Biomaterial comprising a resorbable porous matrix and associated manufacturing process

Abstract

Biomaterial comprising a resorbable porous matrix and associated manufacturing process. The invention relates to a biomaterial (1) comprising a resorbable porous matrix (10) formed of a collagen-containing material, and having an internal volume (100) and an external surface (101). Advantageously, the biomaterial (1) comprises at least one type (110) of living biological cells (11) of a tissue, arranged in the internal volume (100) and, alternatively or in addition, on the surface (101) of the porous matrix (10). The biomaterial (1) forms a tissue substitute closely resembling native tissue, particularly in terms of the biological structure present in the tissue and its physiological functions. Figure for the abstract: Fig. 5B

Description

Title of the invention: Biomaterial comprising a resorbable porous matrix and associated manufacturing method 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 induced by ultraviolet radiation, and chemical burns, for example induced by detergents or acids, ulcers, and in particular 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 sample 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 harvesting and the limited surface area of ​​the donor areas; - total skin grafting does not require a dermatome harvest as with 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 grafts remains limited, particularly in terms of the available surface area of ​​donor areas and the pain induced in the person being removed.

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

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

[0007] It is known, from document WO 88 / 10123 A1, 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] An object of the present invention is therefore to propose a biomaterial which is closer to a native tissue.

[0009] 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

[0010] To achieve this objective, according to a first aspect, a biomaterial is provided comprising a resorbable porous matrix formed from a material comprising collagen, and having an interior volume and an exterior surface.

[0011] 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.

[0012] 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 approaching a native tissue, in particular in terms of biological structure present in the tissue, and physiological functions.

[0013] 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.

[0014] 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.

[0015] A second aspect of the invention relates to a method of manufacturing a biomaterial comprising:

[0016]

[0017]

[0018]

[0019]

[0020] - a supply of a resorbable porous matrix formed from a material comprising collagen, then - seeding of the porous matrix with at least one type of living biological cells from a tissue, then - a cellular growth of at least one type of biological cell. 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. A third aspect concerns the use of a biomaterial, the biomaterial comprising: - a resorbable porous matrix formed from a material comprising collagen, and having an interior volume and a surface area, 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. A fourth aspect concerns the use of a biomaterial for the treatment of lesions, and more particularly the treatment of skin lesions, the biomaterial comprising: - a resorbable porous matrix formed from a material comprising collagen, and having an interior volume and a surface area, 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. A fifth aspect relates to a biomaterial intended to be grafted onto an organism, for example for the treatment of a lesion, and more particularly the treatment of skin lesions, the biomaterial comprising: - a resorbable porous matrix formed from a material comprising collagen, and having an interior volume and a surface area, 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. A combinable or separable aspect relates to a dermo-epidermal substitute comprising a resorbable porous matrix formed from a material comprising collagen, and having an interior volume and an exterior surface, the dermo-epidermal substitute being characterized in that it comprises dermal cells living cells, arranged in the interior volume of the porous matrix, an extracellular matrix arranged in the interior volume, the extracellular matrix being secreted by the dermal cells, and epidermal cells arranged on the exterior surface of the porous matrix. BRIEF DESCRIPTION OF THE FIGURES

[0021] 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:

[0022] [Fig.l] [Fig.l] represents a cross-sectional view of the porous matrix according to one embodiment of the invention.

[0023] [Fig.2] [Fig.2] represents a first seeding of the porous matrix illustrated in [Fig.l] by a first type of biological cells, according to an embodiment of the invention.

[0024] [Fig.3] [Fig.3] represents the cell growth of biological cells following the seeding illustrated in [Fig.2], according to one embodiment of the invention.

[0025] [Fig.4] [Fig.4] represents a second seeding of the porous matrix by a second type of biological cells and a first subsequent cellular undergrowth of the second type of biological cells, following the cellular growth illustrated in [Fig.3], according to an embodiment of the invention.

[0026] [Fig.5A] [Fig.5A] represents a maturation of the second type of biological cells following the first cellular undergrowth illustrated in [Fig.4], according to an embodiment of the invention.

[0027] [Fig.5B] [Fig.5B] represents the biomaterial, according to one embodiment of the invention.

[0028] [Fig.6] [Fig.6] represents the steps of the process for manufacturing the biomaterial, according to one embodiment of the invention.

[0029] [Fig.7] [Fig.7] represents a chronology of the steps of the process for manufacturing the biomaterial, according to one embodiment of the invention.

