Processes for producing a reconstructed skin model, and applications thereof

Abstract

The present patent application relates to the field of skin tissue engineering and aims to generate a reconstructed skin model. The present invention more particularly provides processes to create a three-dimensional construct possessing key features reminiscent of native skin tissue, including capillaries, skin appendages and immune cells. The present invention further relates to this three-dimensional skin construct, and applications thereof.

Description

[0001] PROCESSES FOR PRODUCING A RECONSTRUCTED SKIN MODEL, AND APPLICATIONS THEREOF

[0002] INTRODUCTION

[0003] The present patent application relates to the field of skin tissue engineering and aims to generate a reconstructed skin model. The present invention more particularly provides processes to create a three-dimensional construct possessing key features reminiscent of native skin tissue, including capillaries, skin appendages and immune cells. The present invention further relates to this three-dimensional skin construct, and applications thereof.

[0004] Human skin is the body’s largest and primary protecting organ, serving as a first physical barrier against the environment. It plays crucial roles in the regulation of body temperature, and defense against pathogens, UV radiation, as well as physical injuries. The skin also plays a role in immunologic surveillance, sensory perception, control of insensible fluid loss, and homeostasis in general. To achieve these functions, this complex organ is made up of two primary layers: the upper and thinnest layer is the epidermis, while the thicker layer underneath is the dermis- both varying significantly in their composition and function. A third layer can be found beneath the dermis, named the hypodermis.

[0005] The waterproof barrier and skin color is provided by the epidermis, which contains several strata essentially made of keratinocytes. Keratinocytes found at the very top are continuously shed and replaced by new cells from deeper layers, a process called keratinization. Proliferating keratinocytes can indeed be typically observed in the basal stratum, while differentiated keratinocytes are found suprabasally and dying cells are at the most superficial stratum. Other epidermal cell types include melanocytes, which produce melanin, the pigment responsible for skin color and confers protection from UV radiation, as well as immune cells such as Langerhans cells, which provide a secondary protective barrier.

[0006] The dermis provides strength and elasticity to the skin thanks to the presence of fibroblasts, which produce the key components of the extracellular matrix including collagen and elastin fibers. Immune cells are also present, albeit different in nature as these typically include macrophages and mast cells. In contrast to the epidermis, the dermis does not continuously shed and regenerate, and houses blood vessels, nerves, as well as skin appendages (hair follicles, sweat glands, and sebaceous glands). At last, the hypodermis, also called the subcutaneous tissue, is primarily made of fat and connective tissue, and, as such, plays an important role in glucose homeostasis and lipid metabolism.

[0007] Reconstructed human skin models were developed as far back as the 1980s to substitute for animal experimentation. The aim of such models was to provide a more accurate and ethical solution, not only for the cosmetic industry but also for drug discovery and development of topical compounds and their toxicity evaluation. Such models can also be useful to study human skin development, disease modelling or skin reconstruction. These needs have become more pressing in recent years due to the emergence of new regulatory requirements and guidelines, especially in the European Union where animal testing of cosmetic products is banned and will likely extend to any chemical and pharmaceutical dermal testing.

[0008] To that end, two main types of three-dimensional (3D) human reconstructed skin have been developed from in vitro cultures of native keratinocytes and / or fibroblasts on inert polymer matrixes: those mimicking only the epidermis, and full thickness models typically consisting of a dermal equivalent overlayed by an epidermal equivalent. In the full thickness models, fibroblasts and keratinocytes are configured in a bilayer, embedded in a matrix, in air-liquid interfaces to form stratified layers mimicking the general architecture of native skin. For illustrative purpose, epidermal models are today commercialized under the trademarks SkinEthic™ RHE, EpiSkin™ and EpiDerm™, and full thickness skin models, such as GraftSkin™, EpidDermFT™ or Pheninon™, are also on the market. However, due to their low cellular complexity, these models lack skin appendages as well as the components of the immune system, thereby limiting their use for screening potentially irritating compounds.

[0009] In order to address these shortcomings, organoid technology has been relied on to attempt reproducing the complexity of human skin morphology and function. To this day, the most successful organoid models are derived from stem cells, but require very complex protocols.

