Two-layer support for preparing dermal (epidermal) equivalents or skin equivalents

Abstract

The present invention relates to a method for producing a dermis or skin or epidermis equivalent on a two-layer substrate comprising a layer having a thickness of at least 200 nm and a porosity of less than or equal to 5 μm, formed by electrospinning a composition comprising at least one polymer, and a layer having a thickness of at least 20 μm and a porosity of more than or equal to 20 μm, formed by electrowriting a composition comprising at least one polymer. The application also relates to the skin or dermis or epidermis equivalent obtainable using said method, to the use of the dermis equivalent or skin equivalent or epidermis equivalent for screening compounds, and finally to their use in wound dressings or for skin grafting.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a dermis or skin or epidermis equivalent on a two-layer substrate comprising a layer at least 200 nm thick and having a porosity of 5 μm or less, formed by electrospinning a composition comprising at least one polymer, and a layer at least 20 μm thick and having a porosity of 20 μm or more, formed by electrowriting a composition comprising at least one polymer. The application also relates to the skin or dermis or epidermis equivalent obtainable using said method, to the use of the dermis or skin or epidermis equivalent for screening compounds, and finally to their use in wound dressings or for skin grafting. [Background technology]

[0002] Electrospinning (ES) is a method for producing fibers that uses electrical forces to design threads charged with a polymer solution or melt. This allows for the production of fibers that can be as small as 50 nm in diameter. However, the deposition is random, resulting in a nonwoven fiber mesh. Electrowriting is an emerging technology that uses polymers to control the deposition of electrospun fibers, making it possible to build or assemble complex structures in situ.

[0003] These techniques may be used to fabricate porous substrates for tissue engineering, particularly for reconstructing the dermal and optionally subcutaneous portions of skin tissue.

[0004] Currently, lattice models are the most commonly used in vitro models for gaining new knowledge and for evaluation studies, however, they exhibit poor extracellular matrix neosynthesis and poor mechanical properties due to their high water content.

[0005] Porous substrates have the advantage of allowing extracellular matrix neosynthesis, resulting in a dynamic dermal compartment, and being permissive to multiple cell types. However, for skin tissue reconstruction, porosity is also a limitation because the dermal compartment must be completely filled with matrix so that epidermal cells can be deposited on top and satisfactory epidermal reconstruction can be achieved without keratinocytes invaginating the dermis. This is generally achieved by adding high concentrations of vitamin C to stimulate matrix neosynthesis by dermal fibroblasts, by selecting specific fibroblasts that secrete a high extracellular matrix content, and / or by growing the dermal compartment for a long period of time to achieve complete filling. This becomes a real problem when using fibroblasts from elderly donors and / or exposed to pollutants and / or pathological substances, because they produce less extracellular matrix, making it difficult to fill the dermal compartment and, therefore, epidermal reconstruction. The use of vitamin C in the reconstruction of dermis or skin equivalents is not always desirable for some ingredient evaluation studies because it has a relatively broad range of effects and may cause interference with the molecules being evaluated.

[0006] Furthermore, conventional porous substrates are poorly able to mimic the nanostructured architecture of skin due to manufacturing constraints.

[0007] Electrospinning alone is a solution that allows the creation of porous substrates containing fibers with diameters close to or similar to extracellular matrix fibers, which help enhance cell adhesion, growth, and differentiation. However, due to uncontrolled and low porosity, cell infiltration into the substrate is complicated and mediocre.

[0008] The two-layer substrate according to the invention has a number of advantages over known substrates: - a layer at least 200 nm thick and with a porosity of 5 μm or less is an interface that prevents cell passage, which allows the epidermis to be reconstructed regardless of the composition and amount of the dermal matrix (i.e., it can be reconstructed on an empty or nearly empty dermal compartment), which prevents keratinocyte invagination while allowing the passage of nutrients and proteins and communication between the dermis and epidermis. - Layers with a thickness of at least 20 μm and a porosity of 20 μm or more are highly porous, allowing for neosynthesis and remodeling of the extracellular matrix. Their design influences cell behavior, the composition and organization of the extracellular matrix, and the mechanical properties of the substrate.

[0009] Various two-layer substrates were fabricated, with the most porous layer formed by electrowriting printing using various designs. The inventors observed that epidermal reconstruction was possible on these substrates, achieving expression of key epidermal markers while remaining independent of the filling of the dermal compartment and the design of the most porous layer. Furthermore, the inventors observed that the substrate design affected the content and organization of the extracellular matrix and its response to vitamin C.

[0010] These matrices also have the advantage of being biocompatible and highly customizable, opening new avenues in skin models for new knowledge acquisition, in vitro testing, dressing preparation and grafting. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] FR688226A [Non-patent literature]

[0012] [Non-Patent Document 1] Black et al. (2005) Tissue Eng. 11:723~733 [Non-patent document 2] Mir et al., Biomateriaux polymers synthetiques pour la cicatrisation: une revue, Progress in Biomaterials(2018) [Non-patent document 3] Bibliardi et al., Pansements Bioactifs, Rev. Med. Suisse, 2010 [Non-patent document 4] Tenehaus et al., Agents topiques et pansements pour les soins locaux des brulees, 2021 [Non-Patent Document 5] Yamada (1991) J. Biol. Chem. 266:12809~128012 pages Summary of the Invention [Means for solving the problem]

[0013] Accordingly, the present invention provides a method for producing a dermis equivalent, comprising the steps of: a. forming a layer (i) having a thickness of at least 200 nm and a porosity of less than 5 μm by electrospinning (ES) or electrowriting (EW) of a composition comprising at least one polymer; b. forming a layer (ii) having a thickness of at least 20 μm and a porosity of at least 20 μm by electrowriting (EW) of a composition comprising at least one polymer; c. Seeding layer (ii) with dermal and / or subcutaneous tissue cells wherein steps a and b are performed sequentially in this order (a followed by b) or in the reverse order (b followed by a), and the second electrospinning step is performed longitudinally along the layer produced by the first electrospinning step.

[0014] The present invention further relates to a method for producing a skin equivalent, including the method for producing a dermal equivalent described above, which further comprises the step of seeding layer (i) with epidermal cells after step c) and / or after the two steps a) and b).

[0015] The present invention further provides a method for producing an epidermis equivalent, comprising the steps of: a. forming a layer (i) having a thickness of at least 200 nm and a porosity of 5 μm or less by electrospinning (ES) or electrowriting (EW) of a composition comprising at least one polymer; b. forming a layer (ii) having a thickness of at least 20 μm and a porosity of at least 20 μm by electrowriting (EW) of a composition comprising at least one polymer; c. Seeding epidermal cells into layer (i) wherein steps a and b are performed sequentially in this order (a followed by b) or in the reverse order (b followed by a), and the second electrospinning step is performed longitudinally along the layer produced by the first electrospinning step.

[0016] The present invention also relates to dermis equivalents and skin or epidermis equivalents obtainable using the preparation method according to the invention.

[0017] The present invention also provides a method for forming a skin equivalent or a dermis equivalent or an epidermis equivalent, comprising: b. A layer having a thickness of at least 20 μm and a porosity of 20 μm or more, obtained by electrowriting (EW) of a composition containing at least one polymer. superimposed longitudinally on a. A layer having a thickness of at least 200 nm and a porosity of 5 μm or less, obtained by electrospinning (ES) or electrowriting (EW) of a composition containing at least one polymer. The present invention relates to the use of a substrate comprising:

[0018] The present invention also relates to the use of a dermis equivalent or a skin equivalent or an epidermis equivalent according to the invention for screening compounds, preferably compounds capable of having cosmetic, dermatological or pharmaceutical activity on the skin, preferably cosmetic or dermatological activity after topical application to the skin or injection into the skin.