[0030] [Fig.8] [Fig.8] represents a cross-sectional fluorescence microscopy immunohistological image of the biomaterial, in which the nuclei of biological cells and cytokeratin 14 are labeled.

[0031] [Fig.9] [Fig.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.

[0032] [Fig. 10] [Fig. 10] represents a cross-sectional fluorescence microscopy immunohistological image of the biomaterial, in which the nuclei of biological cells, fibrillin 1 and elastin are labeled.

[0033] 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

[0034] Before beginning a detailed review of embodiments of the invention, optional characteristics are set out below which may possibly be used in association or alternatively for each of the aspects of the invention: - the at least one first type of biological cells may 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 outer surface of the porous matrix, or even only on one 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 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, - the porous matrix material may be free of chitosan, - the porous matrix may be selected from a gel, a powder, and a sponge. Preferably, the porous matrix is ​​selected from a powder and a sponge, - the porous matrix may be a synthetic sponge, and more particularly a hemostatic sponge. Thus, the porous matrix may be commercially available and at a reduced cost, - the porous matrix may 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. In one example, the connective tissue cells are dermal cells called fibroblasts and / or subcutaneous adipose tissue cells called adipose tissue stem cells. In one example, the epithelial cells are epidermis cells including keratinocytes and / or melanocytes. In 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 get even closer to the properties of a native tissue, - a first type of biological cells may be arranged in the interior volume of the porous matrix, and the biomaterial may 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 being furthermore distinct from the first type of biological cells. Thus, the biomaterial may further comprise a cell layer on the surface of the porous matrix forming an external boundary of a tissue, and further approaching 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 selected from 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.

[0035] Optional features of the second aspect of the invention are set out below, which may optionally be used in combination or alternatively: - the method may comprise hydration of the porous matrix so as to balancing 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 method may further comprise 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 may be carried out by depositing a suspension of the at least one type of biological cells on the porous matrix. According to one example, the at least one type of biological cells may be seeded at a cell density of between 0.5.106 cells / cm2 and 1.106 cells / cm2, the cm2 being given with reference to the surface of the porous matrix, the method may further comprise, after a first seeding by 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 by 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 cell, the method may further comprise a drying of the porous matrix prior to the second seeding of the porous matrix, the second seeding may be carried out by depositing a suspension of at least one second type of biological cells on the porous matrix, the biological cells may comprise cells chosen from connective tissue cells and epithelial cells. when the biological cells are connective tissue cells, the cell growth may comprise immersion of the porous matrix in a culture medium. Preferably, the porous matrix is ​​entirely submerged. In addition, the porous matrix may be submerged for the majority of the cell growth, when the biological cells are epithelial cells, the cell growth may comprise a first undergrowth during which the porous matrix is ​​immersed in a culture medium, until a confluence of the biological cells on the surface of the porous matrix, the cell growth may comprise, following the first undergrowth, a second undergrowth, which may equivalently be designated as maturation, during which the porous matrix is ​​placed at an interface between air and a culture medium. Maturation allows the differentiation of epithelial cells to form a more complex, even complete, epidermis and approach a native tissue

[0036] 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.

[0037] By a “biocompatible” matrix, it is meant that the matrix is ​​configured not to interfere with, or even degrade, a biological medium 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.

[0038] 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.

[0039] By porosity of the matrix is ​​meant the proportion of void or gas in the matrix.

[0040] In the remainder of the description, use will be made of terms such as, “transverse”, “ upper” and “lower”. 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. In this example, the upper face corresponds to the face intended to be turned away from the support.

[0041] The method 2 for manufacturing the biomaterial 1 is now described with reference to FIGS. 1 to 6. By way of example, the method is illustrated by [Fig.6], where optional steps are indicated in dotted lines.

[0042] The method 2 comprises the provision 20 of a porous matrix 20. The porous matrix 20 has pores delimiting an interior volume 100, as for example illustrated in [Fig.l]. 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 [Fig.l].

[0043] According to one example, the porosity of the porous matrix 10 is homogeneous, that is to say that the porosity per unit of 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.

[0044] The porous matrix 10 can be prepared for seeding 23 with biological cells 11. For this, the method 2 can comprise a hydration 21 configured to balance the pH and the osmolarity of the porous matrix around values ​​corresponding to the native environment of the cells. 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. During the hydration 21, the porous matrix 10 can be immersed in a buffered solution for several hours, or even several days. The porous matrix 10 can 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 can be a medium equivalent to the medium of the dermis.

[0045] Following its hydration 21, the method 2 may comprise 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 may be carried out by suction of the hydration solution.