[0010] In a first step, Lee, Koehler et al. (Cell Rep., 2018; 22(l):242-254) generated skin organoids with protruding bulb-like structures by inducing the formation and self-organization of skin cell types from mouse stem cells. These bulb-like structures emerged after about 30 days under well-defined culture conditions, and were identified as hair germ-like buds. While no vascular formation or immune cells were reported, sebaceous glands were observed. Moving from mouse to human stem cells proved to be much more cumbersome and time-consuming. To do so, the same authors separated human embryonic stem cells into single cells, placed them into 96-well plates to form uniform aggregates, which were then cultured in a particular differentiation medium for an extended time period. It was only after about 3 to 4 months of incubation that hair pegs appeared followed by sebaceous glands, yet end-stage (5 months) organoids lacked sweat glands, immune cells notably Langerhans cells, or an endothelial network (Lee, Koehler et al., Nature 2020; 582(7812): 399-404). A more recent study published by the same authors proposed an optimized protocol, but critical features of normal human skin were still missing, namely sweat glands, endothelial cells as well as immune cells (Lee, Koehler et al, Nature Protocols 2022; 17: 1266-1305).

[0011] In the meantime, another team (Ebner-Peking et al., Theranostics 2021; 11(17): 8430- 8447) implemented a so-called simplified strategy to produce a floating spheroid skin organoid from the self-assembly of human adult or iPSC-derived fibroblasts, keratinocytes, and endothelial progenitors, through a week-long culture in suspension under the essential control of human platelet lysate (hPL). Prior transfection of FGF-7 mRNA was further required for hiPSC-KC. It was only after in vivo co-transplantation with endothelial cells in presence of hPL that the grafted skin cells did stratify into epidermal and dermal layers and a proper vascularization was detected. Immune cells and melanin pigmentation were lacking from that model, and no skin appendage was reported.

[0012] Thus, despite the tremendous efforts to recapitulate the complex features and functions of human skin features in vitro, there is still a need in the art to provide an improved three- dimensional human skin model having capillaries, skin appendages and immune cells.

[0013] The present invention addresses the above needs in the art.

[0014] SUMMARY OF THE INVENTION

[0015] The present invention is based at least on the Inventor’s discovery that cells constitutive of the human native skin, typically isolated from skin biopsies, when cultured in vitro under well-defined conditions, can form multicellular spheroid aggregates with a basic skin-like structure and composition. Key hallmarks of these spheroids include, inter alia, the selforganization of dermal cells and epidermal cells, notably the emergence of an endothelial network, and the presence of immune cells.

[0016] These spheroids can then be used as building-blocks to engineer an artificial tissue model that recapitulates the complexity and organization of human skin, with mitotically and metabolically active cells (secretion of ECM components such as collagen), a dermal and epidermal stratification, polarization of the epidermis, the presence of vascular-like structures, immune cells and pigmentation (melanin), as well as the formation of sweat glands.

[0017] To this end, the Inventors have implemented processes requiring relatively few steps, and which are described in more details below. These processes address the limitations of previous attempts at generating skin models, which were unable to fully mimic the complex organization and interactions between the numerous critical actors of the skin that are fibroblasts, keratinocytes, endothelial cells and immune cells. The processes of the present invention notably overcome the difficulties reported in the art to reproduce in vitro skin capillarization, skin appendages such as sweat glands, and an immune environment. It is also notable that, when assembling the spheroids into the artificial human skin model of the invention, the viability of the cells is maintained throughout the process. These processes also provide a rapid, scalable and cost-effective way for generating large quantities of skin spheroids and artificial human skin, which can then be used in a wide range of applications, including not only in vitro applications but also therapeutic applications.

[0018] In a first aspect, the present invention relates to a process for the in vitro obtention of a multicellular human skin spheroid, comprising:

[0019] (a) mixing in suspension a first population of cells constitutive of a human epidermis layer with a second population of cells constitutive of a human dermis layer, in a ratio ranging from about 1 : 4 to about 1 : 14 between the number of cells in the first population and the number of cells in the second population; and

[0020] (b) incubating the cell suspension obtained in step (a) in an endothelial cell growth medium.

