BIOMATERIAL WITH A POROUS RESORBABLE MATRIX AND ASSOCIATED MANUFACTURING PROCESS

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of biomaterials comprising collagen and their manufacture. It finds particularly advantageous application in the field of dermo-epidermal substitutes. STATE OF THE ART

[0002] Chronic wounds represent a major public health problem affecting more than 2 million people in mainland France alone. This includes physical burns, for example caused by ultraviolet radiation, and chemical burns, for example caused by detergents or acids, ulcers, including pressure ulcers, venous ulcers, and diabetic ulcers. These chronic wounds generally require the replacement of damaged tissue, for example by a skin graft.

[0003] For the specific case of skin grafts, there are different types of grafts classified according to the thickness of skin taken from the person: The thin skin graft is harvested by an electric dermatome with a thickness of 0.2 to 0.3 mm from a donor area of ​​the patient's body, generally an inner thigh or scalp. This harvest is then transferred to a recipient area (using suture or staple) of the patient for an autologous graft. This technique can be completed with the addition of an artificial dermal substitute such as integra ®< . The main disadvantage lies in the after-effects induced by the harvest and the limited surface area of ​​the donor areas; the total skin graft does not require harvesting from the dermatome as for a thin skin graft. A fusiform incision is made in a fold of skin, for example the supraclavicular fold. The skin is then defatted (to allow better graft take), then sutured to the recipient area. This technique is considered particularly suitable for the face.

[0004] However, the removal of skin tissue for transplants remains limited, particularly in terms of the available surface area of ​​donor areas and the pain induced in the person receiving the transplant.

[0005] Furthermore, since 2013, animal testing of the efficacy and safety of dermo-cosmetic products has been prohibited. To test these substances, the pharmaceutical and cosmetic industries must therefore use tissue substitutes that closely mimic the properties and reactions of native tissue.

[0006] There is therefore a need to develop a tissue substitute as close as possible to native tissue, for example for the treatment of chronic wounds or for pharmacological or cosmetic applications.

[0007] Document WO 88 / 10123 A1 discloses a biomaterial comprising a compound consisting of collagen, acetylated chitosan and glycosaminoglycans. This biomaterial can constitute the dermal layer of artificial skin, and be associated with a biodegradable pseudo-epidermis, for example a chitosan film. However, this biomaterial remains perfectible to mimic native skin.

[0008] US 5,945,101 A describes a material comprising a cross-linked collagen sponge into which fibroblasts and keratinocytes have been seeded and the method of manufacturing a skin model comprising a collagen matrix comprising fibroblasts and stratified layers of differentiated epidermal cells.

[0009] An object of the present invention is therefore to prepare a biomaterial which is closer to a native tissue.

[0010] Other objects, features, and advantages of the present invention will become apparent from the following description and accompanying drawings. It is understood that other advantages may be incorporated. SUMMARY

[0011] To achieve this objective, a biomaterial (unclaimed) is provided comprising a resorbable porous matrix formed from a material comprising collagen, and having an interior volume and an exterior surface.

[0012] Advantageously, the biomaterial comprises at least one type of living biological cells of a tissue, arranged in the interior volume and, alternatively or in addition, on the surface of the porous matrix.

[0013] Thus, the biomaterial comprises a matrix giving it a structure allowing its manipulation, and biological cells of a tissue seeded in the porous matrix. The biomaterial therefore forms a tissue substitute close to a native tissue, particularly in terms of biological structure present in the tissue, and physiological functions.

[0014] Furthermore, since the matrix is ​​resorbable, the porous matrix can be degraded to facilitate the integration of a portion of the biomaterial into the organism, and more particularly of a tissue formed by biological cells, for example following a graft of the biomaterial. The biomaterial therefore forms a tissue substitute that can be grafted onto an organism and then act directly to perform its tissue function, without further requiring the removal of the biomaterial, and more particularly of the porous matrix.

[0015] According to one example, the at least one type of living biological cells of a tissue are arranged in the interior volume of the porous matrix.

[0016] A first aspect of the invention relates to a method of manufacturing a biomaterial according to claim 1 comprising: providing a resorbable porous matrix formed from a material comprising collagen, then seeding the porous matrix with at least one type of living biological cells of a tissue, then cell growth of the at least one type of biological cells.

[0017] The method thus makes it possible to obtain the biomaterial according to the first aspect of the invention. By seeding and growth, it is also possible to form a biomaterial of variable size and shape depending on the dimensions of the porous matrix. A biomaterial is thus obtained forming a tissue substitute close to a native tissue.

[0018] A second aspect relates to the use of a biomaterial manufactured according to the method of claims 1-8, the biomaterial comprising: a resorbable porous matrix formed from a material comprising collagen, and having an interior volume and a surface, and at least one type of living biological cells of a tissue, arranged in the interior volume and, alternatively or in addition, on the surface of the porous matrix, for in vitro application of at least one substance chosen from a pharmaceutical substance and a cosmetic substance. BRIEF DESCRIPTION OF THE FIGURES

[0019] The aims, objects, as well as the characteristics and advantages of the invention will emerge more clearly from the detailed description of an embodiment thereof which is illustrated by the following accompanying drawings in which: There figure 1represents a cross-sectional view of the porous matrix according to one embodiment of the invention. The figure 2 represents a first seeding of the porous matrix illustrated in figure 1 by a first type of biological cells, according to one embodiment of the invention. The figure 3 represents the cell growth of biological cells following seeding illustrated in figure 2 , according to one embodiment of the invention. The figure 4 represents a second seeding of the porous matrix by a second type of biological cells and a subsequent first cell undergrowth of the second type of biological cells, following the cell growth illustrated in figure 3 , according to one embodiment of the invention. The Figure 5A represents a maturation of the second type of biological cells following the first cellular undergrowth illustrated in figure 4, according to one embodiment of the invention. The Figure 5B represents the biomaterial, according to one embodiment of the invention. The figure 6 represents the steps of the process for manufacturing the biomaterial, according to one embodiment of the invention. The figure 7 represents a chronology of the steps of the process for manufacturing the biomaterial, according to one embodiment of the invention. The figure 8 represents a cross-sectional fluorescence microscopy immunohistological image of the biomaterial, in which the nuclei of biological cells and cytokeratin 14 are labeled. figure 9 represents a cross-sectional fluorescence microscopy immunohistological image of the biomaterial, in which the nuclei of biological cells, filaggrin and type 1 collagen are labeled. figure 10represents a cross-sectional fluorescence microscopy immunohistological image of the biomaterial, in which the nuclei of biological cells, fibrillin 1 and elastin are labeled.

