronic acid while those formed in chemically defined medium contained about 1.5 mg of hyaluronic acid.

EXAMPLE 14 Physical test and mechanical properties of cell-matrix construction produced The mechanical properties of the tissue construction of example 1 (cell-matrix construction), example 2 (cell-matrix construction with a keratinocyte layer on it), and example 3 (cell-matrix construction formed on defined medium) were quantified by Inflation tests of the membrane. These tests are similar to the tests used clinically (eg Dermaflex®, Ciberderm Inc., Media, PA, and Cutameter®, Courage Khazaka, Cologne, Germany) but involve higher pressures including pressures capable of tearing the membrane. The sample cell-matrix construction was allowed to stand flat on a polycarbonate block centered on a cylindrical well of 10 mm in diameter filled with normotonic saline solution. A metal plate with a circular hole corresponding to the diameter of the cylindrical well was placed on the sample and fixed to the block. The samples were then inflated by infusing additional saline into the well with a syringe pump. The resulting pressure was measured with a pressure transducer. The pressurization was carried out until the apparatus fails, given the strength that tears the membrane, with averages of approximately 439.02 mm Hg for cell-matrix construction generated by the method of Example 1; 998.52 mm Hg for the samples of the cell-matrix construction with a keratinocyte layer generated by the method of example 2; and, 1542.26 mm Hg for the cell-matrix construction samples formed in defined medium generated according to the method of example 3. To determine the thermal melting point of the dermal matrix, the samples (cell-matrix construction) were they were taken at 21 days and prepared using the procedure described in Example 1. The denaturation temperature samples were determined by analysis with the Mettier Toledo (Highston, NJ) differential selection calorimeter (DSC product # DSCI2E). For the purposes of the inventors, the temperature of Fusion was determined by heating the sample from 45 to 80 ° C at a rate of 1 ° C / minute. The average denaturation temperature for the samples is 60.8 + 1.2 ° C (n = 3). The retention of the suture and the force at the pressure of the epidermalized matrix created using the procedure in Example 1 (cell-matrix construction) and 3 (cell-matrix construction formed in defined medium) were measured to determine the suture of the construction in certain clinical situations. The strength of the suture retention of the human dermal matrix at 21 days of age was determined using methods described in American National Standards for Vascular Graft Prosthesis (Instruments, 1986) using a Mini-Bionex 858 test system (MTS Systems Corporation, Minneapolis, Minn.). For the samples of example 1, (cell-matrix construction), the tensile force was determined to be 365 N / m; for the samples prepared according to example 2 (cell-matrix construction with a keratinocyte layer), the tension force was 2720 N / m. The strength of suture retention for the samples prepared according to example 1 was 0J4 N; for those prepared according to example 2, 0.22 N. The constructions created and described in examples 1, 2 and 3 have been made in both diameters of 24 mm and 75 mm. Constructions made by culture techniques of the 3 methods are cohesive, tissue-like structures that are easily separated from the membrane with minimum force, therefore "easily removable", and capable of being physically handled and manipulated for use and testing without damage occurring.