[0019] Another object of the present invention further relates to a method for screening for active compounds, preferably compounds capable of having a cosmetic, dermatological or pharmaceutical activity on the skin, preferably a cosmetic or dermatological activity after topical application to the skin or injection into the skin, comprising the step of applying a candidate compound to a dermis equivalent according to the invention or to a skin equivalent according to the invention or to an epidermis equivalent according to the invention.

[0020] The present invention also relates to a dermis equivalent according to the invention, a skin equivalent according to the invention, or an epidermis equivalent according to the invention for use in a wound dressing or for skin grafting. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 is a cross-sectional view of a two-layer substrate used in the manufacture of a dermal equivalent, where 1 is a schematic representation of layer (i) and 2 is layer (ii). [Figure 2] Photograph of a two-layer substrate used in the manufacture of a dermal equivalent (bar represents 20 μm). The foreground shows the fibrous portion of layer (ii), and the background shows the less porous layer (i). [Figure 3] Figure 1 shows different types of designs fabricated using electrowriting and tested by the inventors. Scale bar corresponds to 100 μm. From left to right, the figure shows an octagonal design with fibers of diameter = 8.7 ± 0.48 μm, a wavy design with fibers of diameter = 11.71 ± 0.38 μm, a decagonal design with fibers of diameter = 12.38 ± 1.18 μm, and a wavy design with fibers of diameter ≈ 14.5 μm. [Figure 4]FIG. 1 shows a two-layer substrate attached to an Episkin insert using an O-ring. DETAILED DESCRIPTION OF THE INVENTION

[0022] Method for producing a dermal equivalent The present invention provides a method for producing a dermis equivalent, comprising the steps of: a. forming a layer (i) having a thickness of at least 200 nm and a porosity of 5 μm or less by electrospinning (ES) or electrowriting (EW) of a composition comprising at least one polymer; b. forming a layer (ii) having a thickness of at least 20 μm and a porosity of at least 20 μm by electrowriting (EW) of a composition comprising at least one polymer; c. Seeding layer (ii) with dermal and / or subcutaneous tissue cells wherein steps a and b may be performed sequentially in this order (a followed by b) or in the reverse order (b followed by a), and wherein a second electrospinning step is performed longitudinally along the layer produced by the first electrospinning step.

[0023] This method therefore makes it possible to obtain a dermal equivalent, which is a two-layer substrate containing dermal and / or hypodermal cells. A schematic diagram of this two-layer substrate is shown in Figure 1 and a photograph of this type of substrate is shown in Figure 2.

[0024] After steps a and b of the method, a two-layer substrate is obtained. According to a preferred embodiment, the two-layer substrate is a horizontally arranged substantially planar substrate. In this embodiment, the layers are stacked on top of each other.

[0025] Layer (i) can be formed first, and then layer (ii) formed thereon, or vice versa, layer (ii) formed first, and then layer (i) formed thereon.

[0026] The term "electrospinning" (ES) refers to electrohydrodynamic spraying techniques, such as solution or melt electrospinning, melt or solution electrowriting, or meltblowing, which allow polymer fibers to be deposited.

[0027] The term "electrowriting" (EW) refers to electrohydrodynamic spraying techniques, such as solution or melt electrospinning, melt or solution electrowriting, or meltblowing, that deposit fibers along a specific contour to form a predetermined design. Example designs can include octagonal, organic / wavy, or decagonal (see Figure 3).

[0028] These techniques allow for the formation of layers containing pores, also called porous layers, the term "pores" denoting optionally closed hollow spaces of various widths and depths.

[0029] The term "porosity" refers to the ability of a layer to allow elements to cross, in particular through the pores contained in this layer. Porosity is defined here by the pore size, i.e., the maximum distance measured between the fibers that form the pores. The porosity of a layer can be evaluated using techniques well known to those skilled in the art, such as scanning electron microscopy, in particular a Crossbeam 340 SEM, Zeiss, Oberkochen, Germany.

[0030] Thus, a "layer having a porosity of 5 μm or less" refers to a layer that contains pores, the maximum distance measured between the fibers forming each pore being 5 μm or less.

[0031] Similarly, a "layer having a porosity of 20 μm or greater" refers to a layer that contains pores, the smallest distance measured between fibers in each pore being 20 μm or greater.

[0032] Preferably, all cells seeded on the two-layer substrate are sourced from the same human or animal species, particularly mammalian animal species. The seeded cells can be obtained from healthy donors or donors suffering from conditions involving damage to the dermis and / or skin. These cells can be modified using genetic engineering methods (e.g., gene transfer, etc.), particularly to monitor gene expression. Various dermal, dermal, or epidermal equivalents can be produced to study variations related to various factors, such as age, skin type, ethnicity, stress, or pollution.

[0033] The seeded cells may optionally be obtained from immortal cell cultures, or from primary cultures, or from primary cell lines isolated (without prior culture) from skin tissue, preferably all seeded cell types are obtained from primary cultures.

[0034] The term "immortal cells" refers to cells obtained from a tumor, spontaneously immortal cells and / or cells immortalized by the introduction of at least one viral or cellular oncogene. According to a particular embodiment, the cell type or types seeded on the substrate according to the invention are obtained from a culture of cells immortalized by the introduction of at least one viral or cellular oncogene.

[0035] The term "primary culture" refers to a culture of cells obtained directly from tissues and / or cells of an individual. In an alternative embodiment, one or more cell types seeded on the substrate according to the present invention are obtained from primary cultures of tissues and / or cells sampled from individuals of the same species and age, preferably all cell types are obtained from primary cultures of tissues and / or cells sampled from individuals of the same species and age. According to one embodiment of the present invention, one or more, preferably all, cell types are obtained from an adult individual. In this way, the dermis or skin equivalent according to the present invention also makes it possible to study the effects on the dermis or skin of age-related changes or diseases that develop during the lifetime of an individual. Cells obtained from primary cultures, unlike immortalized cells, retain contact inhibition, and therefore the use of these cells allows for limited cell proliferation on the substrate. Furthermore, the use of primary cultures allows for even closer approximation to in vivo conditions.

[0036] The term "individual" refers to a human or animal subject of any animal species, particularly a mammalian animal species.

[0037] In a first particular embodiment, the seeded cells are a disease model cell type.

[0038] In a second particular embodiment, the cells seeded are cells from an individual with a disease, preferably a disease affecting or suspected of affecting the skin.

[0039] The term "disease model cell type" refers to a cell type derived from an animal or human model that reproduces a disease that occurs spontaneously or is induced by genetic engineering methods (e.g., gene transfer) or pharmacological tools to reproduce the cellular characteristics of individuals suffering from these specific conditions. Examples include cell types derived from the shFLG (short hairpin filaggrin) model, which reduces filaggrin protein expression, and is a model for diseases such as atopic dermatitis. Preferably, the disease according to the present invention is a disease that affects or is suspected to affect the skin, such as eczema, lentigines, atopic dermatitis, psoriasis, or solar elastosis.

[0040] Step a: Formation of layer (i) Layer (i) is formed by electrospinning (ES) or electrowriting (EW) of a composition comprising at least one polymer and has a thickness of at least 200 nm and a porosity of 5 μm or less.

[0041] Preferably, this layer is formed by electrospinning (ES) or electrowriting (EW) of a composition comprising at least one molten polymer or one polymer in solution, more preferably a molten polymer.

[0042] Preferably, the porosity of layer (i) is between 0.4 μm and 5 μm. This low porosity prevents cell infiltration but allows the passage of nutrients, growth factors, cytokines, chemokines, etc.

[0043] Preferably, the thickness of layer (i) is between 200 nm and 20 μm, more preferably between 5 μm and 10 μm, which allows the separation of cells while simultaneously realizing their communication, in particular paracrine communication.

[0044] Preferably, the fibers formed by electrospinning of layer (i) have a diameter between 50 nm and 5 μm, more preferably between 100 nm and 500 nm.