[0020] The drawings are given as examples and are not limiting of the invention. They constitute schematic representations of principle intended to facilitate the understanding of the invention and are not necessarily on the scale of practical applications. In particular, the relative dimensions of the biological cells to each other and to the porous matrix are not representative of reality. DETAILED DESCRIPTION

[0021] Before commencing a detailed review of embodiments of the invention, optional features are set out below which may optionally be used in combination or alternatively for each aspect of the invention: the at least one first type of biological cells can be arranged at least in the interior volume of the porous matrix, or even only in the interior volume of the matrix, the at least one first type of biological cells can be arranged at least on an exterior surface of the porous matrix, or even only on a surface of the matrix, and preferably on an upper face of the porous matrix, the porous matrix can be biocompatible, the material of the porous matrix can comprise at least 90%, preferably at least 95%, and more preferably at least 99% by mass of collagen, relative to the dry mass of the porous matrix, the material of the porous matrix can be free of chitosan, the porous matrix is ​​a hemostatic sponge. Thus, the porous matrix can be commercially available and at a reduced cost, the porous matrix can be a product following Good Manufacturing Practices (GMP).The porous matrix may be clinically usable in humans or animals. The biomaterial may thus be clinically usable in humans or animals, the biological cells may comprise cells selected from connective tissue cells, epithelial cells and endothelial cells. According to one example, the connective tissue cells are dermal cells called fibroblasts and / or cells of subcutaneous adipose tissue called adipose tissue stem cells. According to one example, the epithelial cells are epidermis cells including keratinocytes and / or melanocytes. According to one example, the endothelial cells are vascular cells.Preferably, the biological cells are human or animal cells, the at least one type of biological cells can be arranged at least in the interior volume of the porous matrix, or even only in the interior volume of the matrix, the biomaterial can further comprise an extracellular matrix at least in said interior volume, the extracellular matrix being secreted by the at least one first type of biological cells.The biological cells and their extracellular matrix make it possible to form a tissue in the porous matrix, and to further approximate the properties of a native tissue, a first type of biological cells can be arranged in the interior volume of the porous matrix, and the biomaterial can comprise at least a second type of biological cells arranged on the surface, for example the upper face, of the porous matrix, the second type of biological cells can further be distinct from the first type of biological cells. Thus, the biomaterial can further comprise a cell layer on the surface of the porous matrix forming an external boundary of a tissue, and to further approximate a native tissue. According to one example, the second type of biological cells is a type of epithelial cells. Thus, the biomaterial has barrier properties.The biomaterial therefore allows the replacement of the barrier function, for example on a wound that does not heal spontaneously, such as a chronic wound, the first type of biological cells may comprise dermal cells, for example fibroblasts, and the second type of biological cells may comprise epithelial cells, for example keratinocytes, or even further melanocytes. According to one example, the first type of biological cells may further comprise at least one type of biological cells from among endothelial cells, immune cells and adipose tissue stem cells. According to one example, the at least one second type of biological cells may further comprise melanocytes, the biological cells are chosen from among cells derived from standard cell line cultures and preferably primary cells isolated from a biological sample of an organism.In one example, the biomaterial may comprise autologous or allogeneic cells. In one example, the biological cells are isolated from a skin sample of a person.

[0022] Characteristics of the method according to the invention are set out below: the method comprises hydrating the porous matrix so as to balance the pH and osmolarity of the porous matrix, prior to seeding. According to one example, the hydration is carried out with at least one buffered solution, then with a culture medium, the hydration of the porous matrix can be carried out by a hydration solution having a pH of between 6.8 and 7.5, and / or an osmolarity of between 270 and 360 mmol / kg, the method further comprises drying the porous matrix between the hydration of the porous matrix and the seeding of the porous matrix. According to one example, the drying of the porous matrix is ​​carried out by suction, for example of the hydration solution, the seeding of the porous matrix can be carried out by depositing a suspension of the at least one type of biological cells on the porous matrix. According to one example,of the at least one type of biological cells can be seeded at a cell density of between 0.5.10 6< cells / cm 2< and 1.10 6< cells / cm 2< , the cm 2< being given with reference to the surface of the porous matrix, the method may further comprise, after a first seeding with a first type of biological cells, or even after the first seeding and at least part of a cell growth of the first type of biological cells, a second seeding of the porous matrix with at least a second type of biological cells, distinct from a first type of seeded biological cells. The method may further comprise then a cell growth of the at least one second type of biological cells, the method may further comprise drying the porous matrix prior to the second seeding of the porous matrix, the drying, preferably each drying,of the porous matrix may comprise suctioning the solution or medium present in the porous matrix, and placing the porous matrix on an absorbent support, preferably the porous matrix is ​​placed on the absorbent support after suctioning, the absorbent support may comprise a sterile pad, the absorbent support may comprise sterile blotting paper, the sterile pad is preferably placed on sterile blotting paper, the second seeding may be carried out by depositing a suspension of at least a second type of biological cells on the porous matrix, the biological cells may comprise cells selected from connective tissue cells and epithelial cells, when the biological cells are connective tissue cells, the cell growth may comprise immersing the porous matrix in a culture medium. Preferably, the porous matrix is ​​fully submerged. In addition,the porous matrix may be submerged for the majority of cell growth, when the biological cells are epithelial cells, the cell growth may include a first undergrowth during which the porous matrix is ​​submerged in a culture medium, until a confluence of the biological cells at the surface of the porous matrix, the cell growth may include, following the first undergrowth, a second undergrowth, which may equivalently be referred to as maturation, during which the porous matrix is ​​placed at an interface between air and a culture medium. Maturation allows the differentiation of the epithelial cells to form a more complex, or even complete, epidermis and to approach a native tissue,

[0023] It is specified that in the context of the present invention, the term “resorbable matrix” designates the fact that the matrix is ​​biodegradable from cells, for example from an organism.

[0024] By a "biocompatible" matrix, we mean that the matrix is ​​configured not to interfere with, or even degrade, a biological environment in contact with which the matrix is ​​placed. More particularly, the matrix is ​​tolerated by an organism in or on which the matrix is ​​in contact, for example by grafting. More particularly, the organism exhibits a low level of inflammatory reaction, or even an absence of inflammatory reaction induced by contact with the matrix, for example by grafting.