EXAMPLE 15 In Vitro Formation of a Collagen Matrix by Fibroblasts of the Neonatal Prepuce of Humans in Chemically Defined Media Human neonatal foreskin fibroblasts were expanded using the procedure described in Example 1. The cells were resuspended at a concentration of 3 x 10 6 cells / ml, and seeded on treated membrane inserts for tissue culture 0.4 microns in size. pore, and 24 mm diameter in a six-well box at a density of 3 x 106 cells / TW (6.6 x 10 5 cells / cm2). The cells in this example were cultured in chemically defined medium throughout. The medium contained: a DMEM 3: 1 base mix, half Hams F-12 (Quality Biologics, Gaithersburg, MD), GlutaMAX 4mM (Gibco BRL, Grand Island NY) and additives: 5 ng / ml epidermal growth factor (Upstate Biotechnology, Lake Placid, NY), ethanolamine 1 X 10"4 M (Fluka, Ronkonkoma, NY cat. # 02400 ACS grade), o-phosphoryl-ethanolamine 1 x 10" 4 M (Sigma, St. Louis, MO), and 5 μg / ml transferrin (Sigma, St. Louis, MO), triiodothyronine 20 (Sigma, St. Louis, MO), and 6.78 ng / ml selenium (Sigma Aldrich Fine chemicals Company, Milwaukee, Wl), 50 ng / ml L-ascorbic acid (WAKO Chemicals USA, Inc.), 0.2 μg / ml L-proline (Sigma, St. Louis, MO), OJ μg / ml glycine (Sigma, St. Louis, MO). To the above basic medium, other components were added under these separate conditions: 1. 5 μg / ml insulin (Sigma, St. Louis, MO), 0.4 μg / ml hydrocortisone (Sigma, St. Louis, MO), 0.05% polyethylene glycol (PEG) (Sigma, St. Louis, MO). 2. 5 μg / ml insulin (Sigma, St. Louis, MO), 0.4 μg / ml hydrocortisone (Sigma, St. Louis, MO). 3. 375 μg / ml insulin (Sigma, St. Louis, MO), 6 μg / ml hydrocortisone (Sigma, St. Louis, MO). The samples were fixed in formalin and processed for hematoxylin and eosin staining for light microscopy analysis. Visual histological evaluation showed that condition 2 lacking PEG demonstrates a matrix comparably similar to that of condition 1 containing PEG. The biochemical analyzes of the collagen content of the construction show approximately the same amount of collagen in both: 168.7 + 7.98 μg / cm2 for condition 1 with PEG compared to 170.88 + 9.07 μg / cm2 for condition 2 without PEG. Condition 3 that contains high levels of insulin and hydrocortisone shows higher matrix expression, including collagen, at a earlier time than in the other two conditions. In addition to the endogenously produced fibrillar collagen, decorin and glycosaminoglycans are also present in the construction cell-matrix in all conditions. The cultivated dermal construction formed by the method of condition 2 of this example is shown in Fig. 2. Fig. 2 shows a photomicrograph of a section of a fixed cell-matrix construct, embedded in paraffin, stained with hematoxylin and eosin formed at from human dermal fibroblasts cultured in chemically defined medium at 21 days. The porous membrane appears as a thin translucent band under construction and it can be seen that the cells grow on the surface of the membrane and do not envelop it by integrating the membrane with the matrix. Figure 3 shows transmission electron microscopy (TEM) images of two amplifications of the cultivated dermal construct formed by the method of condition 2 of this example at 21 days. Figure 3A is a 7600X amplification showing the alignment of endogenous collagen fibers between the fibroblasts. Figure 3B is a 19000X amplification of fully formed endogenous collagen fibers demonstrating fibrillar arrangement and packaging. In all the conditions of this example, the cultured dermal constructs formed comprise dermal fibroblasts and matrix produced endogenously. All have fully formed collagen fibers in packaged arrangement organization between cells. The fibrous qualities, thickness, and cohesive integrity give the construction considerable strength to allow it to be removed from the membrane. cultivated and handled as if transferred to a patient to be treated with the construction, in a graft or implant.