[0021] In a preferred embodiment, the first population of cells constitutive of the human epidermis layer comprises keratinocytes, melanocytes, and immune cells of the epidermis, and optionally epidermal stem cells; and / or the second population of cells constitutive of a human dermis layer comprises fibroblasts, endothelial cells, and immune cells of the dermis, and optionally dermal stem cells.

[0022] In a preferred embodiment, the first population of cells and / or the second population of cells are primary cells, preferably adult primary cells. In a preferred embodiment, the above described process comprises, prior to step (a): separating an isolated human skin sample into an epidermis layer and a dermis layer; and dissociating the cells from each separated layer, thereby obtaining a first population of cells constitutive of a human epidermis layer and a second population of cells constitutive of a human dermis layer, respectively.

[0023] In a preferred embodiment, in step (a), the ratio between the number of cells of the first population and the number of cells of the second population is about 1 : 9.

[0024] In a preferred embodiment, in step (b), the cell suspension is incubated under low- adherent or non-adherent culture conditions.

[0025] In a preferred embodiment, the endothelial cell culture medium used in step (b) comprises ascorbic acid, hydrocortisone, and growth factors EGF (Epidermal Growth Factor), FGF (Fibroblast Growth Factor), IGF (Insulin Growth Factor) and VEGF (Vascular Endothelial Growth Factor); and optionally heparin, serum, or any combination thereof.

[0026] In a preferred embodiment, the cell suspension is incubated in step (b) for a time period of at least about 5 days to about 10 days, preferably at least about 7 days.

[0027] In a preferred embodiment, the cell suspension is incubated in step (b) for a time period of at most about 15 days to about 25 days, preferably at most about 20 days.

[0028] In another aspect, the present invention pertains to a multicellular human skin spheroid, obtainable or obtained by the process as defined in any one of the preceding claims.

[0029] In a preferred embodiment, the multicellular human skin spheroid comprises a network of endothelial cells.

[0030] In another aspect, the present invention is directed to a method for the in vitro assembly of multicellular human skin spheroids into an artificial human skin construct, comprising:

[0031] (1) placing a plurality of multicellular human skin spheroids of the invention on or in a porous scaffold optionally comprising extracellular matrix components; and

[0032] (2) culturing the plurality of multicellular human skin spheroids placed on or in the scaffold in step (1), in presence of an organoid growth medium.

[0033] In a preferred embodiment, the above described method comprises, prior to step (1): (0) obtaining a plurality of multicellular human skin spheroids according to the above-described process, especially wherein the cell suspension is incubated in step (b) of that process for a time period of at least about 5 days to about 10 days, preferably at least 7 days. In a preferred embodiment, the plurality of multicellular human skin spheroids used in step (1) comprises from about 5 to about 50 multicellular skin spheroids.

[0034] In a preferred embodiment, the extracellular matrix components are extracellular matrix components of the skin.

[0035] In a preferred embodiment, the porous scaffold is in form of a hydrogel comprising extracellular matrix components.

[0036] In a preferred embodiment, the porous scaffold comprises at least one synthetic polymer, such as a poly(epsilon-caprolactone) (PCL).

[0037] In a preferred embodiment, the porous scaffold comprises at least one synthetic polymer, such as a poly(epsilon-caprolactone) (PCL), in combination with a hydrogel comprising extracellular matrix components.

[0038] In a preferred embodiment, the organoid growth medium is an epidermalization medium.

[0039] In a preferred embodiment, the plurality of multicellular human skin spheroids is cultured, in step (2), at an air-liquid interface and / or under low-adherent or non-adherent culture conditions.

[0040] In a preferred embodiment, the plurality of multicellular human skin spheroids is cultured, in step (2), for a time period of at least about 10 days to about 30 days.

[0041] In another aspect, the present invention relates to an artificial human skin construct, obtainable or obtained by the above-described method.

[0042] In a preferred embodiment, the artificial human skin construct comprises immune cells, one or more vascular vessel-like structures and one or more skin appendages, especially sweat gland-like structures.

[0043] In another aspect, the present invention pertains to the use of a multicellular human skin spheroid or artificial human skin construct, as described herein, in in vitro assays.