[0025] A parameter "substantially equal / greater / less than" a given value means that this parameter is equal / greater / less than the given value, to within plus or minus 10%, or even plus or minus 5%, of this value.

[0026] By matrix porosity we mean the proportion of void or gas in the matrix.

[0027] In the remainder of the description, terms such as "transverse", "upper" and "lower" will be used. These terms must be interpreted relatively in relation to the normal position of use of the biomaterial. For example, in the case where the porous matrix is ​​intended to be deposited on a support, the lower face corresponds to the face intended to be turned towards the support. According to this example, the upper face corresponds to the face intended to be turned away from the support.

[0028] The process 2 for manufacturing the biomaterial 1 is now described with reference to the figures 1 to 6 . As an example, the process is illustrated by the figure 6 , where optional steps are indicated in dotted lines.

[0029] The method 2 comprises providing 20 a porous matrix 20. The porous matrix 20 has pores delimiting an interior volume 100, as for example illustrated in figure 1 . Equivalently, the interior volume 100 represents the volume of void or gas inside the matrix 20, and therefore the porosity of the matrix. The porous matrix 20 has an exterior surface 101, comprising an upper face 1010, and a lower face 1012. The exterior surface 100 of the porous matrix 10 may further comprise at least one lateral face 1011, or even a plurality of lateral faces 1011, as for example illustrated in figure 1 .

[0030] According to one example, the porosity of the porous matrix 10 is homogeneous, that is to say that the porosity per unit volume is substantially identical in any portion of the same determined size of the porous matrix 10. Thus, it is excluded that the interior volume is arranged in the porous matrix in the form of a cavity. The dimension of the pores of the porous matrix 10 is preferably greater than the dimension of biological cells 11, and in particular of eukaryotic biological cells. Thus, the pores of the porous matrix 10 allow the migration of the biological cells 11 into the interior volume 100. Additional characteristics of the porous matrix 10 are described later.

[0031] The porous matrix 10 can be prepared for seeding 23 with biological cells 11. For this, the method 2 comprises a hydration 21 configured to balance the pH and osmolarity of the porous matrix around values ​​corresponding to the native environment of the cells. This makes it possible to obtain a homogeneous osmolarity in the porous matrix 10, and thus a better migration and colonization of the seeded cells 11. This is particularly advantageous when the porous matrix 10 is thick, for example when the porous matrix 10 is a sponge. For example, for the manufacture of a dermo-epidermal substitute, this pH can be between 6.8 and 7.5, and the osmolarity can be between 270 and 360 mmol / kg. For comparison, in vivothe cellular osmolarity is substantially equal to 290 mmol / kg and the cellular pH is substantially equal to 7.4. During hydration 21, the porous matrix 10 may be immersed in a buffered solution for several hours, or even several days. The porous matrix 10 may further be immersed in a culture medium 3 configured to correspond to the native environment of the cells. For example, for the manufacture of a dermo-epidermal substitute, the culture medium 3 may be a medium equivalent to the medium of the dermis.

[0032] Following its hydration 21, the method 2 comprises a drying 22 of the porous matrix 2 to promote the adhesion of the biological cells 11 to the porous matrix during their seeding 23, and therefore their subsequent cell growth 24. For example, the drying of the porous matrix 10 can be carried out by suction of the hydration solution.

[0033] The method 2 then comprises a first seeding 23 of the porous matrix with living biological cells 11 of a tissue, and more particularly with at least a first type 110 of biological cells 11. Thus, biological cells 11 are added to the porous matrix 10 to form a biomaterial 11 approaching a native tissue, compared to a biomaterial formed solely from an inert material.

[0034] The biological cells 11 may be eukaryotic, human or animal cells. In the following, reference is made to the non-limiting example where the biological cells 11 are human cells.

[0035] The biological cells 11 may be selected from cells derived from standard cell line cultures and cells isolated from a biological sample of an organism. A biomaterial 1 comprising cells 11 derived from standard cell line cultures makes it possible to form a high-performance tissue substitute, while remaining low in cost, which is particularly advantageous for pharmaceutical or cosmetic applications. The standard cell line cells may be commercially available from vendors known to those skilled in the art.

[0036] According to one example, the biological cells may be autologous or allogeneic. The allogeneic biological cells may, for example, be isolated from surgical residues, in particular independently of a skin graft. A biomaterial 1 comprising autologous cells 11 can be personalized for a given person, which is particularly advantageous for medical applications such as, for example, a graft of the biomaterial 1. In the case of autologous cells, the biological cells may be isolated from a biological sample of a person, for example following one or more biopsies and in particular a skin biopsy.

[0037] The biological cells 11 may comprise cells selected from connective tissue cells and epithelial cells. For example, connective tissue includes the dermis, subcutaneous adipose tissue, corneal stroma, bones, and epithelial tissue includes the epidermis and corneal epithelium. According to one example, the connective tissue cells are dermal cells called fibroblasts. According to one example, the epithelial cells are epidermal cells called keratinocytes. Thus, and as described in more detail below, the biomaterial 1 may form a dermal, or even dermo-epidermal, substitute for applications in vitro Or in vivo.

[0038] In the following, reference is made to the non-limiting example in which a first type of cell comprises, or is made up of, dermis cells, and more particularly fibroblasts, and a second type of cell comprises, or is made up of, epidermis cells, and more particularly keratinocytes. Note that for the manufacture of a dermal, or even dermo-epidermal, substitute, it may be provided that the first type of biological cells comprises, in addition to the dermis cells, at least one type of biological cell from among: endothelial cells, to obtain a vascularized dermis, immune cells, to obtain an immunocompetent dermis, adipose tissue stem cells, to obtain a subcutaneous adipose structure.

[0039] In one example, the second type of biological cells may also include melanocytes, to obtain a pigmented epidermis. The biomaterial is thus made more complex and even closer to native skin, particularly in terms of the diversity of biological cells present in the tissue, and physiological functions.