EXAMPLE 16 Construction of full-thickness skin Using a 21-day dermal construct formed by human dermal fibroblasts under chemically defined conditions according to the condition 2 method (without PEG) described in Example 15, above, epidermal keratinocytes from neonatal foreskin of normal human were seeded on the upper surface of the cell-matrix construction to form the epidermal layer of the skin construction. The medium was removed aseptically from the culture insert and its surroundings. Normal human epidermal keratinocytes were generated up to step 4 from subcultures grown to confluence of frozen storage cells. The cells were then released from the culture boxes using trypsin-versen, pooled, centrifuged to form a cell pellet, resuspended in epidermalization medium, counted and seeded on the upper part of the membrane at a density of 4.5 X 104 cells / cm2. The constructs were then incubated for 90 minutes at 37 + 1 ° C, 10% C02 to allow the keratinocytes to anchor. After incubation the constructions were immersed in epidermalization medium. The means of epidermalization is composed of: a 3: 1 base mixture of Dulbecco's Modified Eagle Medium (DMEM) (which contains no glucose or calcium, BioWhittaker, Waikersville, MD), and Hams F-12 medium (Quality Biologics, Gaithersburg, MD), supplemented with 0.4 μg / ml hydrocortisone (Sigma, St. Louis, MO), 1 × 104 M ethanolamide (Fluka, Ronkonkoma, NY), or 1 × 104 M phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 μg / ml insulin (Sigma, St. Louis, MO), 5 μg / ml transferrin (Sigma, St. Louis, MO), 20 pM triiodothyronine (Sigma, St. Louis, MO), 6.78 ng / ml selenium (Aldrich), 24.4 μg / ml adenine (Sigma Aldrich Fine Chemicals Company), 4 mM L-glutamine (BioWhittaker, Waikersville, MD), 50 μg / ml sodium ascorbate salt, 16 μM linoleic acid (Sigma, St. Louis, MO), 1 μM tocopherol acetate (Sigma, St. Louis, MO) and 50 μg / ml gentamicin sulfate (Amersham, Ariington Heights, IL) Constructs were cultured in the epidermalization medium for 2 days at 37 ± 1 ° C, C02 to 10 ± 1% After 2 days the medium was exchanged with fresh medium compound as stated above, and returned to the incubator at 37 ± 1 ° C, C02 at 10 + 1% for two days. After 2 days, the vehicle containing the construction was transferred aseptically to new culture devices with sufficient medium to achieve a level of fluid just to the surface of the carrier membrane to keep the construction under development at the air-liquid interface. The air that comes in contact with the surface of the epidermal layer in formation allows the stratification of the epidermal layer. The constructs were incubated at 37 ± 1 ° C, 10% C02, and a low humidity, in the middle with medium changes every 2-3 days for 7 days. This medium contained a 1: 1 mixture of Dulbecco's modified Eagle's medium (DMEM) (which does not contain glucose or calcium, BioWhittaker, Waikersville, MD), and Hams F-12 medium (Quality Biologics, Gaithersburg, MD), supplemented with 0.4 μg / ml hydrocortisone (Sigma, St. Louis, MO), 1 × 104 M ethanolamide (Fluka, Ronkonkoma, NY), 1 × 104 M o-phosphoryl-ethanolamine (Sigma, St. Louis, MO), 5 μg / ml insulin (Sigma, St. Louis, MO), 5 μg / ml transferrin (Sigma, St. Louis, MO), 20 pM triiodothyronine (Sigma, St. Louis, MO), 6.78 ng / ml selenium ( Aldrich), 24.4 μg / ml adenine (Sigma Aldrich Fine Chemicals Company), 4 mM L-glutamine (BioWhittaker, Waikersville, MD), 2.65 μg / ml calcium chloride, 16 μM linoleic acid (Sigma, St. Louis, MO), 1 μM tocopherol acetate (Sigma, St. Louis, MO), 1.25 mM serine (Sigma, St. Louis, MO), 0.64 mM choline chloride (Sigma, St. Louis, MO) and 50 μg / ml of gentamicin sulfate (Amersham, Arington Heights, IL) Crops were fed every 2-3 days, for 14 days. The samples, in triplicate, were subjected to 10, 12, and 14 days after the construction was elevated to the air-liquid interface for hematoxylin and eosin processing as described in Example 1 to determine the appearance of the low thickness light microscopy. The resulting construction was a bilayer skin construct consisting of a lower dermal layer consisting of dermal fibroblasts surrounded by matrix superimposed by an epidermal layer of stratified and differentiated keratinocytes. The bilayer leather construction of this example is shown in Figure 4. Figure 4 is a photomicrograph of a section of a fixed, paraffin-embedded cultured skin construct stained with hematoxylin and eosin formed in chemically defined medium in the absence of exogenous matrix components comprising a cell-matrix construct formed from human dermal fibroblasts cultured in chemically defined medium with a multilayer, differentiated epidermis formed from human keratinocytes cultured in chemically defined medium.