[0044] In another aspect, the present invention is directed to a multicellular human skin spheroid or artificial human skin construct, as described herein, for use as a medicament.

[0045] LEGENDS TO THE FIGURES

[0046] Figure 1. Methods according to the invention. (A) Native epidermal (E) and dermal (D) cells are mixed at day 0 (DO) with a 1 : 9 ratio. Process (1): Cells spontaneously self-organize into spheroids in ultra-low attachment plate wells and mature for 7 days or 20-21 days. Process (2): After 7 days of self-organization, the neoformed spheroids are placed on a non-adherent insert coated with dECM and put in an Air-Liquid interface for additional ~20 days to allow their fusion and maturation. (B) Composition of the native cells constitutive of the epidermis and dermis used in process (1). To characterize this composition, cell populations obtained after digestion of skin biopsies were analyzed by flow cytometry. Each cell population was characterized by a combination of markers specific of skin cell types consisting of keratinocytes, melanocytes, fibroblasts, endothelial cells, Langerhans cells, monocytes / macrophages, dendritic cells, neutrophils, eosinophils, lymphocytes T, and lymphocytes B.

[0047] Figure 2. Multicellular skin spheroid according to the invention. (A) Phase contrast images of a spheroid formation from day 5 to day 26, using the EGM2 medium. Cells forming an aggregate are indicated by a white marking. (B) Confocal immunofluorescence microscope images of immunostained spheroids at day 7, using the EGM2 medium: in addition to DAPI staining, (Bl) shows the staining for cytokeratin 14 (basal epidermal cells) and cytokeratin 10 (suprabasal epidermal cells), (B2) the staining for CD31 (endothelial cells), and (B3-B4) the staining for CD68 (macrophages) and CDl lc (dendritic cells). (C) Confocal immunofluorescence microscope images of immunostained spheroids at day 20, using the EGM2 medium: in addition to DAPI staining, (Cl) shows the staining for cytokeratin 14 and cytokeratin 10, (C2) the staining for CD31, and (C3) the staining for CD68 and CD11c. At last, spheroids are stained with a pan-cytokeratin antibody to highlight all keratinocytes regardless of differentiation (C4).

[0048] Figure 3. Spheroids assembly according to the invention (embodiment using Skin dECM hydrogel). Several spheroids assemblies were cultured for 14 to 21 days, at the air-liquid interface (supplemented 3dGRO™ medium) in a non-adherent insert coated with Skin dECM hydrogel. (A) Phase contrast microscope image of a spheroid assembly. (B-I) Histological images of spheroid assemblies. For histological analysis, each spheroid assembly was embedded in agarose, followed by inclusion in paraffin, then sectioned and stained. The hematoxylin eosin (HE) staining shows the ongoing fusion of about 30 spheroids into 3 main zones (each encircled), with the self-organization of epidermal cells (lighter pink) and dermal cells (darker pink) (B). The establishment of an epidermis / dermis interface is clearly apparent from the vimentin staining (C), the dermis being essentially in the center (dark brown) surrounded, intermittently, by the epidermis. Upon magnification, HE staining shows the formation of an epidermal layer with typical epithelial differentiation, basal layer in depth and Malpighian differentiation of large cells towards the surface (arrow) (D), as well as the presence of brown pigmented melanin (arrows) (E). Immunostaining with the anti-vimentin antibody shows the dermal compartment (dark brown) underneath the epidermal layer (F), while staining with the anti-Ki67 antibody shows the nuclei (dark brown) of proliferating cells (arrows) at the dermis-epidermis interface (G). The formation of a vascular vessel-like structure is apparent: vascular lacunas (gaps lined with endothelial cells) were detected after staining with the anti- CD31 antibody (arrows) (H) with proliferating cells (positive for Ki67) at the edge of the lacunas (arrows) (I). The presence of immune cells was detected by anti-CD45 antibody staining (arrows) (J), dendritic cells and macrophages being more specifically detected by anti- CDl lc (M) and anti-CD68 (N) staining, respectively (arrows). Finally, Masson Trichrome staining shows the presence of several mature collagen fibers (one fiber is highlighted by the arrow) (K), and Periodic acid Schiff staining the presence of sweat glands (arrows) (L). (D-N) Scale bar: 50pm.