[0040] The fibroblasts 1100 can be seeded 23 by depositing 230 a cell suspension, for example drop by drop on the surface 101 of the porous matrix, and in particular on its upper face 1010 according to the example illustrated in figure 2. According to one example, the fibroblasts 1100 may be seeded at a cell density of between 500,000 and 1,000,000 cells / cm 2< , or even substantially 800,000 cells / cm 2< . Note that depending on the type of biological cells seeded, the cell density may be adapted. Thus, the fibroblasts 1100 may penetrate into the interior volume 100 of the porous matrix 10. To further facilitate the adhesion of the seeded fibroblasts 1100 23, the method may further comprise a step of adhesion of the cells 11, during which the porous matrix 10 is left in the open air for a determined time, for example several minutes, or even an hour, or even preferably in an atmosphere containing 5% CO 2 .

[0041] The method 2 then comprises a cell growth 24 of the seeded fibroblasts 1100 23. The fibroblasts 1100 can multiply by cell division at least in the interior volume 100 of the porous matrix 10 to colonize at least part of the interior volume 100, or even its entirety, as illustrated by the figure 3 . During cell growth 24, the fibroblasts can further synthesize 241, or equivalently secrete, an extracellular matrix 112 at least in the interior volume 100. The biological cells 11, and in particular the fibroblasts 1100, and their extracellular matrix 112 make it possible to form a tissue in the porous matrix 10, and to further approach the properties of a native tissue, and in particular of native skin. The fibroblasts 1100 in the interior volume 100 of the porous matrix 10, or even the fibroblasts 1100 and their extracellular matrix 112, thus form a dermal substitute equivalent to the native dermis.

[0042] To enable cell growth 24 of the fibroblasts 1100, the porous matrix 10 with the seeded fibroblasts 1100 23 may be immersed at least partially, and preferably completely, in a culture medium 3 configured to mimic the native environment of the fibroblasts 1100, as illustrated in figure 3 . The matrix 10 being porous, the culture medium 3 is thus distributed homogeneously in the porous matrix 10 to facilitate cell growth 24. For example, the culture medium is a medium equivalent to the medium of the dermis, that is to say whose properties, or even composition, is substantially equivalent to the medium of the dermis. During cell growth 24, the culture medium 3 can also be renewed 242, typically at a frequency of several times per week. Following a first immersion in a culture medium 3 free of ascorbic acid and epidermal growth factor (abbreviated EGF, from the English Epidermal Growth Factor ), subsequent renewals 242 of medium can be carried out with a culture medium 3 comprising ascorbic acid and EGF. According to one example, the first renewal 242 of media takes place 24 to 48 hours after seeding 23 of the fibroblasts 1100.

[0043] Note that depending on the type of other cell types that can be seeded with the 1100 fibroblasts, the medium can be configured to adapt and mimic the environment more favorable to the cell types seeded.

[0044] The method 2 may further comprise a second seeding 26, for example carried out after one or more weeks of cell growth 24 of the fibroblasts 1100. Prior to the second seeding 26, the porous matrix 10 may be dried 25 in a manner similar to the drying 22 described previously. Then, a second type 111 of biological cells 11 may be seeded 26, including in particular keratinocytes 1110.

[0045] The drying operations 22, 25 prior to seeding make it possible to promote the cellular adhesion of the cells 11 seeded on the porous matrix, and therefore their subsequent cellular growth, as indicated previously for the drying 22. In addition, the culture medium can be renewed or changed. The drying of the porous matrix 10 may comprise suction of the solution or cellular medium present in the porous matrix 10. According to one example, during drying, the porous matrix 10 may also be deposited on an absorbent support. The porous matrix 10 may for example be deposited on a sterile compress, preferably deposited on sterile blotting paper. Preferably, the porous matrix 10 is deposited on the absorbent support after suction. The suction makes it possible to remove the solution or medium present in the porous matrix 10 in a controlled and precise manner.The deposition on the absorbent support makes it possible to finalize the drying of the porous matrix 10 and thus to further improve the cellular adhesion of the cells 11 seeded on the porous matrix 10.

[0046] The keratinocytes 1110 can be seeded 26 by the deposition 260 of a cell suspension, for example drop by drop on the surface 101 of the porous matrix 10, and in particular on its upper face 1010 according to the example illustrated in figure 4. Preferably, the deposition of the cell suspension on the lateral faces 1011 of the porous matrix 10 is avoided to prevent the keratinocytes 1110 from spreading on these faces. The keratinocytes 1110 can be seeded at a cell density of between 500,000 and 1,000,000 cells / cm 2< , or even substantially 800,000 cells / cm 2< . Note again that depending on the type of biological cells seeded, the cell density can be adapted. The seeded keratinocytes 1110 26 remain on the outer surface 101 of the porous matrix 10 thanks to the presence of extracellular matrix secreted by the fibroblasts and by the presence of the fibroblasts themselves.To further facilitate the adhesion of the seeded keratinocytes 1110 26, the method may further comprise a step of adhesion of the cells 11, during which the porous matrix 10 is left for a determined time, for example several minutes, or even an hour, and preferably in an atmosphere containing 5% CO 2 and at 37°C.

[0047] The method 2 then comprises a cell growth 27 of the seeded keratinocytes 1110 26. Thus, the keratinocytes 1110 multiply by cell division on the outer surface 101 of the porous matrix 10 until the confluence 2702 of the keratinocytes 1110, as illustrated by the figure 4 .

[0048] The keratinocytes 1110 on the surface 101 of the porous matrix 10 thus form an epidermal substitute equivalent to the native epidermis. It is understood that with the fibroblasts 1100, or even with their extracellular matrix 112, the biomaterial 1 can form a dermo-epidermal substitute equivalent to the native skin.

[0049] For this, the cell growth 27 of the keratinocytes 1110 may comprise a first sub-growth 270 during which the porous matrix 10 with the seeded cells 23, 26 is immersed in a culture medium 3 configured to mimic the native environment of the keratinocytes 1110, for example a medium called MC2, adapted from the “Green” medium as described later. The addition of the culture medium 3 is preferably carried out without the incident medium flow 3 directly touching the surface 101 of the porous matrix 10, so as not to disturb the adhered keratinocytes 1110, and until covering the porous matrix and the cells, including the keratinocytes 1110. The matrix 10 being porous, the culture medium 3 is thus distributed homogeneously in the porous matrix 10 to facilitate the cell growth 27.Furthermore, during cell growth 26, the culture medium 3 can further be renewed 2701, typically at a frequency of several times per week.