[0045] Layer (i) may be substantially flat or may comprise undulations. The term undulations refers to bumps and / or depressions. If the layer comprises undulations, the undulations have a height of less than 2 mm, preferably less than 1.5 mm. The undulations may be uniformly distributed over the surface of the layer.

[0046] Step b: Formation of layer (ii) Layer (ii) is formed by electrowriting (EW) of a composition comprising at least one polymer and has a thickness of at least 20 μm and a porosity of 20 μm or more.

[0047] In one embodiment, this layer is formed by electrowriting (EW) of a composition comprising at least one molten polymer or one polymer in solution, more preferably a molten polymer.

[0048] The term "molten polymer" refers to a polymer that is normally solid at room temperature and becomes liquid upon increasing the temperature.

[0049] The term "polymer in solution" refers to a composition in which a polymer is dissolved in a solvent.

[0050] Thus, for example, a 15% polycaprolactone (PCL) solution prepared in a mixed dichloromethane and dimethylformamide solvent is a composition comprising a solution-like polymer, whereas PCL pellets preheated above 60°C, i.e., above the melting point of PCL, form a molten polymer.

[0051] Preferably, the thickness of this layer is between 20 μm and 5 mm. This type of thickness makes it possible to reproduce the compartments of the dermis and subcutaneous tissue.

[0052] Preferably, the porosity of layer (ii) is between 20 μm and 600 μm, which allows for three-dimensional integration of living cells and neosynthesis and organization of extracellular matrix without imposing excessive stress on the cells.

[0053] Preferably, the fibers formed by electrospinning of layer (ii) have a diameter between 50 nm and 100 μm, more particularly between 1 μm and 20 μm, which makes it possible to mimic the diameter of the fibers or fibrils or fiber bundles observed in vivo in the extracellular matrix.

[0054] Layer (ii) may be substantially flat or may comprise undulations. The term undulations refers to bumps and / or depressions. If the layer comprises undulations, the undulations have a height of less than 2 mm, preferably less than 1.5 mm. The undulations may be uniformly distributed over the surface of the layer.

[0055] In a first embodiment, the design of this layer may be outlined by substantially straight or curved fibers, or a mixture of both, or a superposition of both, so that some fibers may be superimposed or suspended above the cavities (see Figure 3).

[0056] In a second embodiment, the design of this layer may be defined by a mixture or superposition of different substantially straight fibers, the term "different" indicating that their orientation is different. Some fibers may therefore be superimposed or suspended above the cavity.

[0057] In a third embodiment, the design of this layer may be outlined by a mixture or superposition of different curves, the term "different" indicating different orientations and / or different curvatures. Some fibers may therefore be superimposed or suspended above the cavities.

[0058] In a preferred embodiment of the present invention, layer (ii) has a wave-type or octagonal-type or decagonal-type design.

[0059] A composition comprising at least one polymer for forming layers (i) and (ii). The at least one polymer of the composition used to form layers (i) and (ii) can be synthetic or natural. The at least one polymer of the composition used to form layers (i) and (ii) is preferably bioerodible, bioabsorbable, biocompatible, bioresorbable and / or biodegradable, preferably biocompatible and bioresorbable.

[0060] The composition comprising at least one polymer used to form layer (i) may be the same as or different from the composition comprising at least one polymer used to form layer (ii).Similarly, the at least one polymer used to form layer (i) may be the same as or different from the composition used to form layer (ii).

[0061] Examples of suitable polymers include, but are not limited to, poly(alpha-hydroxy acid), polylactide (PLA), polyglycolide (PG), polyethylene glycol (PEG), conjugates of poly(alpha-hydroxy acid), poly(orthoester) (POE), polyaspirin, polyphosphagen, triethyl-2-acetylcitrate, collagen, elastin-type peptides, starch, pregelatinized starch, chitosan, alginate, albumin, fibrin, silk, homopolymers or copolymers of caprolactone, preferably polycaprolactone. lactone, PLCL (poly(lactide-co-caprolactone), poly(2-ethyl-2-oxazine) (PEtOzi), dextran, vinylpyrrolidone, polyvinyl alcohol (PVA), PVA-g-PLGA, PEGT-PBT copolymer (polyactive), polyethylene oxide-polypropylene oxide-poly(acrylic acid) (PEO-PPO-PAA) copolymer, PLGA-PEO-PLGA copolymer, PEG-PLG copolymer, PLA-PLGA copolymer, poloxamer 407, PEG-PLGA-PEG triblock copolymer, or combinations thereof.

[0062] In various embodiments, the composition comprises poly(lactic-co-glycolic acid) copolymer (PLGA), polylactide (PLA), polyglycolide (PGA), a homopolymer or copolymer of D-lactide, D,L-lactide, L-lactide, D,L-lactide-co-ε-caprolactone, D,L-lactide-co-glycolide-co-ε-caprolactone, L-lactide-co-ε-caprolactone, or a poly(ester)amide, or a mixture thereof.

[0063] Preferably, said at least one polymer of the composition used to form layers (i) and (ii) is selected from: natural polymers, such as proteins and polypeptides, glycosaminoglycans, proteoglycans, such as collagen, elastin, hyaluronic acid, dermatan sulfate, gelatin, or mixtures or composites thereof; synthetic polymers, such as biodegradable synthetic polymers, such as polylactic acid, polyglycolide, poly(lactic-co-glycolic acid) copolymers ("PLGA"), polycaprolactone ("PCL"), poly(dioxanone), poly(trimethylene carbonate) copolymers, polyglyconate, poly(propylene fumarate), poly(ethylene terephthalate), poly(butylene terephthalate), polyethylene glycol, polycaprolactone copolymers, polyhydroxybutyrate, polyhydroxyvalerate, polycarbonates derived from tyrosine and any random or (multi)block copolymers, or mixtures thereof; - and mixtures thereof.

[0064] Polyethylene glycol methyl ether (mPEG) can be used to impart malleability to the polymer.

[0065] In some embodiments, these polymers may also be applied to the formed fibers to provide the desired release profile.

[0066] Step c, seeding layer (ii) Dermal and / or subcutaneous tissue cells are preferably seeded onto the free surface of layer (ii), and the seeded cells are then allowed to migrate into layer (ii).

[0067] The term "dermal and / or subcutaneous tissue cells" refers to any cell type present or migrating in the dermis or subcutaneous tissue, including, but not limited to, fibroblasts, adipocytes, endothelial cells, neuronal cells, hair follicle cells, sweat gland cells, sebaceous gland cells, apocrine gland cells, immune cells (e.g., macrophages, monocytes, mast cells, lymphocytes, neutrophils, eosinophils, dendritic cells, etc.), nerve cells, vascular smooth muscle cells, specialized muscle cells, stem cells, or induced stem cells (e.g., cells derived from iPS and ES cells), of human, animal, or other origin, optionally genetically modified. In this application, stem cells should be understood to exclude human embryonic stem cells.

[0068] In a particular embodiment, the seeded dermal and / or subcutaneous tissue cells comprise in particular fibroblasts, typically dermal fibroblasts, in particular human fibroblasts, more particularly primary human fibroblasts.

[0069] In a preferred embodiment, the concentration of fibroblasts seeded in the substrate is between 0 and 100,000 cells / mm 3 , more preferably 1500 to 25,000 cells / mm 3 is.

[0070] Seeding can be carried out using any suitable technique known to those skilled in the art, under culture conditions suitable for the growth of dermal and / or subcutaneous tissue cells, in particular fibroblasts.

[0071] The term cell seeding refers to controlled or random manual or machine-assisted (using a dispenser or bioprinting) cell deposition.