EXAMPLE 17 Formation of a collagen matrix by human buccal fibroblasts The purpose of this experiment is to produce a cell-matrix construction from buccal fibroblasts isolated from human internal cheek tissue. The mouth explant was grown in T-150 bottles in DMEM medium containing 10% NBCS. After 7 days, to expand the number of cells further, the buccal cells were harvested and transferred to nine T-150 bottles at 4.0x10d cells in DMEM medium containing 10% NBCS and cultured to confluence at which time the cells they were harvested To harvest the cells, the medium was aspirated from the culture bottles, to rinse the monolayer, sterile filtered phosphate-buffered saline was added to the bottom of each culture bottle and then aspirated. from the bottles. The cells were released from the bottles by adding 5 ml of trypsin-verseno glutamine (Bio Whittaker, Waikersville, MD) to each bottle and gently turned to ensure complete coverage of the monolayer. The cultures were returned to the incubator. As soon as the cells were released 5 ml of SBTI (soybean trypsin inhibitor) was added to each bottle and mixed with the suspension to stop the action of trypsin-versen. The cell suspension was removed from the bottles and divided equally between sterile, conical centrifuge tubes. The cells were collected by centrifugation at approximately 800-1000 x g for 5 minutes. Cells were resuspended using fresh medium at a concentration of 3.0 × 10 6 cells / ml, and seeded on 0.4 μm-diameter, 24 mm diameter pore size treated tissue culture inserts (TRANSWELL®, Corning Costar) in a box of six wells at a density of 3.0x106 cells / insert (6.6x105 cells / cm2). The cells were maintained in an incubator at 37 + 1 ° C with an atmosphere of 10 + 1% C02 and were fed with medium containing: a 3: 1 base mixture of DMEM medium and Hams F-12 (Quality Biologics Gaithersburg, MD), 4 mM, GlutaMAX-1 ™ (Gibco BRL, Grand Island, NY) and additives: 5 ng / ml human recombinant epidermal growth factor (Upstate Biotechnology Lake Placid, NY), 0.4 ug / ml hydrocortisone ( Sigma St. Louis, MO), ethanolamine 1 x 104 M (Fluka, Ronkonkoma, NY ACS grade), o-phosphoryl-ethanolamine 1 x 10"4 M (Sigma, St. Louis,), 5 μg / ml insulin ( Sigma, St. Louis, MO), 5 μg / ml transferrin (Sigma, St. Louis, MO), 20 pM triiodothyronine (Sigma, St. Louis, MO), and 6.78 ng / ml selenium (Sigma Aldrich Fine Chemicals Co., Milwaukee, Wl), 50 ng / ml L-ascorbic acid (WAKO Chemicals USA Inc.), 0.2 μg / ml L-proline (Sigma, St. Louis, MO), OJ μg / ml glycine (Sigma, St. Louis, MO) and 0.05% polyethylene glycol (PEG) (Sigma, St. Louis, MO). At day 1 post-seeding, the medium was replaced with serum-free production medium, changed every 2-3 days for 21 days. On day 21, the samples were fixed in formalin for histology. Three samples were used for analysis of protein and collagen production. The production of collagen for constructions of 24 mm diameter averaged 519 μg per construction after 21 days in culture. The total protein production of the 24 mm diameter constructions averaged 210 μg per construct after 21 days in culture. Morphologically, the cell-matrix construction of buccal fibroblast, a cultured tissue construct of oral connective tissue, shows buccal fibroblasts surrounded by matrix although physically, the construction has physical integrity and dimension. Although the above invention has been described in some detail by way of illustration and examples for purposes of clarity and 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 (30)