[0050] Following a first immersion in a culture medium 3 free of ascorbic acid and EGF, subsequent renewals 2701 of medium may be carried out with a culture medium 3 comprising ascorbic acid and EGF. According to one example, the first renewal 2701 of media takes place 24 to 48 hours after seeding 26 of the keratinocytes 1110.

[0051] Following the first undergrowth, the cellular growth 27 of the keratinocytes 1110 may include a maturation 271 during which the porous matrix 10 is placed at an interface 5 between air 4 and a culture medium 3. This configuration makes it possible to induce a differentiation 2710 of the keratinocytes 1110' to form a complex, or even complete, epidermis, characterized by multi-stratification and differentiation as illustrated in Figure 5A. In addition, during and following maturation, it is possible to observe the expression of specific proteins or lipids linked to the differentiation of keratinocytes 1110, such as cytokeratin 1 or 10, loricin, involucrin or filaggrin, or to their proliferative state, such as cytokeratin 5 or 14, ki67. Biomaterial 1 is still closer to native skin, particularly in terms of the diversity of biological cells present in the tissue, and physiological functions. More particularly, keratinocytes 1110' can differentiate to form at least part, or even all of the following layers of the epidermis, given from the inside out: stratum germinativum: composed of a single layer of keratinocytes at the junction with the dermis, these cells being capable of proliferating, stratum spinosum: composed of 5 to 6 layers of polygonal keratinocytes, stratum granulosum: composed of 2 to 4 layers of flattened and spindle-shaped keratinocytes, stratum corneum.

[0052] According to the example illustrated in Figure 5A , the porous matrix 10 with the seeded cells 23, 26 can be arranged on an absorbent support 7, placed on a grid 6, for example made of stainless steel and sterilizable by autoclave. A suitable culture medium 3, designated Air / liquid medium, can then be added so as to be flush with the absorbent support 7, for example blotting paper. Thus, the porous matrix is ​​kept hydrated while allowing the induction of the differentiation of the keratinocytes 1110. Preferably, the level of the culture medium 3 is adjusted so as to avoid the presence of bubbles under the grid 6, or even between the absorbent support 7 and the grid 6. In addition, during the maturation 271, the Air / liquid culture medium, preferably supplemented only with vitamin C, can also be renewed 2711, typically at a frequency of several times per week.

[0053] As an example, a chronology of the steps of manufacturing process 2 is given with reference to the figure 7 . In a first phase A, the porous matrix 10 is provided 20, then prepared by hydration 21 and drying 22. The fibroblasts 1100 can then be seeded 23 on a day designated D0. In a second phase B, the cell growth 24 of the fibroblasts can be carried out, after which the keratinocytes 1110 can be seeded 26, 14 days to 21 days after D0. In a third phase C, the first undergrowth 270 of the keratinocytes 1110 can be carried out. 21 to 28 days after D0, the porous matrix 10 can be placed at the interface 5 between the air and the culture medium 5 to allow cell differentiation 2710 in a phase D, for example for 7 to 21 days. Note that the indicated durations can vary according to the needs and the quantity of cells available.

[0054] From the characteristics of the method 2 described, it is understood that the biomaterial 1 can be obtained according to the first aspect of the invention, comprising in particular at least one type 110 of living biological cells 11 of a tissue, arranged in the interior volume 100. Alternatively or in addition, the type 110 of living biological cells 11 of a tissue can be arranged on the surface 101 of the porous matrix 10. In particular, the arrangement of the cells 11 in the porous matrix 10 can depend on the nature of the biological cells 11 seeded. In the case of the cornea, stromal cells of the cornea can be deposited in the porous matrix 10 to form the stroma, and keratocytes can be deposited on the surface of the porous matrix 10 to form the epithelium of the cornea. More particularly, biomaterial 1 can form a dermo-epidermal substitute for native skin, as illustrated in Figure 5B .

[0055] Biomaterial 1 can in particular be used for an application in vitro of pharmaceutical substances or cosmetic substances. During the development of the invention, it was demonstrated that the described method makes it possible to obtain the material 1 in a reproducible manner. Furthermore, the biomaterial 1 can be used as a model tissue, for example for basic and dermatological research.

[0056] Due to the characteristics described above, biomaterial 1 exhibits regenerative activity. Biomaterial 1 is thus particularly suitable for the treatment of lesions and / or for a graft, for example on the skin, on the eye or on a bone.

[0057] Since biomaterial 1 can form a reproducibly obtained dermo-epidermal substitute for native skin, biomaterial 1 is particularly suitable for testing new products in the pharmaceutical industry. For example, skin reconstructed from patient cells can represent an alternative model for modeling dermatological diseases, such as vitiligo, psoriasis, atopic dermatitis and Xeroderma Pigmentosum, and for the development of therapeutic molecules.

[0058] Biomaterial 1 forming a dermo-epidermal substitute for native skin, biomaterial 1 can in particular be used for: treat the after-effects of burns, treat an acute skin wound, treat a chronic skin wound, treat skin donor areas once a tissue sample has been taken, perform a skin graft, for example following a burn, an ulcer, skin trauma, treat any loss of dermal or dermo-epidermal substance.

[0059] Biomaterial 1, forming a dermo-epidermal substitute for native skin, is particularly suitable for the treatment of chronic wounds or burns because it minimizes, or even avoids, the addition of iatrogenic trauma, linked to the removal of skin from a donor area. In addition, since the biological cells 11 can be autologous, biomaterial 1 minimizes the risk of immune rejection of biomaterial 1.

[0060] To obtain the autologous cells 11, a skin biopsy can be performed on a healthy surface area that is reduced compared to the surface area required for treating a wound or burn. The cells 11 can be multiplied by cell proliferation in the manufacturing method 2 in order to obtain a sufficient quantity to allow cell growth 24 and / or 27. It is therefore possible to obtain a biomaterial 1 having a surface area adapted to the surface area of ​​the injured area. Thus, the biomaterial 1 and its manufacturing method 2 make it possible to minimize the risks and trauma associated with taking skin from a person, compared to existing solutions.

[0061] In addition, biomaterial 1 can be used as a skin substitute for the test in vitroof pharmaceutical substances, for example dermatological drugs, and dermo-cosmetic products. Tests of the efficacy and safety of dermatological drugs and dermo-cosmetic products can thus be carried out on a biomaterial close to native skin.