[0049] Figure 4. Spheroids assembly according to the invention (embodiment using a 3D-PCL scaffold coated with Skin dECM hydrogel). A spheroid assembly was cultured for 21 days, at the air-liquid interface (supplemented 3dGRO™ medium) using a 3D-PCL scaffold coated with Skin dECM hydrogel and placed on a non-adherent insert. (A-H) Histological images of the spheroid assembly. For histological analysis, the spheroid assembly was embedded in agarose, included in paraffin, then sectioned and stained. Hematoxylin eosin (HE) staining of the whole spheroid assembly (A) (left side: longitudinal section; right side: transversal section). Upon magnification, HE staining shows the self-organization of epidermal cells (arrows) with a layer-like structure on top of dermal cells (B), as well as the presence of a sweat gland (F). Ki67-positive staining shows the presence of many proliferating cells across the spheroid assembly (dark spots, e.g. as showed by arrows) (C). Vascular lacunas (gaps lined with endothelial cells) can be observed after anti-CD31 antibody staining (arrow) (D). Masson Trichrome staining detects the presence of mature collagen fibers (highlighted by the arrow) (E). The presence of dendritic cells and macrophages was detected by anti-CDllc (G) and anti-CD68 (H) antibodies staining, respectively (arrows). (B-H) Scale bar: 50pm.

[0050] Figure 5. Spheroids assembly according to the invention (embodiment using a 3D-PCL scaffold). A spheroid assembly was cultured for 21 days, at the air-liquid interface (supplemented 3dGRO medium) using a 3D-PCL scaffold placed on a non-adherent insert. (A-G) Histological images of the spheroid assembly. The spheroid assembly was embedded in agarose, included in paraffin, then sectioned and stained. Upon magnification, HE staining shows the self-organization of epidermal cells (arrows) with a layer-like structure on top of dermal cells (A), as well as the initial development of a sweat gland (B). Ki67-positive staining shows the presence of many proliferating cells across the spheroid assembly (dark spots, e.g. as showed by arrows) (C). Masson Trichrome staining detects the presence of collagen fibers (highlighted by the arrow) (D). The presence of dendritic cells and macrophages was detected by anti-CD68 (E) and anti-CDllc (F) antibodies staining, respectively (arrows). The initial organization of vascular lacunas (gaps lined with endothelial cells) can be observed after anti- CD31 antibody staining (arrow) (G). Scale bar: 50pm.

[0051] DETAILED DESCRIPTION OF THE INVENTION

[0052] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, nomenclatures used herein, and techniques of cell culture and tissue engineering are those well-known and commonly used in the art.

[0053] The term "comprising?" which is synonymous with "including" or "containing", is inclusive or open-ended and does not exclude additional, unrecited element or method step.

[0054] By contrast, the term "consisting of excludes the presence of any additional, unrecited element or method step.

[0055] The term "consisting essentially of or "comprising substantially" means that additional, unrecited element or method step can be present, under the proviso that such element or step does not materially affect the essential characteristics of the described or claimed subject-matter.

[0056] In some embodiments not otherwise explicitly recited, any instance of “ comprising ' may be replaced by "consisting of or, alternatively, by "consisting essentially of .

[0057] By "about? it is meant that the measured value may vary within a certain range depending on the margin of error of the method or device used to evaluate the parameter of interest. In the context of the present invention, it preferably means that the measured value may be 10% away, advantageously 5%, 4%, 3%, 2%, or 1% away, from the given numerical value and that the recited range includes both endpoints.

[0058] The present invention may be understood more readily by reference to the following detailed description, included preferred embodiments of the invention, and examples included herein.

[0059] The present invention primarily provides a two-step process for generating multicellular skin spheroids, which are useful as building-block units for engineering a functional tissue skin model. As demonstrated in the experimental section, this process essentially aims at mixing and promoting the aggregation of two key groups of cells into a spherical structure in which the multiple cells types are self-organized, without using any exogenous scaffold (e.g. hydrogel, etc.). In other words, it is immediately apparent that this initial process is a scaffold-free process.