NOVELTY OF THE INVENTION CLAIMS

1. A cultured tissue construct comprising fibroblast cells grown under conditions that produce an extracellular matrix layer that is synthesized and assembled by cultured fibroblast cells, with cultured fibroblast cells contained within the synthesized extracellular matrix layer, wherein the extracellular matrix comprises: (i) fibrillar collagen showing a packaged organization of fibrils and bundles of fibrils exhibiting a 67 nm banding pattern in four planes; (ii) decorin; and (iii) glycosaminoglycans; and wherein said extracellular matrix is produced by cultured fibroblast cells in the absence of exogenous matrix or synthetic member components during culture conditions.

2. The cultured tissue construction according to claim 1, further characterized in that the fibroblasts are derived from tissue selected from the group consisting of foreskin, dermis, tendon, lung, urethra, umbilical cord, corneal stroma, oral mucosa, and intestine of a neonate male.

3. The construction of cultured tissue according to claim 1, further characterized in that said cells are dermal fibroblasts.

4. The construction of cultured tissue according to claim 1, further characterized in that said cells are cultured from dermal papilla of hair follicles.

5. The construction of cultured tissue according to claim 1, further characterized in that said layer has cells cultured from the hair follicle dermal papilla located on said layer.

6. The cultured tissue construction according to claim 1, further characterized in that said cultured cells are cultured in chemically defined medium.

7. The cultured tissue construction according to claim 1, further characterized in that said cultured cells are derived from human tissue and cultured in medium that does not contain non-human components.

8. A cultured tissue construct comprising cultured dermal fibroblast cells cultured under conditions to produce an extracellular matrix layer which is synthesized and assembled by cultured fibroblast cells, with cultured fibroblast cells contained within the layer of synthesized extracellular matrix, wherein the extracellular matrix comprises: (i) fibrillar collagen type I and type III showing an organization of fibrils in packing and fibrillar bundles that exhibit a banding pattern of 67 nm in four planes; (ii) decorin, (iii) fibronectin (iv) tenascin; and, (v) glycosaminoglycans; and where said Extracellular matrix is produced by cultured fibroblast cells in the absence of exogenous components of the matrix or of synthetic limbs during culture conditions.

9. A cultured tissue construct having at least two layers, comprising: (a) a first layer of cultured fibroblast cells, cultured under conditions to produce an extracellular matrix layer that is synthesized and assembled by the fibroblast cells cultured, with cultured fibroblast cells contained within the synthesized extracellular matrix layer, wherein the extracellular matrix comprises: (i) fibrillar collagen showing a packaged organization of fibrils and bundles of fibrils exhibiting a 67 nm banding pattern in four planes; (I) decorina; and (iii) glycosaminoglycans; and wherein said extracellular matrix is produced by cultured fibroblast cells in the absence of exogenous matrix or synthetic member components during culture conditions; and, (b) a second layer of cells comprising epithelial cells disposed on the first layer.

10. The construction of tissue bilayer cultured according to claim 9, further characterized in that the epithelial cells are selected from the group consisting of keratinocytes, corneal epithelial cells, epithelial cells from oral mucosa, esophageal epithelial cells, and uroepithelial cells.

11. The construction of tissue cultured in bilayer according to claim 9, further characterized in that the cells of fibroblasts contained within said first layer derive from tissue selected from the group consisting of the foreskin, dermis, tendon, lung, cartilage, urethra, corneal stroma, oral mucosa, and neonate gut.

12. The construction of tissue cultured in bilayer according to claim 9, further characterized in that said fibroblast cells contained within said first layer are dermal fibroblasts.

13. The construction of tissue cultured in bilayer according to claim 12, further characterized in that said fibroblast cells contained within said layer are from dermal papilla of hair follicles.