[0062] Characteristics of biomaterial 1 were described during the description of process 2. In the following, additional characteristics of biomaterial 1 are detailed.

[0063] The porous matrix 10 of the biomaterial 1 is now described in detail. The porous matrix 10 may be elastically deformable, for example under the pressure of a user's finger. Thus, the use of the biomaterial 1 is facilitated, for example for its application to an injured area. The barrier properties of the biomaterial 1 may, for example, be improved.

[0064] The porous matrix 20 is resorbable. The porous matrix 20 may further be biocompatible, in particular by being made of biocompatible materials. The porous matrix may more particularly be formed from a material comprising collagen. The collagen may be bovine or porcine type I collagen. The material of the porous matrix 10 may comprise at least 90%, preferably at least 95%, and more preferably at least 99% by mass of collagen, relative to the dry mass of the porous matrix 10. Thus, the integration into the body of a portion of the biomaterial 1, and more particularly of the tissue formed by the biological cells 11, is facilitated.

[0065] Furthermore, the material of the porous matrix 10 may be chitosan-free. Chitosan has a lower degradation rate in an organism than collagen. Since the biomaterial 1 comprises biological cells 11, it is not necessary to wait for a person's cells to colonize the biomaterial following its implantation, for example by grafting, as is the case with existing solutions. Therefore, it is possible to use a porous matrix based on a material having a faster degradation rate. It may even be advantageous to accelerate the degradation of the porous matrix to accelerate the integration of the tissue formed by the biological cells.

[0066] The porous matrix 10 is in the form of a sponge. Thus, the porous matrix can be shaped in two or even three dimensions as required. The porous matrix 10 is a hemostatic sponge. A hemostatic sponge can easily be shaped by cutting and has a hardness allowing it to retain the desired shape, while remaining sufficiently deformable to be able to be applied to a support, for example on a person's body. A hemostatic sponge is a synthetic sponge generally composed of materials of biological origin, and resorbable by the body. The porosity of these sponges allows for a high absorption of incoming fluid, typically up to 35 times their weight. A hemostatic sponge is a dry, flexible, porous material and is commercially available in various forms and trademarks. Thus, the porous matrix 10 can be commercially available and at a reduced cost.

[0067] A hemostatic sponge can absorb blood from the human and animal body, for example from a wound on which the biomaterial is deposited. A hemostatic sponge can absorb a weight of blood substantially greater than or equal to 10 times, preferably substantially greater than or equal to 20 times, and more preferably substantially greater than or equal to 30 times the weight of the sponge before absorption. The hemostatic sponge thus produces pressure on the bleeding site, inducing platelet aggregation and therefore activating the fibrin coagulation pathways to achieve hemostasis. However, one might expect that the aspirated blood would degrade the cells of the biomaterial.However, during the development of the invention, it was surprisingly found that the aspiration of blood into the biomaterial 1 did not harm the cells 11 seeded in the biomaterial 1, in particular when the biomaterial 1 comprises an extracellular matrix 112 secreted by the cells 11.

[0068] The porous matrix 10 provided 20 is preferably sterile, in order to avoid the proliferation of unwanted organisms in the biomaterial 1. According to one example, the porous matrix 10 is a product following Good Manufacturing Practices (abbreviated GMP, and which can be translated into English as Good Manufacturing Practices,(abbreviated GMP). Good manufacturing practices are regulatory texts established by States, the European Commission or the World Health Organization, and apply in particular to the manufacture of medicinal products for human or veterinary use. Similarly, the porous matrix 10 can be used clinically on an animal or a person, for example for its application to a wound. Thus, the process of placing the biomaterial on the market is simplified.

[0069] For example, the porous matrix 10 is a GMP hemostatic sponge selected from the following brand hemostatic sponges: Spongostan ™< , CuraSpon ®< , Gelfoam ®< and Surgifoam ®< .

[0070] As an example, immunohistological characterizations by fluorescence microscopy of biomaterial 1 (unclaimed) are illustrated by the figures 8 to 10. For these characterizations, antibodies specific to certain cellular structures, and coupled to a fluorescent probe are applied to the biomaterial 1.

[0071] There figure 8 represents a cross-sectional fluorescence microscopy image of the biomaterial, in which the nuclei 11' of the biological cells 11 and the cytokeratin 14 1111 are labeled. The arrangement of the fibroblasts in the porous matrix 10 and those of the keratinocytes above the upper face 1010 of the porous matrix 10 can be observed. The differentiated keratinocytes 1110' further have a cytokeratin cytoskeleton 1111.

[0072] There figure 9represents a cross-sectional fluorescence microscopy image of the biomaterial 1, in which the nuclei 11' of the biological cells 11, the filaggrin 1120 and the type I collagen 1121 are labeled. The secreted extracellular matrix 112 can be observed, by the labeling of the stated proteins, respectively below and above the upper face 1010 of the porous matrix 10.

[0073] There figure 10 represents a cross-sectional fluorescence microscopy image of the biomaterial 1, in which the nuclei 11 of the biological cells, fibrillin 1 1122 and elastin 1123 are labeled. The extracellular matrix 112 secreted by the fibroblasts 1100 can be observed in the porous matrix 10, by the labeling of the stated proteins. Example of the operating mode of the biomaterial manufacturing process

[0074] By way of example, an operating mode of the method 2 for manufacturing the biomaterial 1 is now described according to a particular embodiment.

[0075] The composition of the culture media 3 used is given in the following tables. The abbreviation "qsp" means quantity sufficient for the desired volume of solution. The percentages are given by volume relative to the desired volume of solution. MC1 culture medium

[0076] Table 1 Components Concentrations DMEM / glutamax medium qsp Calf serum 5% Fetal bovine serum 5% EGF 10 ng / mL Penicillin 100 units / mL Streptomycin 100 µg / mL Amphotericin B 1 µg / mL Vitamin C 82.2µg / mL MC2 culture medium adapted from GREEN medium

[0077] Table 2 Components Concentrations Alternatives for the clinic DMEM / glutamax medium qsp HAM F12 qsp Fetal bovine serum 10 % Hydrocortisone 0.4 µg / mL Insulin 5 µg / mL Umuline ® < 0.12 IU / ml Choleratoxin 10 -10< mol / L Isuprel ® < 0.4µg / ml Adenine 24.3 µg / mL Tri lodo Thyronine 2 nM EGF 10 ng / ml Amphotericin B 100 units / mL Penicillin 100 µg / mL Streptomycin 1 µg / mL Vitamin C 82.2 µg / mL