[0072] Once seeded, the dermal and / or subcutaneous tissue cells are typically cultured in a suitable culture medium, preferably suitable for fibroblast culture, for example, FHN2D medium (corresponding to DMEM medium supplemented with 2 mM glutamine, antibiotics, and 10% fetal bovine serum, with or without ascorbic acid). They may be cultured submerged or at an air-liquid interface, preferably under conditions suitable for their growth, more particularly at 37°C and 5% CO2. Once seeded, the dermal and / or subcutaneous tissue cells may be cultured for up to 6 months, preferably 5 to 40 days.

[0073] Method for producing a skin equivalent The free surface of layer (i) can be seeded with epidermal cells, and cell seeding into layers (i) and (ii) can be carried out simultaneously or sequentially.

[0074] The present invention therefore relates to a method for producing a skin equivalent, including a method for producing a dermal equivalent according to the invention, which further comprises the step of seeding layer (i) with epidermal cells after step c. and / or after the two steps a. and b.

[0075] The term "epidermal cells" refers to any cell type present or migrating in the epidermis, including keratinocytes, neuronal cells, melanocytes, Merkel cells, stem cells or induced stem cells (e.g., cells derived from iPS and ES cells), and immunocompetent cells, such as Langerhans cells.

[0076] The term cell seeding refers to controlled or random manual or machine-assisted (using a dispenser or bioprinting) cell deposition.

[0077] In one embodiment, the seeded epidermal cells are of the keratinocyte type, optionally including melanocytes. Preferably, the keratinocytes and melanocytes are cultured separately before being seeded in or on the substrate.

[0078] In certain embodiments using keratinocytes, layer (i) may further comprise, as a bioactive agent in and / or on the fibers, collagen 4 or collagen 7. Advantageously, the thickness of the collagen on layer (i) does not exceed 10 μm.

[0079] The epidermal cells are preferably seeded on the free surface of layer (i).

[0080] In a preferred embodiment, the concentration of keratinocytes seeded on the substrate is between 0 and 2 million cells / cm. 2 , more preferably 150,000 to 400,000 cells / cm 2 is.

[0081] Once seeded, epidermal cells are typically cultured in a suitable culture medium, preferably adapted for the culture of keratinocytes, such as G7F expansion medium and G3F differentiation medium with or without ascorbic acid (Black et al. (2005) Tissue Eng. 11:723-733). They may be cultured submerged or at the air-liquid interface at 37°C and 5% CO for up to 6 months, preferably 5 to 40 days.

[0082] Following an incubation period of preferably 0 to 7 days, even more preferably 3 to 7 days, the skin equivalent is preferably maintained at an air / liquid interface, for example by suspending the insert onto a metal grid or any other cell culture device known to those skilled in the art that provides an air / liquid interface.

[0083] Incubation is then continued until a skin equivalent exhibiting the characteristics of skin, i.e., a dermis equivalent covered by an epidermis equivalent exhibiting the four standard cell layers, i.e., basal and suprabasal layers, granular layer, and stratum corneum, is obtained. Incubation is thus continued for a period preferably between 7 and 21 days, and even more preferably between 7 and 14 days.

[0084] Natural or synthetic compounds can be added to the free surface of layer (i) or to the cell-seeded epidermal surface, such as water-resistant compounds (e.g., synthetic polymers, such as silicones), UV filters, bactericides (as described by Mir et al., Biomateriaux polymeres synthetiques pour la cicatrisation: une revue, Progress in Biomaterials (2018), Bibliardi et al., Pansements Bioactifs, Rev. Med. Suisse, 2010, or Tenehaus et al., Agents topiques et pansements pour les soins locaux des brulures, 2021), alone or in mixtures, or cellulose.

[0085] Method for producing an epidermal equivalent The present invention further provides a method for producing an epidermis equivalent, comprising the steps of: a. forming a layer (i) having a thickness of at least 200 nm and a porosity of 5 μm or less by electrospinning (ES) or electrowriting (EW) of a composition comprising at least one polymer; b. forming a layer (ii) having a thickness of at least 20 μm and a porosity of at least 20 μm by electrowriting (EW) of a composition comprising at least one polymer; c. Seeding epidermal cells into layer (i) wherein steps a and b are performed sequentially in this order (a followed by b) or in the reverse order (b followed by a), and the second electrospinning step is performed longitudinally along the layer produced by the first electrospinning step.

[0086] The term "epidermal cells" refers to any cell type present or migrating in the epidermis, including keratinocytes, neuronal cells, melanocytes, Merkel cells, stem cells or induced stem cells (e.g., cells derived from iPS and ES cells), and immunocompetent cells, such as Langerhans cells.

[0087] The term cell seeding refers to controlled or random manual or machine-assisted (using a dispenser or bioprinting) cell deposition.

[0088] In one embodiment, the seeded epidermal cells are of the keratinocyte type, optionally including melanocytes. Preferably, the keratinocytes and melanocytes are cultured separately before being seeded in or on the substrate.

[0089] In certain embodiments using keratinocytes, layer (i) may further comprise, as a bioactive agent in and / or on the fibers, collagen 4 or collagen 7. Advantageously, the thickness of the collagen on layer (i) does not exceed 10 μm.

[0090] The epidermal cells are preferably seeded on the free surface of layer (i).

[0091] In a preferred embodiment, the concentration of keratinocytes seeded on the substrate is between 0 and 2 million cells / cm. 2 , more preferably 150,000 to 400,000 cells / cm 2 is.

[0092] Once seeded, epidermal cells are typically cultured in a suitable culture medium, preferably adapted for the culture of keratinocytes, such as G7F expansion medium and G3F differentiation medium with or without ascorbic acid (Black et al. (2005) Tissue Eng. 11:723-733). They may be cultured submerged or at the air-liquid interface at 37°C and 5% CO for up to 6 months, preferably 5 to 40 days.

[0093] Following an incubation period of preferably 0 to 7 days, even more preferably 3 to 7 days, the skin equivalent is preferably maintained at an air / liquid interface, for example by suspending the insert onto a metal grid or any other cell culture device known to those skilled in the art that provides an air / liquid interface.

[0094] Incubation is then continued until an epidermal equivalent exhibiting the desired characteristics is obtained, i.e., an epidermal equivalent having the four standard cell layers, i.e., basal, suprabasal, granular and stratum corneum. Incubation is thus continued for a period preferably between 7 and 21 days, even more preferably between 7 and 14 days.

[0095] Optionally, the method may comprise an additional step before step c, which comprises loading or coating layer (ii) with one or more bioactive agents, optionally in the form of a hydrogel that mimics the dermal matrix.

[0096] In a preferred embodiment, prior to step c, layer (i) is treated with ethanol.

[0097] Furthermore, natural or synthetic compounds can be added to the free surface of layer (i) or to the cell-seeded epidermal surface, such as water-resistant compounds (e.g., synthetic polymers, such as silicones), UV filters, bactericides (as described by Mir et al., Biomateriaux polymeres synthetiques pour la cicatrisation: une revue, Progress in Biomaterials (2018), Bibliardi et al., Pansements Bioactifs, Rev. Med. Suisse, 2010, or Tenehaus et al., Agents topiques et pansements pour les soins locaux des brulures, 2021), alone or in mixtures, or cellulose.