[0046] 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.

[0047] 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.

[0048] The biological cells 11 may be chosen 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 available on the market from vendors known to those skilled in the art.

[0049] According to one example, the biological cells may be autologous or allogeneic. The allogeneic biological cells may, for example, be isolated from residues surgical procedure, 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 can be isolated from a biological sample of a person, for example following one or more biopsies and in particular a skin biopsy.

[0050] The biological cells 11 may comprise cells chosen from connective tissue cells and epithelial cells. For example, connective tissue may include the dermis, subcutaneous adipose tissue, corneal stroma, bones, and epithelial tissue may include the epidermis and the epithelium of the cornea. 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 in vitro or in vivo applications.

[0051] In the following, reference is made to the non-limiting example in which a first type of cell comprises, or even consists of, dermal cells, and more particularly fibroblasts, and a second type of cell comprises, or even consists of, epidermal 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 dermal cells, at least one type of biological cell from among: - endothelial cells, to obtain a vascularized dermis, - immune cells, to obtain an immunocompetent dermis, - stem cells from adipose tissue, to obtain a subcutaneous adipose structure. 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.

[0052] 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 [Fig.2]. According to one example, the fibroblasts 1100 can be seeded at a cell density of between 500,000 and 1,000,000 cells / cm2, or even substantially 800,000 cells / cm2. Note that depending on the type of biological cells seeded, the cell density can be adapted. Thus, the fibroblasts 1100 can penetrate into the interior volume 100 of the porous matrix 10. To further facilitate the adhesion of the fibroblasts 1100 seeded 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 preferably in an atmosphere containing 5% CO2.

[0053] 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 [Fig. 3]. During the 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 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.

[0054] 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 [Fig. 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 may further 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), 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.

[0055] Note that depending on the type of other cell types that may be seeded with the fibroblasts 1100, the medium can be configured to adapt and mimic the environment more favorable to the cell types seeded.

[0056] 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 fi broblasts 1100. Prior to the second seeding 26, the porous matrix 10 can be dried 25 in a manner similar to the drying 22 described previously. Then, a second type 111 of biological cells 11 can be seeded 26, including in particular keratinocytes 1110.

[0057] The keratinocytes 1110 may be seeded 26 by depositing 260 a cell suspension, for example drop by drop onto the surface 101 of the porous matrix 10, and in particular onto its upper face 1010 according to the example illustrated in [Fig.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 onto these faces. The keratinocytes 1110 may be seeded at a cell density of between 500,000 and 1,000,000 cells / cm2, or even substantially 800,000 cells / cm2. Note again that depending on the type of biological cells seeded, the cell density may be adapted. The seeded keratinocytes 1110 26 remain on the outer surface 101 of the porous matrix 10 due 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 keratinocytes 1110 seeded 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% CO2 and at 37°C.

[0058] 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 [Fig.4].

[0059] 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.

[0060] 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 it covers 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.

[0061] 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 the seeding 26 of the keratinocytes 1110.

[0062] Following the first undergrowth, the cellular growth 27 of the keratinocytes 1110 may comprise 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 a multi-stratification and a differentiation as illustrated in [Fig.5A]. In addition, during and following maturation, it is possible to observe the expression of specific proteins or lipids linked to the differentiation of the 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 even closer to native skin, particularly in terms of the diversity of biological cells present in the tissue, and physiological functions.More specifically, 1110' keratinocytes can differentiate to form at least some, 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 fusiform keratinocytes, - stratum corneum.

[0063] According to the example illustrated in [Fig.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 metal 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.

[0064] As an example, a chronology of the steps of the manufacturing method 2 is given with reference to [Fig.7]. In a first phase A, the porous matrix 10 can be 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, following 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 durations indicated may vary depending on the needs and the quantity of cells available.

[0065] 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 [Fig.5B].

[0066] The biomaterial 1 can in particular be used for in vitro application of pharmaceutical substances or cosmetic substances. During the development of the invention, it was demonstrated that the method described makes it possible to obtain the material 1 in a reproducible manner, moreover, the biomaterial 1 can be used as a model tissue, for example for fundamental and dermatological research.

[0067] Due to the characteristics described above, the biomaterial 1 has a regenerative activity. The 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.

[0068] Since biomaterial 1 can form a dermo-epidermal substitute for native skin, obtained in a reproducible manner, 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.

[0069] Biomaterial 1 forming a dermo-epidermal substitute for native skin, biomaterial 1 can in particular be used for: - treating the after-effects of burns, - treating 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.