14. The construction of tissue bilayer cultivated according to claim 9, further characterized in that said first layer has cells cultured from dermal papilla hair follicles located on said first layer.

15. The construction of tissue bilayer cultivated according to claim 9, further characterized in that it comprises a third layer of cells arranged on the second layer of epithelial cells.

16. A cultured skin construction having at least two layers, comprising: a) a first layer of cultured dermal fibroblast cells, cultured under conditions to produce a layer of extracellular matrix that is synthesized and assembled by cultured fibroblast cells, with cultured fibroblast cells contained within the synthesized extracellular matrix layer, wherein the extracellular matrix comprises: (i) type I and type III fibrillar collagen showing a organization of bundled fibrils and fibrillar bundles that exhibit a 67 nm banding pattern in four planes; (ii) decorin, (iii) fibronectin (iv) tenascin; and, (v) glycosaminoglycans; and wherein said extracellular matrix is produced by cultured fibroblast cells in the absence of exogenous components of the matrix or of synthetic members during culture conditions and b) a second layer of keratinocyte cells disposed on the first layer to form a cell layer epidermal, where the epidermal cell layer is multilayer, stratified, differentiated and exhibits a basal lamina, superbasal lamina, a granular lamina and a stratum corneum; and wherein the skin construction constructed in bilayer has a basement membrane present at the junction of the first and second layers.

17. The construction according to any of claims 1, 8, 9 and 16, further characterized in that the cultured fibroblast cells are genetically modified to produce components of the extracellular matrix.

18. The construction according to claim 17, further characterized in that the cultured fibroblast cells are genetically modified to produce a growth factor, hormone, peptide, or protein.

19. A method for producing a cultured tissue construct, comprising, a) seeding fibroblast cells capable of synthesizing an extracellular matrix on a porous membrane in a culture vessel in a first cell culture medium; b) culturing the fibroblast cells from step (a) in the first cell culture medium from about 80% to about 100% confluence on the porous membrane; c) stimulating the fibroblast cells to synthesize, secrete and organize components of the extracellular matrix under culture conditions in a second culture medium; and d) continuing to culture the fibroblast cells until the cells from the synthesized extracellular matrix layers of at least about 30 microns thick, with the cultured fibroblast cells contained within the synthesized extracellular matrix layer, where the extracellular matrix comprises: i) collagen fibers showing a packed organization of fibrillar fibers and bundles exhibiting a 67 nm banding pattern in four planes; ii) decorin; and iii) glycosaminoglycans; and wherein said cellular matrix is produced by cultured fibroblast cells in the absence of exogenous matrix or synthetic member components during culture conditions.

20. The method according to claim 19, further characterized because either the first culture medium, or the second culture medium, or both culture media first and second culture are chemically defined.

21. The method according to claim 19, further characterized in that the first and second culture medium do not contain non-human components.

22. The method according to claim 19, further characterized in that in step (a) the fibroblast cells are seeded at a density between about 1 x 10 5 cells / cm 2 to about 6.6 x 10 5 cells / cm 2.

23. The method according to claim 19, further characterized in that the fibroblast cells are derived from tissue selected from the group consisting of the foreskin of the neonate, umbilical cord, dermis, tendon, lung, urethra, stroma corneal, oral mucosa, and intestine.

24. A method for producing a bilayer cultured tissue construct, comprising: a) seeding fibroblast cells capable of synthesizing an extracellular matrix on a porous membrane in a culture vessel in a first cell culture medium; b) culturing the fibroblast cells from step (a) in the first cell culture medium at about 80% to about 100% confluence on the porous membrane; c) stimulating the fibroblast cells to synthesize, secrete and organize components of the extracellular matrix under culture conditions in a second culture medium; and d) continue to cultivate fibroblast cells until the cells from the synthesized extracellular matrix layers of at least about 30 microns thick, with the cultured fibroblast cells contained within the synthesized extracellular matrix layer, wherein the extracellular matrix comprises: collagen showing a packed organization of fibrillar fibers and bundles exhibiting a 67 nm banding pattern in four planes; ii) decorin; and iii) glycosaminoglycans; and wherein said cellular matrix is produced by cultured fibroblast cells in the absence of exogenous matrix or synthetic member components during culture conditions; e) seeding epithelial cells on the surface of the synthesized extracellular matrix of step (d), and, f) stimulating the epithelial cells of step (e) under culture conditions to form a bilayer tissue construct of an extracellular matrix, with the cultured fibroblast cells contained within the synthesized extracellular matrix layer, and a second layer of epithelial cells.