[0098] Supplements to layers (i) and (ii) In some embodiments of the method according to the present invention, layer (i) and / or layer (ii) can further comprise one or more bioactive agents in and / or on the fibers. Bioactive agent or bioactive compound is used herein to refer to a compound or entity that modifies, inhibits, activates, or affects a biological or chemical event. For example, bioactive agents can include, but are not limited to, cell-extracellular matrix interaction modulators, including proteins or peptides, antibiotics, antivirals, enzyme inhibitors, hormones, cell growth inhibitors, and anti-adhesion molecules, vasodilators, DNA, RNA, or protein synthesis inhibitors, anti-inflammatory agents, anti-angiogenic factors, angiogenic factors, anti-secretory factors, anticoagulants and / or antithrombotic agents, and prostaglandins, preferably of the skin. Preferably, the bioactive agent is different from the polymer forming the composition used to form layers (i) and (ii). For example, the bioactive agent can be collagen, gelatin, peptides, ethanol, growth factors, poly-lysine, optionally thiolated RGD peptide, optionally thiolated GRGDS peptide (SEQ ID NO: 1), and the proteins and peptides described in Yamada (1991) J. Biol. Chem. 266:12809-128012, such as fibronectin or fibronectin-derived peptides, such as type I and II peptides, laminin or laminin-derived peptides, such as peptides YIGSR (SEQ ID NO: 2), PDSGR (SEQ ID NO: 3), F9, LGTIPG (SEQ ID NO: 4), p20 or PA22-2, vitronectin, fibrinogen, von Willebrand factor, entactin, circumsporozoite protein, thrombospondin, or amyloid P component, and mixtures thereof. In some embodiments, the bioactive agent can include a dietary supplement, such as one or more vitamins, such as ascorbic acid, zinc, calcium, or combinations thereof.

[0099] The presence of collagen, gelatin, fibronectin, laminin or fibrin typically promotes adhesion of cells, particularly keratinocytes and fibroblasts.

[0100] In certain embodiments using fibroblasts, the collagen used is collagen 1.

[0101] In certain embodiments using keratinocytes, the collagen used is collagen 4 or collagen 7.

[0102] In one embodiment, the bioactive agent can be a cell growth promoting agent, such as a sugar, or a combination thereof.

[0103] In some embodiments, the bioactive agent is a pharmaceutical agent.

[0104] Bioactive agents further include RNA, such as siRNA or shRNA. In some embodiments, the bioactive agent is a growth factor, cytokine, extracellular matrix molecule, or a fragment or derivative thereof, such as a cell binding site, such as an RGD or GRGDS sequence.

[0105] The bioactive agent in and / or on the fiber can be deposited onto the fiber using different techniques known to those skilled in the art, for example by: - Immersion, optionally followed by draining or drying, - spraying or atomizing; - Injecting, especially with a pipette.

[0106] Thus, in the method according to the invention, after steps a and b, layer (i) can be filled or coated with one or more bioactive agents, optionally in the form of a hydrogel that mimics the matrix of the dermal-epidermal junction, and / or layer (ii) can be filled or coated with one or more bioactive agents, optionally in the form of a hydrogel that mimics the dermal matrix, which steps, if carried out, are preferably carried out before the step of seeding said layers with cells.

[0107] The term "loaded" indicates that at least 50% of the free volume of the layer is loaded with said active agent, preferably at least 70% of the free volume is loaded, and more preferably at least 90% of the free volume is loaded. Loading techniques include, inter alia, immersion in a bath containing said bioactive agent, injection or deposition of said bioactive agent into said layer.

[0108] The terms "coated" or "covered" as used herein indicate that a bioactive agent is deposited on the surface of the fibers forming a layer. The void spaces of the layer are not filled with said bioactive agent. Methods for depositing a bioactive agent, and thus coating or covering a layer, include, for example, immersion in a solution containing the bioactive agent followed by drying or draining.

[0109] The term "hydrogel" refers to a gel in which the swelling agent is water. The matrix of a hydrogel is generally a polymer network.

[0110] Such hydrogels can include, for example, collagen, gelatin, fibrin, elastin-type peptides, agar, alginate, decellularized dermal extracellular matrix, and mixtures thereof.

[0111] For example, collagen 4 or 7 or perlecan (glycosaminoglycan) are used to mimic the matrix of the dermal-epidermal junction, while collagen 1, hyaluronic acid, hyaluronic acid (acrylate or methacrylate), elastin-like peptides (acrylate or methacrylate), gelatin (methacrylate or acrylate), and mixtures thereof are used to mimic the dermal matrix.

[0112] In some embodiments of the method according to the present invention, layer (i) and / or layer (ii) may be treated and / or modified to increase cell adhesion with any compound or treatment (e.g., a chemoattractant for fibroblasts, melanocytes, keratinocytes, or other skin cells) that enables cell adhesion, growth, differentiation, proliferation, or migration of any cell type present or migrating in the skin, such as fibroblasts, adipocytes, endothelial cells, neuronal cells, hair follicle cells, sweat gland cells, sebaceous gland cells, apocrine gland cells, sudoral cells, immune cells (e.g., macrophages, monocytes, mast cells, lymphocytes, neutrophils, eosinophils, dendritic cells, etc.), nerve cells, vascular smooth muscle cells, specialized muscle cells, stem cells, or induced stem cells (e.g., cells derived from iPS and ES cells), of human, animal, or other origin, optionally genetically modified. These treatments include, for example, NaOH treatment, ethanol treatment, plasma treatment after layer formation, or shish-kebab formation during layer formation.

[0113] In some embodiments, the fibers of layer (i) and / or layer (ii) may be chemically modified, for example with methacrylates or acrylates, acrylamides or methacrylamides, which may be covalently crosslinked after or during fiber formation.

[0114] Equivalents obtainable using the method according to the invention The present invention also relates to a dermis equivalent obtainable using the method according to the invention.

[0115] The present invention also relates to a skin equivalent obtainable using the method according to the invention.

[0116] The present invention also relates to an epidermis equivalent that can be obtained using the method according to the invention.

[0117] 2 layer base material The two-layer substrate can be configured on an insert, nacelle, suspension system with or without an adapter for placing it on the relevant platform (culture plate adapted to the insert (or using an adapter or support element)), or it can rest on any type of device (cotton wool, grid with legs, etc.) or be placed in a Petri dish if the suspension system has legs or a lifting system for achieving an air-liquid interface. Commercially available examples are: CellCrown™ from Sigma, SnapWell™ from Corning, NetWell™ or TransWell™ from Costar, support plates used with Nunc™ inserts, or EPISKIN inserts with O-rings (Episkin insert: Episkin nacelle + O-ring (Patent FR688226A), see Figure 4). In this case, the liquid preferably consists of a suitable culture medium.

[0118] Incubation is then preferably continued until a dermis equivalent, skin equivalent, or epidermis equivalent is obtained, preferably until the equivalent has the desired characteristics. For example, for skin equivalents, incubation is then preferably continued until an equivalent exhibiting the characteristics of skin is obtained, i.e., a dermis equivalent covered by an epidermis equivalent exhibiting the four standard cell layers, i.e., the basal layer, suprabasal layer, granular layer, and stratum corneum.

[0119] Incubation is thus continued for a period preferably lasting between 7 and 21 days, even more preferably between 7 and 14 days.

[0120] Use according to the present invention The present invention relates to a method for forming a skin equivalent, a dermis equivalent, or an epidermis equivalent, b. A layer having a thickness of at least 20 μm and a porosity of 20 μm or more, obtained by electrowriting (EW) of a composition containing at least one polymer. superimposed longitudinally on a. A layer having a thickness of at least 200 nm and a porosity of 5 μm or less, obtained by electrospinning (ES) or electrowriting (EW) of a composition containing at least one polymer. The present invention relates to the use of a substrate comprising:

[0121] Of course, said use is an in vitro use.

[0122] A layer having a thickness of at least 20 μm and a porosity of 20 μm or more may be formed by electrowriting a composition comprising at least one molten polymer or at least one polymer in solution, preferably the composition comprises at least one molten polymer.

[0123] The present invention also relates to the use of a dermal equivalent according to the invention or a skin equivalent according to the invention or an epidermal equivalent according to the invention for gaining new knowledge about the dermis or skin or epidermis, respectively. Indeed, these equivalents may serve as models for studying the biology of these tissues, for example related to skin aging, the effects of ultraviolet radiation, environmental influences such as dryness or pollution, or for studying diseases.