[0070] The 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, the biomaterial 1 minimizes the risk of immune rejection of the biomaterial 1.

[0071] To obtain the autologous cells 11, a skin biopsy can be performed, on a healthy surface and reduced compared to the surface necessary for the treatment of a wound or a burn. The cells 11 can be multiplied by cell proliferation in the manufacturing method 2 in order to obtain a sufficient quantity to allow the cell growths 24 and / or 27. It is therefore possible to obtain a biomaterial 1 having a surface adapted to the surface of the injured area. Thus, the biomaterial 1 and its manufacturing method 2 make it possible to minimize the risks and trauma linked to the removal of skin from a person, compared to existing solutions.

[0072] Furthermore, the biomaterial 1 can be used as a skin substitute for in vitro testing of 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.

[0073] Characteristics of the biomaterial 1 were described during the description of the method 2. In the following, additional characteristics of the biomaterial 1 are detailed.

[0074] 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 can for example be improved.

[0075] 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.

[0076] Furthermore, the material of the porous matrix 10 may be free of chitosan. 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 the cells of a person to colonize the biomaterial following its implantation, for example by grafting, as for 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.

[0077] The porous matrix 10 may be in the form of a gel, and more particularly a powder or a sponge. Thus, the porous matrix can be shaped in two or even three dimensions as required. According to one example, the porous matrix 10 is a synthetic sponge, and more particularly a hemostatic sponge. A synthetic 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 commercially available in various forms and trademarks. Thus, the porous matrix 10 can be commercially available at a reduced cost.

[0078] 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). 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. This simplifies the process of placing the biomaterial on the market.

[0079] For example, the porous matrix 10 is a BPF hemostatic sponge chosen from the hemostatic sponges of the following brands: Spongostan™, CuraSpon®, Gelfoam® and Surgifoam®.

[0080] By way of example, immunohistological characterizations by fluorescence microscopy of the biomaterial 1 according to one embodiment are illustrated by FIGS. 8 to 10. For these characterizations, antibodies specific to certain cellular structures, and coupled to a fluorescent probe are applied to the biomaterial 1.

[0081] [Fig. 8] shows 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.

[0082] [Fig.9] represents 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.

[0083] [Fig. 10] shows 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

[0084] By way of example, an operating mode of the method 2 for manufacturing the biomaterial 1 is now described according to a particular embodiment.

[0085] 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.

[0086] MCI culture medium

[0087] [Tables 1] Components Concentrations Medium DMEM / glutamax qsp Calf serum 5% Fetal calf serum 5% EGF 10 ng / mL Penicillin 100 units / mL Streptomycin 100 pg / mL Amphotericin B 1 pg / mL Vitamin C 82.2 pg / mL

[0088] MC2 culture medium adapted from GREEN Medium

[0089] [Tables2] Components Concentrations Alternatives for clinical use DMEM / glutamax medium qs HAM F12 qs Fetal bovine serum 10% Hydrocortisone 0.4 pg / mL Insulin 5 pg / mL Umuline® at 0.12 IU / ml Choleratoxin 10 10 mol / L Isuprel® at 0.4 pg / ml Adenine 24.3 pg / mL Tri lodo Thyronine 2 nM EGF 10 ng / ml Amphotericin B 100 units / mL Penicillin 100 pg / mL Streptomycin 1 pg / mL Vitamin C 82.2 pg / mL

[0090] Note that for clinical use, the MC2 medium can be adapted by those skilled in the art to be compatible with clinical use.

[0091] A / L culture medium

[0092] [Tables3] Components Concentrations Medium DMEM / glutamax qsp HAM F12 qsp Bovine serum albumin (BSA) 8 mg / mL Hydrocortisone 0.4 pg / mL Insulin 5 pg / mL Amphotericin B 1 pg / mL Penicillin 100 units / mL Streptomycin 100 pg / mL Vitamin C 82.2 pg / mL