25. The method according to claim 24, further characterized in that said fibroblast cells capable of synthesizing an extracellular matrix are derived from tissues selected from the group consisting of foreskin, dermis, tendon, lung, cartilage, urethra, stroma corneal, oral mucosa, and neonatal male intestine.

26. The method according to claim 24, further characterized in that the epithelial cells are selected from the group consisting of keratinocytes, corneal epithelial cells, cells epithelial from oral mucosa, epithelial cells of the esophagus and uroepithelial cells.

27. A method for producing a bilayer cultured skin construction comprising a dermal layer and an epidermal layer disposed over the first layer in the absence of a structural support or exogenous matrix components, wherein the method comprises the steps of: a ) seeding fibroblast cells capable of synthesizing an extracellular matrix on a porous membrane in a culture vessel in a first cell culture medium; b) culturing the fibroblast cells from step (a) in the first cell culture medium from about 80% to about 100% confluence on the porous membrane; c) stimulating the fibroblast cells of step (a) to synthesize, secrete and organize components of the extracellular matrix under culture conditions in a second culture medium; and d) continuing to culture the fibroblast cells until the cells from the synthesized extracellular matrix layers of at least about 30 microns thick, with the cultured fibroblast cells contained within the synthesized extracellular matrix layer, wherein the The extracellular matrix comprises: i) collagen type I and III that show a packaging organization of fibers and fibrillar bundles that exhibit a banding pattern of 67 nm in four planes; ii) decorin; and iii) glycosaminoglycans; and wherein said cellular matrix is produced by cultured fibroblast cells in the absence of exogenous matrix or synthetic member components during culture conditions; e) planting keratinocytes in the upper surface of the extracellular matrix synthesized from step (d); and f) culturing the keratinocytes under culture conditions to form an epidermal layer, wherein the epidermal cell layer is a multilayer, stratified and differentiated layer of keratinocytes exhibiting a basal layer, a suprabasal layer, a granular layer and a stratum corneum; and wherein the skin construction bilayer cultivated has a base membrane present at the junction of the first and second layers.

28. A method for transplanting or implanting a tissue culture construct to a patient that comprises transplanting or implanting a cultured tissue construct as claimed in any of claims 1, 8, 9 or 16 to a patient in need the treatment of it.

29. A method for producing a cultured tissue construct, comprising, a) seeding fibroblast cells capable of synthesizing an extracellular matrix on a porous membrane in a culture vessel in a first cell culture medium at approximately 80% up to 100% confluence; b) stimulating the fibroblast cells to synthesize, secrete and organize components of the extracellular matrix under culture conditions in a second culture medium; and c) continue to culture the fibroblast cells until the cells from the extracellular matrix layers synthesized at least about 30 microns thick, with the cultured fibroblast cells contained within the synthesized extracellular matrix layer, where the extracellular matrix it comprises: i) collagen fibers showing a packed organization of fibers and fibrillar bundles exhibiting a 67 nm banding pattern in four planes; ii) tenascin; and iii) glycosaminoglycans; and wherein said cellular matrix is produced by cultured fibroblast cells in the absence of exogenous matrix or synthetic member components during culture conditions.

30. The construction according to any of claims 1-18, further characterized because the construction is cohesive to have physical unitary integrity and handling properties similar to the fabric.