[0124] The present invention also relates to the use of a dermis equivalent according to the invention, or a skin equivalent, or an epidermis equivalent according to the invention, for screening compounds, preferably compounds capable of having cosmetic, dermatological or pharmaceutical activity on the skin, preferably cosmetic or dermatological activity after topical application to the skin or injection into the skin.

[0125] The term "compound capable of having cosmetic, dermatological or pharmaceutical activity on the skin" refers to a compound that is known to have cosmetic, dermatological or pharmaceutical activity on the skin or that is suspected to have such activity.

[0126] The present invention further relates to a method for screening for active compounds, preferably compounds capable of having a cosmetic, dermatological or pharmaceutical activity on the skin, preferably a cosmetic or dermatological activity after topical application to the skin or injection into the skin, comprising the step of applying a candidate compound to a dermis equivalent according to the invention or a skin equivalent according to the invention or an epidermis equivalent according to the invention. Of course, said screening method is an in vitro method.

[0127] The present invention also relates to a dermis equivalent according to the invention or a skin equivalent according to the invention or an epidermis equivalent according to the invention for use in a wound dressing or for skin grafting.

[0128] The term "wound" refers to a lesion in which the skin is abraded, cut, lacerated, burned or damaged.

[0129] Skin grafting is used especially in cases of burns, diseases such as diabetes, and plastic and cosmetic surgery.

[0130] For example, in the case of the use of an epidermal equivalent according to the invention in a wound dressing, layer (ii) forms an area that provides for the treatment of human fibroblasts, which can then migrate and proliferate into this part of the equivalent in order to reconstruct healthy skin at the wound site. Furthermore, layer (ii) of the epidermal equivalent can contain a bioactive agent or a treatment agent that promotes fibroblast migration and adhesion.

[0131] Similarly, in the case of the use of the dermal equivalent according to the present invention in wound dressings, layer (i) forms an area that provides the treated human keratinocytes, which can then migrate, proliferate and colonize layer (i) to reconstruct healthy skin at the wound site. Furthermore, layer (i) of the dermal equivalent can contain a bioactive agent or a treated agent that promotes keratinocyte migration and / or proliferation.

[0132] The present invention also relates to a method of treatment comprising the step of applying a dermal equivalent according to the invention, or a skin equivalent according to the invention, or an epidermal equivalent according to the invention. Preferably, the present invention relates to a method for treating a wound, comprising the step of administering a dermal equivalent according to the invention, or a skin equivalent according to the invention, or an epidermal equivalent according to the invention to an individual in need thereof.

[0133] The invention will now be described in more detail in the following examples. [Example]

[0134] We obtained a full-thickness in vitro skin model using: - Layers (ii) of different designs (straight fibers with octagonal and decagonal designs, as well as wavy fibers) - Different porosity distribution: Layer (i): 0.01 μm 2 to 0.3 μm 2 Between Linear octagonal fused electrowriting (MEW) layer (ii): 10 μm 2 to 500 μm 2 Between Wavy MEW layer (ii): 20 μm 2 to 1000 μm 2 Between - Different seeded cell concentrations (fibroblasts: 58,000 to 1 million cells / cm 2 , keratinocytes 150,000 to 400,000 cells / cm 2 ) - Different fiber coatings (coating with ethanol, fibronectin, poly-L-lysine (PLL)), and plasma treatment - different fiber diameters (9 to 14 μm were tested for layer (ii)), - Different incubation times (11, 18, 28 and 36 days).

[0135] Under all conditions tested, fibroblasts and keratinocytes were able to attach to and reconstitute the dermis (fibroblasts only) or the complete skin model (fibroblasts and keratinocytes). Keratinocytes formed a fully differentiated epidermis with all layers (basal, spinous, granular, and stratum corneum) and expressed relevant markers (keratin 10 for the spinous and granular layers, and filaggrin for the granular and stratum corneum).

[0136] Thanks to layer (i), the inventors did not observe any invagination, infiltration or migration of keratinocytes into the dermis, regardless of the filling of the dermal portion. Keratinocytes can be seeded simultaneously with fibroblasts, which is not possible in any porous substrate without a barrier between the epidermis and the dermis.

[0137] Depending on the design, we observed differences. Indeed, extracellular matrix neosynthesis depends on the design: - Elastin is preferentially expressed in designs with straight (and less porous) fibers; - Collagen 1 content appears to be increased in designs with straight fibers.

[0138] In general, the organization of the outer cell membrane correlates with design.

[0139] Example 1 Formation of a two-layer substrate having a first layer (ii) with a wavy design material Polycaprolactone (PURASORB PC, Corbion Inc., Gorinchem, Netherlands) Episkin insert: Episkin nacelle and O-ring (described in patent FR688226A) Solution electrospinning syringe: Henke-Sass, Wolf GmbH; Tuttlingen, Germany Molten Electrolighting Syringe: Nordson EFD; Pforzheim, Germany Scanning electron microscope, TM3030Plus, Hitachi; Tokyo, Japan Crossbeam 340 scanning electron microscope, Zeiss; Oberkochen, Germany Fused Electrowriting (MEW) printer (pink), custom-made; University of Wurzburg, Germany EM ACE600 sputter coating system, Leica; Wetzlar, Germany Syringe pump, World Precision Instruments; Sarasota, FL, USA Laser cutting machine, Rayjet; Plymouth, Michigan, USA

[0140] Electrospinning of layer (i): A 15% by weight solution of medical-grade polycaprolactone (PCL) (Corbion, PC-12) was prepared in a mixed dichloromethane and dimethylformamide solvent (3:2 DCM:DMF ratio). The vial was sealed and the solution was left stirring overnight. A 5 ml syringe was loaded with the prepared solution and attached to a 27 G nozzle. A flow rate of 0.5 ml / h was used for electrospinning the polymer fibers. A voltage difference of 18 kV was applied between the nozzle and collector. A nozzle-collector distance of 14 cm was used. The electrospun fibers were collected for 30 minutes on a glass strip attached to a rotating collector. The room temperature and humidity were 20.8 °C and 43%, respectively.

[0141] The fiber-coated glass strip was then used as a substrate for melt electrospinning of layer (ii).

[0142] Layer (ii) electrolighting, wavy design: The syringe filled with PCL pellets was preheated at 75°C for at least 24 hours. To print the sinusoidal grid, a 25G nozzle was used, and the nozzle-collector distance was set at 3.75 mm. A nozzle temperature slightly below 70°C was used, and a voltage difference of 6 kV (+4.5 kV at the nozzle and -1.5 kV at the collector) was applied to initiate jetting. The pressure used to extrude the polymer was 1.5 bar. A total of 36 layers (12 layers in each direction; 3 directions) were deposited, with each layer deposited at an angle of 120° relative to the previous layer. The wavelength of the sinusoidal wave was set to 2 mm, and the amplitude alternated between 500 μm and 250 μm every 3 layers. Fiber diameters of approximately 9 to 15 μm, preferably 10 μm, were obtained, resulting in a total scaffold height of approximately 400 μm.