[0093] The preparation of the reagents for manufacturing the culture media is now described: - preparation of trio-iodo-L-thyronine (abbreviated T3 in the following): • preparation of a solution A at 2x104M by weighing 13.6mg of T3, then dissolving them in 1.5ml of a sodium hydroxide solution NaOH at 0.02 mol / L, complete to 100ml with sterile water, • preparation of the 2x106 M stock solution by carrying out a dilution to 1 / 100th of solution A, • filter on a filter of dimension 0.22 pm, • aliquot into 550pL portions and freeze at -20°C, • use 500pl of the 2x10 6M solution for 500 mL of MC2 medium to obtain a final concentration of 2x109 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 on a 0.22pm 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 pg / mL, - preparation of EGF: • prepare a 10 pg / mL solution by taking 200 pg of lyophilized EGF with sterile water to qsp 20 mL, • filter on a 0.22 pm porosity filter, • aliquot into 500pL portions and freeze at -20°C, • use 500 pL for 500 mL of medium when using the culture medium, to obtain a final concentration of 10 ng / mL, Preparation of vitamin C: • weigh 2.5g of powdered vitamin C 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 on a 0.22 pm porosity filter, • aliquot into 0.5ml portions and freeze at -20°C • Use 200 pL in 100 mL of medium to obtain a final concentration of 82.2 pg / mL, preparation of BSA: • weigh 4 g of BSA and dissolve them in 20 mL of DMEM medium at 37°C, • filter on a 0.22 pm porosity filter, • use 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, • prepare a solution C at 200 pg / mL by taking 0.4 mL of solution B in 9.6 mL (qsp 10 mL), • aliquot solution C into portions of 1 ml, • use ImL for 500 mL of medium, to obtain a final concentration of 0.4 pg / mL, preparation of insulin: • resuspend 50 mg of insulin with 500 pL of a 0.1 mol / L HCL solution, • dilute the suspension obtained in sterile water qsp 10 mL, to obtain a concentration of 5 mg / mL, • filter on a 0.22 pm porosity filter, • aliquot into 500 pL portions, • use 500pL for 500mL of medium, to obtain a final concentration of 5 pg / mL, preparation of choleratoxin: • resuspend 1 mg of choleratoxin in 12 mL of sterile water to obtain a concentration of 10.107 mol / L, • use 500 pL of the 10.107 solution for 500 mL of medium for obtain a final concentration of 10 10 mol / L.

[0094] The steps of the operating procedure are now detailed.

[0095] 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 a sterile dispase II solution. The dermis and epidermis are then separated using sterile forceps and then rinsed in sterile PBS IX.

[0096] 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 sieve with a porosity of 70 μm 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 / cm2.

[0097] For the extraction of keratinocytes, the pieces of epidermis 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 evaluation of the count and viability. The keratinocytes are seeded at a density of 8000 cells / cm2.

[0098] 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.

[0099] For the preparation of the porous matrix, the porous matrix is ​​hydrated for 6 to 48 hours prior to seeding the cells by a PBS 1X bath whose volume is sufficient to cover the upper face of the matrix. The PBS can then be aspirated. A volume of MCI medium, without antibiotics, EGF, or vitamin C, the volume being sufficient to immerse and cover the upper face 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, it is possible to hydrate the porous matrix by a 2 to 3 hour bath in PBS 1X.

[0100] For the seeding of fibroblasts, the fibroblasts are detached and dissociated by enzymatic action and counted. A cell suspension to obtain a cell density of 0.5.106 cells / cells / cm2 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% CO2.

[0101] A sufficient volume to cover the upper face of the porous matrix of MCI medium with antibiotics but without EGF or vitamin C is added for growth of the fibroblasts. The medium is renewed 3 times a week for 2-3 weeks with an MCI medium with the addition of antibiotics, EGF and vitamin C.

[0102] For keratinocyte seeding, the keratinocytes are detached by enzymatic action and counted. A cell suspension of 0.5.106 cells (cm2) is prepared. After aspirating the culture medium from the formed dermal substitute, the latter can be transferred to a sterile compress to absorb the maximum amount of moisture.

[0103] 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% CO2.

[0104] For the first undergrowth, a volume sufficient to cover the upper face of the MC2 medium matrix with antibiotics but without EGF or vitamin C is added to the matrix very delicately 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.

[0105] 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.

[0106] In view of the above description, it is clear that the invention provides a biomaterial which is closer to a native tissue.

[0107] The invention is not limited to the embodiments previously described and extends to all the embodiments covered by the claims.

[0108] It may in particular be provided that the growth times and / or the composition of the media are adapted for other types of biological cells.