[0060] The present invention thus relates, in a first aspect, to a process for the in vitro obtention of a multicellular human skin spheroid, comprising:

[0061] (a) mixing in suspension a first population of cells constitutive of a human epidermis layer with a second population of cells constitutive of a human dermis layer, in a ratio ranging from about 1 : 4 to about 1 : 14 between the number of cells in the first population and the number of cells in the second population; and

[0062] (b) incubating the cell suspension obtained in step (a) in an endothelial cell growth medium.

[0063] The term “layer”, “cell layer”, “stratum” or “cell stratum” typically refers to a sheet of cells that are associated together and that are arranged relative to each other in a spatially- defined architecture. A layer may be in the form of a monolayer (i.e. a single sheet), as in e.g. a 2D cell culture, or in the form of a multilayer (i.e. a plurality of sheets), as e.g. in a tissue of an organism. A layer may form a contiguous, substantially contiguous, or non-contiguous sheet of cells.

[0064] By “epidermal layer”, it is meant the outer layer of the skin in an organism, which is typically a stratified squamous epithelium. In humans, the epidermal layer is a multilayer. This skin layer is made essentially of, but not only, keratinocytes. The term “dermal layer" represents the inner layer of skin in an organism, that is comprised between the epidermis and subcutaneous tissues, and which essentially consists of connective tissue. In humans, the dermal layer is a multilayer. This skin layer is made primarily of, but no only, fibroblasts.

[0065] When referring to “a population of cells constitutive of an epidermis or a dermis layer ”, this is meant to pertain to a population or group of cells isolated from an epidermis layer or dermis layer, said layer being taken as a whole. In other words, this term designates the multiple cell types that naturally reside in an epidermal layer or in a dermal layer, respectively. Advantageously, this term may also further refer to the respective natural proportion of these cells within that layer.

[0066] In step (a), it shall be understood that the two populations of cells are typically mixed in a single suspension and / or that the cells of those mixed populations are dispersed within said suspension.

[0067] In a preferred embodiment, the population of cells constitutive of a human epidermis layer (or human epidermal cells), as used in the instant process, comprises, or consists essentially of, keratinocytes, melanocytes, and immune cells of the epidermis (such as Langerhans cells, neutrophils, eosinophils, and / or lymphocytes such as lymphocytes T), and optionally epidermal stem cells.

[0068] In a preferred embodiment, the population of cells constitutive of a human dermis layer (or human dermal cells), as used in the instant process, comprises, or consists essentially of, fibroblasts, endothelial cells, and immune cells of the dermis (such as dendritic cells, macrophages, monocytes, neutrophils, eosinophils, and / or lymphocytes such as lymphocytes T / lymphocytes B), and optionally dermal stem cells.

[0069] The first population of cells constitutive of a human epidermis layer and the second population of cells constitutive of a human dermis layer are used herein to generate a multicellular human skin spheroid.

[0070] The term “multicellular skin spheroid” designates a three-dimensional aggregate or cluster of multiple cell types (heterotypic), that has a spherical closed or confined shape, and in which the cells are self-organized. Cellular self-organization is a well-known spontaneous biological process by which interacting cells organize and arrange themselves spatially in higher order tissue-like structure, pattern and / or morphology. This self-organization phenomenon is commonly observed in in vitro tissue engineering, where the cells can display morphological and structural features reminiscent of their native tissue.

[0071] In the case of the present invention, self-organization (also referred herein as autoorganization) means that the cells self-assemble to form one or more key features of skin structure, pattern and / or morphology within the skin spheroid. As observed herein, these cells can notably organize into at least two main poles or regions in the generated spheroid. One pole or region can comprise, or consist essentially of, cells constitutive of a dermis layer, especially fibroblasts; this pole or region is referred herein as the dermal pole or region. The other pole or region can comprise, or consist essentially of, cells constitutive of an epidermis layer, especially keratinocytes; this pole or region is referred herein as the epidermal pole or region. For example, one can observe fibroblasts aggregating with each other and / or keratinocytes aggregating with each other. It should be further understood that each pole is not necessarily exclusively constituted of cells of a dermis layer or cells of an epidermis layer. The dermal pole may indeed comprise a few isolated cells constitutive of an epidermis layer, and / or the epidermal pole may comprise a few isolated cells constitutive of a dermis layer.