[0143] Example 2 Formation of a two-layer substrate having a second wavy design layer (ii) material Polycaprolactone (PURASORB PC, Corbion Inc., Gorinchem, Netherlands) Episkin insert: Episkin nacelle and O-ring (described in patent FR688226A) Solution electrospinning syringe: Henke-Sass, Wolf GmbH; Tuttlingen, Germany Molten Electrolighting Syringe: Nordson EFD; Pforzheim, Germany Scanning electron microscope, TM3030Plus, Hitachi; Tokyo, Japan Crossbeam 340 scanning electron microscope, Zeiss; Oberkochen, Germany Fused Electrowriting (MEW) printer (pink), custom-made; University of Wurzburg, Germany EM ACE600 sputter coating system, Leica; Wetzlar, Germany Syringe pump, World Precision Instruments; Sarasota, FL, USA Laser cutting machine, Rayjet; Plymouth, Michigan, USA

[0144] Electrospinning of layer (i): A 15% by weight solution of medical-grade polycaprolactone (PCL) (Corbion, PC-12) was prepared in a mixed dichloromethane and dimethylformamide solvent (3:2 DCM:DMF ratio). The vial was sealed and the solution was left stirring overnight. A 5 ml syringe was loaded with the prepared solution and attached to a 27 G nozzle. A flow rate of 0.5 ml / h was used for electrospinning the polymer fibers. A voltage difference of 18 kV was applied between the nozzle and collector. A nozzle-collector distance of 14 cm was used. The electrospun fibers were collected for 30 minutes on a glass strip attached to a rotating collector. The room temperature and humidity were 20.8 °C and 43%, respectively.

[0145] The fiber-coated glass strip was then used as a substrate for melt electrospinning of layer (ii).

[0146] Layer (ii) electrolighting, wavy design: The syringe filled with PCL pellets was preheated at 80°C for at least 24 hours. To print the sinusoidal grid, a 25G nozzle was used, the nozzle-collector distance was set at 3.6 mm, and the collector speed was 180 mm / min. A nozzle temperature slightly below 80°C was used, and a voltage difference of 6 kV (+4.5 kV at the nozzle and -1.5 kV at the collector) was applied to initiate jetting. The pressure used to extrude the polymer was 2 bar. A total of 30 layers (15 layers in each direction; 2 directions) were deposited, with each layer deposited at a 90° angle relative to the previous layer. Fiber diameters of approximately 13 to 15 μm, preferably 14 μm, were obtained, resulting in a total scaffold height of approximately 400 μm.

[0147] Example 3 Formation of a two-layer substrate having a linear design layer (ii) Electrospinning of layer (i) A 15% solution of medical-grade polycaprolactone (PCL) (Corbion, PC-12) was prepared in a mixed dichloromethane and dimethylformamide solvent (3:2 DCM:DMF ratio). The glass vial was sealed and the solution was left stirring overnight. A 5 ml syringe was loaded with the prepared solution and attached to a 27 G nozzle. A flow rate of 0.5 ml / h was used for electrospinning the polymer fibers. A voltage difference of 18 kV was applied between the nozzle and collector. A nozzle-collector distance of 14 cm was used. The electrospun fibers were collected for 30 minutes on a glass strip attached to a rotating collector. The room temperature and humidity were 20.8 °C and 43%, respectively.

[0148] Electrolighting of layer (ii) Decagonal Design: A syringe was filled with PCL pellets and preheated at 77 °C for at least 24 hours. A 22G nozzle was attached to the loaded syringe. A 1.5 bar air pressure was applied to the syringe, and a 6 kV voltage difference (+4.5 kV at the nozzle and -1.5 kV at the collector) was applied to initiate liquid ejection. A stabilized print was performed before printing the decagonal structure. The decagonal design consisted of 30 layers of fibers printed in a grid, with each layer printed / electrowritten at a 72° (360 / 5) rotation angle relative to the previous layer. A fiber spacing of 150 μm was used for each layer. A nozzle-to-collector distance of 3.6 mm was used for all prints. Fiber diameters of approximately 8 to 13 μm, with 10 μm being preferred, were obtained, and the total height of the resulting layers was approximately 400 μm.

[0149] Example 4 Obtaining a dermal equivalent using a two-layer substrate with a linearly designed layer (ii) The bilayer substrates were treated with ethanol to sterilize and increase cell attachment.

[0150] To achieve culture at the air-liquid interface, the two-layer substrate was attached to the culture insert with an O-ring according to the following orientation: layer (i) inside the insert and layer (ii) at the bottom. Layer (ii) has an octagonal design.

[0151] The bilayer substrate was washed with phosphate-buffered saline and then incubated in fibroblast culture medium ("FHN2D": DMEM containing 2 mM glutamine, antibiotics, and 10% fetal bovine serum).

[0152] Fibroblasts (NHFs) were isolated from skin tissue obtained from plastic surgery after patients provided informed consent and expanded in FHN2D medium.

[0153] NHFs were trypsinized (trypsin EDTA 0.05%) (4–6 min at 37°C), counted, and pelleted by centrifugation at 190 g for 5 min.

[0154] The pellet was resuspended in fibroblast medium ("FHN3D": DMEM containing 2 mM glutamine, antibiotics, and 10% calf serum and 1 mM ascorbic acid) for a final NHF concentration of 6 million NHFs per ml of FHN3D medium.

[0155] The culture inserts were placed in the Petri dishes with the two-layer substrate facing up (layer (ii) on top).

[0156] For NHF seeding, 100 μL of cell solution was added per insert, i.e., 600,000 NHFs / cm. 2 The cells were then seeded onto layer (ii) on the insert at a ratio of 0.01 to 0.01, and the insert containing the cells was then incubated at 37°C for 1 hour to allow cell attachment.

[0157] For the dermal equivalent culture process, the inserts were suspended in 6-well plates with FHN3D medium on top and two-layer substrate on the bottom.

[0158] The inserts were then incubated in this FHN3D medium at 37°C and 5% CO2 for 11 days. The resulting dermal equivalents were observed by light microscopy and multiphoton microscopy. We observed the specific spindle shape of fibroblasts histologically (eosin, hematoxylin, saffron) along with the adhesion of these cells and the filling of the substrate with extracellular matrix (ECM). Multiphoton microscopy showed an organization of collagen 1 similar to that of human skin.

[0159] Example 5 Obtaining a dermal equivalent with a wavy design layer (ii) In this example, the same protocol as in Example 3 is applied, except that the two-layer substrate used has a layer with a wavy design for layer (ii).

[0160] The resulting dermal equivalents were observed under a light microscope and by multiphoton microscopy.

[0161] The inventors observed that between the dermal equivalents of Example 4 and Example 5, the adhesion of these cells and the filling of the extracellular matrix (ECM) of the substrate, as shown histologically (eosin, hematoxylin, and saffron) along with the particular spindle shape of the fibroblasts, were more uniform in Example 4, which may be due to the heterogeneous porosity in Example 5. Furthermore, multiphoton microscopy showed a different organization of collagen 1 fibers between Example 4 and Example 5. These results demonstrate that the change in design induces changes in the organization and ECM filling of the substrate.

[0162] Example 6 Obtaining a complete skin equivalent with a linearly designed layer (ii) In this example, layer (ii) has a decagonal design. The substrate was prepared as in Example 4.

[0163] In this example, the NHF pellet was resuspended in FHN3D medium, with a final NHF concentration of 4 million NHFs per ml of FHN3D medium.

[0164] The culture inserts were placed in Petri dishes with the two-layer substrate facing up (layer (ii) on top). For NHF seeding, 100 μL of cell solution was added per insert, i.e., 400,000 NHFs / cm. 2 The cells are seeded onto the inserts at a ratio of 0.1:1, and the inserts containing the cells are then incubated at 37°C for 1 hour to promote cell attachment.

[0165] For the dermal equivalent culture process, inserts were suspended in 6-well plates with FHN3D medium on top and two-layer substrate on the bottom. The dermal equivalents were then incubated in FHN3D medium at 37°C, 5% CO for 4 days (for a total of 18 days of culture) or 14 days (for a total of 36 days of culture).

[0166] Keratinocytes (NHKs) were isolated from skin tissue obtained from plastic surgery after patients provided informed consent, and then NHKs were expanded in G7F expansion medium (Black et al. (2005) Tissue Eng. 11:723-733) using feeder cells.

[0167] The resulting NHKs were trypsinized (trypsin-EDTA 0.05% for 8–10 min at 37°C), counted, and pelleted by centrifugation at 190 g for 5 min, and the pellet was then resuspended in G7F amplification medium (containing 0 to 1 mM ascorbic acid).