[0078] Note that for clinical use, the MC2 medium can be adapted by those skilled in the art to be compatible with clinical use. A / L culture medium

[0079] Table 3 Components Concentrations DMEM / glutamax medium qsp HAM F12 qsp Bovine serum albumin (BSA) 8 mg / mL Hydrocortisone 0.4 µg / mL Insulin 5 µg / mL Amphotericin B 1 µg / mL Penicillin 100 units / mL Streptomycin 100 µg / mL Vitamin C 82.2 µg / mL

[0080] The preparation of reagents for making culture media is now described: preparation of trio-iodo-L-thyronine (abbreviated T3 in the following): ∘ preparation of a solution A at 2×10 -4< M by weighing 13.6 mg of T3, then dissolve them in 1.5 ml of a sodium hydroxide NaOH solution at 0.02 mol / L, make up to 100 mL with sterile water, ∘ preparation of the stock solution of 2×10 -6< M by carrying out a dilution to 1 / 100 th< of the solution A, ∘ filter on a filter of dimension 0.22 µm, ∘ aliquot into portions of 550 µL and freeze at -20°C, ∘ use 500 µl of the solution at 2×10 -6< M for 500 mL of MC2 medium to obtain a final concentration 2×10 -9< M, preparation of adenine:∘ Dissolve 0.486g of adenine in 3mL of a 0.4 mol / L NaOH solution. Add 10 mL of sterile water then 10 mL of a 1 mol / L hydrochloric acid HCl solution. Mix and make up to 200 mL with sterile water, ∘ Filter through a 0.22µm porosity filter, ∘ Aliquot into 5 mL portions and freeze at -20°C ∘ Use 5 mL for 500 mL of medium to obtain a final concentration of 24.3 µg / mL, EGF preparation : ∘ prepare a 10 µg / mL solution by taking 200 µg of lyophilized EGF with sterile water in qsp 20 mL, ∘ filter on a filter with porosity 0.22 µm, ∘ aliquot into portions of 500 µL and freeze at -20 °C, ∘ use 500 µL for 500 mL of medium at the time of use of the culture medium, to obtain a final concentration of 10 ng / mL, Preparation of vitamin C:∘ Weigh 2.5g of vitamin C powder and gradually dissolve it in 60.8 mL of DMEM medium at 37°C to obtain a concentration of 41.1 mg / mL or equivalently 142 mmol / L, ∘ Filter through a 0.22 µm porosity filter, ∘ Aliquot into 0.5ml portions and freeze at -20°C ∘ Use 200 µL in 100mL of medium to obtain a final concentration of 82.2 µg / mL, BSA preparation: ∘ weigh 4 g of BSA and dissolve them in 20 mL of DMEM medium at 37°C, ∘ filter through a 0.22 µm porosity filter, ∘ use the 20 mL for 500 mL of medium, preparation of hydrocortisone: ∘ prepare a 5 mg / mL solution B by diluting 25 mg in 5 mL of 95% ethanol, o prepare a 200 µg / mL solution C by taking 0.4 mL of solution B in 9.6 mL (qsp 10 mL), o aliquot solution C into 1 mL portions, o use 1 mL for 500 mL of medium, to obtain a final concentration of 0.4 µg / mL, insulin preparation: ∘ resuspend 50 mg of insulin with 500 µL of a 0.1 mol / L HCL solution, o dilute the suspension obtained in sterile water qsp 10 mL, to obtain a concentration of 5 mg / mL, o filter through a 0.22 µm porosity filter, o aliquot into 500 µL portions, o use 500µL for 500mL of medium, to obtain a final concentration of 5 µg / mL, preparation of choleratoxin: ∘ Re-suspend 1 mg of choleratoxin in 12 mL of sterile water to obtain a concentration of 10.10 -7< mol / L, o Use 500 µL of the 10.10 -7< solution for 500 mL of medium to obtain a final concentration of 10 -10< mol / L.

[0081] The steps of the operating procedure are now detailed.

[0082] For the extraction of fibroblasts and keratinocytes from a patient's skin biopsy, the skin biopsy is incubated for 3 hours at 37°C in sterile dispase II solution. The dermis and epidermis are then separated using sterile forceps and rinsed in sterile 1X PBS.

[0083] For the extraction of fibroblasts, the pieces of dermis obtained are then placed in a sterile solution of collagenase A at 0.5 mg / mL, with stirring, for 4 to 6 hours at 37°C. Then the solution is filtered through a 70 µm porosity sieve and centrifuged at 1200 rotations per minute (abbreviated rpm in the following) for 5 to 10 minutes. After resuspension of the pellet, the cell count and viability are evaluated. The fibroblasts are seeded at a density of 8,000 cells / cm 2 < .

[0084] For keratinocyte extraction, epidermal pieces are incubated in 0.25% trypsin-EDTA at 37°C for 15 minutes. After adding trypsin inhibitor solution, the keratinocyte suspension is then filtered through a 70 µm porosity sieve, centrifuged at 1200 rpm for 5 to 10 minutes and the cell pellet thus obtained is resuspended in culture medium for assessment of cell count and viability. Keratinocytes are seeded at a density of 8000 cells / cm 2 < .

[0085] Fibroblasts and keratinocytes are cultured at 37°C in a humid atmosphere with 5% CO2, the media are renewed 3 times per week and the cells are passed to sub-confluence by the action of EDTA trypsin.

[0086] For the preparation of the porous matrix, the porous matrix is ​​hydrated for 6 to 48 hours prior to seeding the cells by a 1X PBS bath whose volume is sufficient to cover the upper surface of the matrix. The PBS can then be aspirated. A volume of MC1 medium, without antibiotics, EGF, or vitamin C, the volume being sufficient to immerse and cover the upper surface of the matrix, is added in order to balance the pH and osmolarity of the matrices. As an alternative to this preferred method of preparation, the porous matrix can be hydrated by a 2 to 3 hour bath in 1X PBS.

[0087] For fibroblast seeding, the fibroblasts are detached and dissociated by enzymatic action and counted. A cell suspension to obtain a cell density of 0.5.10 6< cells / cells / cm 2< is prepared. After aspirating the culture medium to dry the porous matrix, the fibroblast suspension is deposited drop by drop homogeneously on the upper face of the porous matrix. The fibroblasts are left to adhere for 1 hour in the incubator at 37°C and in an atmosphere of 5% CO 2 .