[0109] LIST OF DIGITAL REFERENCES 1 Biomaterial 10 Porous matrix 100 Interior volume 101 Surface 1010 Upper face 1011 Side face 1012 Underside 11 Biological Cells 11' Cores 110 First type 1100 Fibroblasts 111 Second type 1110 Keratinocyte 1110' Differentiated keratinocytes 1111 Cytokeratin 14 112 Extracellular matrix 1120 Filaggrine 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 sowing 230 Deposit of a biological cell suspension 24 First cell growth 240 Immersion of the porous matrix in a culture medium 241 Synthesis of extracellular matrix 242 Renewal of the culture medium 25 Drying of the porous matrix 26 Second sowing 260 Deposit of a biological cell suspension 27 Second cell growth 270 First undergrowth 2700 Immersion of the porous matrix in a culture medium 2701 Renewal of the culture medium 2702 Confluence of biological cells 271 Maturation 2710 Cell differentiation 2711 Renewal of the culture medium 3 Culture medium 4 Air 5 Air / culture medium interface 6 Grid 7 Porous support

Claims

Claims

1. Biomaterial (1) comprising a resorbable porous matrix (10) formed from a material comprising collagen, and having an interior volume (100) and an exterior surface (101), the biomaterial (1) being characterized in that it comprises at least one type (110) of living biological cells (11) of a tissue, arranged in the interior volume (100) and alternatively or additionally on the surface (101) of the porous matrix (10) and that the porous matrix (10) is a hemostatic sponge.

2. Biomaterial (1) according to the preceding claim in which the material of the porous matrix (10) comprises 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).

3. Biomaterial (1) according to any one of the preceding claims wherein the material of the porous matrix (10) is free of chitosan.

4. Biomaterial (1) according to any one of the preceding claims, wherein the biological cells (11) comprise cells selected from connective tissue cells and epithelial cells.

5. Biomaterial (1) according to any one of the preceding claims, wherein the at least one type (110) of biological cells (11) is arranged at least in the interior volume (100) of the porous matrix (10), the biomaterial (1) further comprising an extracellular matrix (112) in said interior volume (100), the extracellular matrix (112) being secreted by the at least one first type (110) of biological cells (11).

6. Biomaterial (1) according to any one of the preceding claims, wherein a first type (110) of biological cells (11) is arranged in the interior volume (100) of the porous matrix (10), and the biomaterial (1) comprises at least a second type (111) of biological cells (11) arranged on the surface (101) of the porous matrix (10), the second type (111) of biological cells (11) being distinct from the first type (110) of biological cells (11).

7. Biomaterial (1) according to the preceding claim, wherein the first type (110) of biological cells (11) comprises fi- broblasts (1100), and the second type (111) of biological cells (11) includes keratinocytes (1110).

8. Biomaterial (1) according to any one of the preceding claims, wherein the biological cells (11) are selected from cells derived from standard cell line cultures and cells isolated from a biological sample of an organism.

9. Method (2) for manufacturing a biomaterial (1) according to any one of claims 1 to 8, comprising: • a supply (20) of a resorbable porous matrix (10) formed of a material comprising collagen, the porous matrix (10) being a hemostatic sponge and having an interior volume (100) and an exterior surface (101), then • a hydration (20) of the porous matrix (10) so as to balance the pH and the osmolarity of the porous matrix (10), then • a seeding (23) of the porous matrix (10) with at least one type (110) of living biological cells (11) of a tissue, then • a cell growth (24) of the at least one type (110) of biological cells (10).

10. Method (2) according to the preceding claim, further comprising drying (22) the porous matrix (10) between hydration (21) of the porous matrix (10) and seeding (23) the porous matrix (10).

11. Method (2) according to any one of claims 9 to 10, in which the seeding (23) of the porous matrix (10) is carried out by depositing (230) a suspension of the at least one type (110) of biological cells (11) on the porous matrix (10).

12. Method (2) according to any one of claims 9 to 11, further comprising, after a first seeding (23) by a first type (110) of biological cells (11), or even after the first seeding (23) and at least part of a cell growth (24) of the first type (110) of biological cells (11), a second seeding (26) of the porous matrix (10) by at least a second type (111) of biological cells (11), distinct from the first type (110) of biological cells (11) seeded, then a cell growth (27) of the at least a second type (111) of biological cells (11).

13. Method (2) according to the preceding claim, further comprising drying (25) of 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).

14. A method (2) according to any one of claims 9 to 13, wherein the biological cells (11) comprise cells selected from connective tissue cells and epithelial cells.

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

16. Method (2) according to any one of the two preceding claims, wherein, when the biological cells (10) are epithelial tissue cells, the cell 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) at the surface of the porous matrix (10).

17. Method (2) according to the preceding claim, in which 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).

18. Use of a biomaterial (1), the biomaterial (1) comprising: • a resorbable porous matrix (10) formed from a material comprising collagen, the porous matrix (10) being a hemostatic 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, arranged 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 chosen from a pharmaceutical substance and a cosmetic substance.

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