[0168] After centrifugation of the keratinocytes, they were suspended in G7F medium to a final NHK concentration of 300,000 NHK per ml of G7F medium, and then manually seeded into the inserts to a final concentration of approximately 150,000 NHK / cm. 2 It was.

[0169] The whole skin equivalents were then submerged in G7F medium containing 0 to 1 mM ascorbic acid at 37°C, 5% CO2 and incubated for 3 days for a total of 18 days of culture or 7 days for a total of 36 days of culture.The whole skin equivalents were then floated at the air-liquid interface in G3F differentiation medium (Black et al. (2005) Tissue Eng. 11:723-733 + 0 to 1 mM ascorbic acid) at 37°C, 5% CO2 for 11 days for a total of 18 days of culture or 15 days for a total of 36 days of culture.

[0170] We observed satisfactory differentiation of the epidermis with all expected layers (basal, suprabasal, granular, and stratum corneum). Furthermore, the dermis retained satisfactory fibroblast adhesion, and no epidermal invagination into the dermis was observed. Histologically (eosin, hematoxylin, and saffron) and immunofluorescence studies revealed the synthesis of dermal extracellular matrix (ECM), such as collagen 1, fibrillin, or elastin, as well as ECM at the dermal-epidermal junction, such as collagen 4 and perlecan, with the amounts of these proteins increasing between 18 and 36 days of total culture. Furthermore, the organization of collagen fibers around the fibers of the scaffold was observed by multiphoton microscopy. In response to vitamin C, an increase in ECM loading, particularly of collagen 1 (labeled with saffron or observed by multiphoton microscopy), was observed in the scaffold.

[0171] Example 7 Obtaining a full skin equivalent with a wavy design layer (ii) In this example, the same protocol as in Example 6 was applied, except that the two-layer substrate used had a layer with a wavy design for layer (ii).

[0172] After centrifugation of the keratinocytes, they were suspended in G7F medium to a final NHK concentration of 300,000 NHK per ml of G7F medium, and then manually seeded into the inserts to a final concentration of approximately 150,000 NHK / cm. 2 It was.

[0173] The whole skin equivalents were then submerged in G7F medium containing 0 to 1 mM ascorbic acid and incubated for 7 days at 37°C, 5% CO. The whole skin equivalents were then floated at the air-liquid interface in G3F differentiation medium (Black et al. (2005) Tissue Eng. 11:723-733 + 0 to 1 mM ascorbic acid) for 14 days at 37°C, 5% CO.

[0174] The inventors observed histological (eosin, hematoxylin, and saffron) and immunofluorescence analyses of the skin equivalents of Example 6 and Example 7 that dermal extracellular matrix (ECM), such as collagen 1, fibrillin, or elastin, and dermal-epidermal junction ECM, such as collagen 4 and perlecan, were synthesized, and the amounts of these proteins increased between 18 and 36 days of total culture. Furthermore, collagen organization, with fibers wrapped around the fibers of the substrate, was observed with multiphoton microscopy. Increased ECM packing in the substrate, particularly of collagen 1 (labeled with saffron or observed with multiphoton microscopy), was observed in response to vitamin C. Differences between Example 6 and Example 7 include a more heterogeneous organization of matrix proteins with the presence of void areas in Example 7, lower elastin expression in Example 7, and a different organization of collagen 1 fibers (seen with multiphoton microscopy) with larger collagen 1 fiber bundles in Example 7. These results demonstrate that changes in design and porosity result in changes in the organization, loading, and content of ECM in the substrate.

Claims

1. 1. A method for producing a dermis equivalent, comprising: a. forming a layer (i) having a thickness of at least 200 nm and a porosity of 5 μm or less by electrospinning (ES) or electrowriting (EW) of a composition comprising at least one polymer; b. forming a layer (ii) having a thickness of at least 20 μm and a porosity of at least 20 μm by electrowriting (EW) of a composition comprising at least one polymer; c. Seeding layer (ii) with dermal and / or subcutaneous tissue cells wherein steps a and b are performed sequentially in this order or in the reverse order, and the second electrospinning step is performed longitudinally along the layer produced by the first electrospinning step.

2. A method for producing a skin equivalent, comprising the method for producing a dermal equivalent according to claim 1, further comprising the step of seeding layer (i) with epidermal cells after step c) and / or after the two steps a) and b).

3. 3. The method of claim 1 or 2, wherein layer (i) is filled or coated with one or more bioactive agents, optionally in the form of a hydrogel that mimics the matrix of the dermal-epidermal junction, and / or layer (ii) is filled or coated with one or more bioactive agents, optionally in the form of a hydrogel that mimics the dermal matrix.

4. 4. The method according to claim 1, wherein the dermal and / or subcutaneous tissue cells are cultured in a suitable culture medium in a liquid or at an air-liquid interface.

5. 1. A method for producing an epidermis equivalent, comprising: a. forming a layer (i) having a thickness of at least 200 nm and a porosity of 5 μm or less by electrospinning (ES) or electrowriting (EW) of a composition comprising at least one polymer; b. forming a layer (ii) having a thickness of at least 20 μm and a porosity of at least 20 μm by electrowriting (EW) of a composition comprising at least one polymer; c. Seeding epidermal cells into layer (i) wherein steps a and b are performed sequentially in this order or in the reverse order, and the second electrospinning step is performed longitudinally along the layer produced by the first electrospinning step.

6. 6. The method according to claim 1, wherein layer (ii) is formed by electrowriting a composition comprising at least one molten polymer (melt electrowriting MEW) or a composition comprising at least one polymer in solution.

7. The method of any one of claims 2 to 6, wherein the epidermal cells are cultured in a suitable culture medium in a liquid or at an air-liquid interface.

8. A dermis equivalent obtainable using the method according to any one of claims 1, 3-4 and 6.

9. A skin equivalent obtainable using the method according to any one of claims 2 to 4 and 6 to 7.

10. 8. An epidermis equivalent obtainable using the method according to any one of claims 5 to 7.

11. For forming a skin equivalent or a dermis equivalent or an epidermis equivalent, b. A layer having a thickness of at least 20 μm and a porosity of 20 μm or more, obtained by electrowriting (EW) of a composition containing at least one polymer. superimposed longitudinally on a. A layer having a thickness of at least 200 nm and a porosity of 5 μm or less, obtained by electrospinning (ES) or electrowriting (EW) of a composition containing at least one polymer. Use of a substrate comprising:

12. 12. Use of a substrate according to claim 11, wherein a layer having a thickness of at least 20 μm and a porosity of at least 20 μm is formed by electrowriting (EW) of a composition comprising at least one molten polymer (melt electrowriting MEW) or by electrowriting (EW) of a composition comprising at least one polymer in solution.

13. Use of a dermis equivalent according to claim 8, a skin equivalent according to claim 9 or an epidermis equivalent according to claim 10 for screening compounds.

14. Use of a dermis equivalent as described in claim 13 or a skin equivalent as described in claim 13 or an epidermis equivalent as described in claim 13 for screening compounds that may have cosmetic, dermatological or pharmaceutical activity on the skin.

15. A method for screening for active compounds, comprising the step of applying a candidate compound to a dermis equivalent as described in claim 8, or a skin equivalent as described in claim 9, or an epidermis equivalent as described in claim 10.

16. A method for screening compounds according to claim 15, wherein the compound is capable of having cosmetic, dermatological or pharmaceutical activity on the skin.

17. 9. A dermis equivalent according to claim 8 for use in wound dressings or for skin grafting.

18. 10. The skin equivalent of claim 9 for use in wound dressings or for skin grafting.

19. 11. The epidermal equivalent of claim 10 for use in wound dressings or for skin grafting.

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