[0088] A sufficient volume to cover the upper surface of the porous matrix with MC1 medium with antibiotics but without EGF or vitamin C is added for fibroblast growth. The medium is renewed 3 times per week for 2-3 weeks with MC1 medium with the addition of antibiotics, EGF and vitamin C.

[0089] For keratinocyte seeding, the keratinocytes are detached by enzymatic action and counted. A cell suspension at 0.5.10 6< cellscm 2< ) is prepared. After aspirating the culture medium from the formed dermal substitute, the latter can be transferred to a sterile compress to absorb as much moisture as possible.

[0090] The keratinocyte cell suspension is deposited drop by drop homogeneously on the surface of the matrix, taking care that the cell suspension does not overflow onto the lateral faces of the porous matrix. The keratinocytes are left to adhere for 1 hour in the incubator at 37°C and in an atmosphere of 5% CO 2 .

[0091] For the first undergrowth, a sufficient volume to cover the upper surface of the MC2 medium matrix with antibiotics but without EGF or vitamin C is added to the matrix very gently without touching the samples. The culture medium is renewed 3 times per week for 1 week with MC2 medium with the addition of antibiotics, EGF and vitamin C.

[0092] For maturation, the porous matrix is ​​deposited after aspiration of the culture medium and drying on a sterile compress on sterile blotting paper itself placed on a metal grid. The volume of A / L medium supplemented with antibiotics and vitamin C is adjusted so that it is flush with the level of the blotting paper, avoiding the presence of bubbles trapped under the metal grid. The medium is renewed 3 times a week with the A / L medium containing antibiotics and vitamin C.

[0093] In view of the foregoing description, it is clear that the invention provides a biomaterial that is closer to native tissue.

[0094] The invention is not limited to the embodiments previously described and extends to all embodiments covered by the claims.

[0095] In particular, it can be expected that growth times and / or the composition of the media are adapted for other types of biological cells. REFERENCES

[0096] 1 Biomaterial 10 Porous matrix 100 Interior volume 101 Surface 1010 Upper face 1011 Lateral face 1012 Lower face 11 Biological cells 11 Nuclei 110 First type 1100 Fibroblasts 111 Second type 1110 Keratinocyte 1110 Differentiated keratinocyte 1111 Cytokeratin 14 112 Extracellular matrix 1120 Filaggrin 1121 Type I collagen 1122 Fibrillin 1123 Elastin 2 Process 20 Provision of a porous matrix 21 Hydration of the porous matrix 22 Drying of the porous matrix 23 First seeding 230 Deposition of a biological cell suspension 24 First cell growth 240Immersion of the porous matrix in a culture medium 241Synthesis of extracellular matrix 242Renewal of the culture medium 25Drying of the porous matrix 26Second seeding 260Deposition of a biological cell suspension 27Second cell growth 270First undergrowth 2700Immersion of the porous matrix in a culture medium 2701Renewal of the culture medium2702Confluence of biological cells 271Maturation 2710Cell differentiation 2711Renewal of the culture medium 3Culture medium 4Air 5Air / culture medium interface 6Grid 7Porous support

Claims

1. A method (2) for manufacturing a biomaterial (1), comprising: • supply (20) of a resorbable porous matrix (10) formed of a material comprising collagen, the porous matrix (10) being a haemostatic sponge, and having an interior volume (100) and an exterior surface (101), then • hydration (20) of the porous matrix (10) so as to balance the pH and osmolarity of the porous matrix (10), • following hydration (20) of the porous matrix (10), drying (22) of the porous matrix (10), • following drying (22) of the porous matrix (10), seeding (23) of the porous matrix (10) with at least one type (110) of living biological cells (11) of a tissue, then • a cellular growth (24) of at least one type (110) of biological cells (10), to form a biomaterial (1) comprising: • the porous matrix (10), • at least one type (110) of living biological cells (11) of a tissue, disposed in the interior volume (100) and alternatively or additionally on the surface (101) of the porous matrix (10).

2. The method (2) according to the preceding claim, wherein the seeding (23) of the porous matrix (10) is carried out by depositing (230) a suspension of at least one type (110) of biological cells (11) on the porous matrix (10).

3. The method (2) according to any one of the preceding claims, further comprising, after a first seeding (23) with a first type (110) of biological cells (11), or even after the first seeding (23) and at least part of a cellular growth (24) of the first type (110) of biological cells (11), a second seeding (26) of the porous matrix (10) with at least a second type (111) of biological cells (11), distinct from the first type (110) of seeded biological cells (11), then a cellular growth (27) of the at least one second type (111) of biological cells (11).

4. The method (2) according to the preceding claim, further comprising drying (25) the porous matrix (10) prior to the second seeding (26) of the porous matrix (10), the second seeding (26) being carried out by depositing (260) a suspension of at least one second type (111) of biological cells (11) on the porous matrix (10).

5. The method (2) according to any one of the preceding claims, wherein the biological cells (11) comprise cells selected from connective tissue cells and epithelial cells.

6. The method (2) according to the preceding claim, wherein, when the biological cells (10) are connective tissue cells, the cellular growth (24) comprises an immersion (240) of the porous matrix (10) in a culture medium (3).

7. The method (2) according to any one of the two preceding claims, wherein, when the biological cells (10) are epithelial tissue cells, the cellular growth (27) comprises a first undergrowth (270) during which the porous matrix is immersed (270a) in a culture medium (3), until a confluence of the biological cells (10) on the surface of the porous matrix (10).

8. The method (2) according to the preceding claim, wherein the growth (27) comprises, following the first undergrowth (270), a maturation (271) during which the porous matrix (10) is placed at an interface between air (4) and a culture medium (3).

9. A use of a biomaterial (1) manufactured by the method according to any one of the preceding claims, the biomaterial (1) comprising: • a resorbable porous matrix (10) formed from a material comprising collagen, the porous matrix (10) being a haemostatic sponge, and having an interior volume (100) and a surface (101), and • at least one type (110) of living biological cells (11) of a tissue, disposed in the interior volume (100) and / or on the surface (101) of the porous matrix (10), for an in vitro application of at least one substance selected from a pharmaceutical substance and a cosmetic substance.