[0072] The spherical shape of the multicellular skin spheroid is nearly homogeneous.

[0073] Indeed, each pole or region may take the form of a multicellular bud, bulge or protuberance. For example, as shown in the Figures, one may observe a “tail” and a “head”, representing the epidermal pole and the dermal pole, respectively.

[0074] Alternatively, one pole may surround the other one. For example, as shown in the Figures, one may observe an “inner core” surrounded by an “outer shell”, representing the epidermal pole and the dermal pole, respectively.

[0075] Regardless of their morphology, these poles or regions can be detected by conventional means in the art, for instance, by histology and / or microscopy. For example, a whole spheroid or a portion thereof can be stained for well-known biological markers by immunohistochemistry (IHC). Non-limiting examples of such markers include the marker vimentin for fibroblasts and endothelial cell, the marker CK14 (cytokeratin 14) or CK10 (cytokeratin 10) for keratinocytes, the generic marker CD45 for immune cells, the marker CDl lc for dendritic cells, and / or the marker CD68 for macrophages. Appropriate antibodies specific for those markers are known and commercially available. The distribution and organization of various cells in the stained spheroid or section thereof can then be visualized by confocal microscopy. In some cases, confocal microscopy might be sufficient to detect the poles or regions, especially when these poles or regions form well defined multicellular buds, bulges or protuberances. In such cases, one pole may sometimes be larger in size than the other pole, typically the dermal pole, while remaining in contact with one another.

[0076] Because very diverse cell types are used in the present process, one can also observe the self-organization of cells other than fibroblasts and / or keratinocytes in the spheroid of the invention. Endothelial cells are notably able to form a network resembling vascular capillaries. This network can be detected by conventional means in the art, for instance, by immunostaining of endothelial markers such as the biological marker CD31.

[0077] The ratio between the number of cells of the first population and the number of cells of the second population used in step a) can also be used to control self-organization of the cells. Said ratio ranges herein from about 1 : 4 to about 1 : 14. For example, the ratio between the number of cells of the first population and the number of cells of the second population can be about 1 : 4, about 1 : 5, about 1 : 6, about 1 : 7, about 1 : 8, about 1 : 9, about 1 : 10, about 1 : 11, about 1 : 12, about 1 : 13, or about 1 : 14. Preferably, the ratio between the number of cells of the first population and the number of cells of the second population can be from about 1 : 6 to about 1 : 12, such as about 1 : 6, about 1 : 7, about 1 : 8, about 1 : 9, about 1: 10, about 1 : 11, or about 1 : 12. More preferably, the ratio between the number of cells of the first population and the number of cells of the second population can be from about 1 : 8 to about 1 : 10, such as about 1 : 8, about 1 : 9, or about 1 : 10. A particularly preferred ratio according to the invention is about 1 : 9, as it allows for an optimal self-organization of the cells.

[0078] It can also be appreciated that the present process does not preferably make use of a human embryo which requires to be destroyed, or even of human stem cells induced or not, to provide the populations of cells constitutive of a human epidermis layer and of a human dermis layer.

[0079] Instead, for time-saving purpose and ease of use, to provide the populations of cells used in step (a), the present process preferably relies on human primary cells typically isolated from the skin, especially adult human primary cells. Primary cells are particularly advantageous as they closely mimic the physiology of cells in vivo and retain the original tissue genetic makeup upon division, without drifting over time like immortalized cell lines. A promising source for primary cells includes a human skin tissue sample, such as a skin biopsy which may have been taken on any body part such as the abdomen. A particularly preferred skin tissue sample of the invention is a full thickness skin sample excised during abdominoplasty or abdominal dermolipectomy. Methods for collecting a skin tissue sample are well known in the art, and will thus not be detailed herein. It will also be appreciated that the skin tissue sample may be a healthy tissue sample or a diseased tissue sample such as a skin tumor sample.

[0080] Methods for preparing cells constitutive of the human epidermis and dermis layers from a human skin sample are well-known to the skilled practitioner. Generally, the layers of a skin sample are separated from each other, and the cells of each layer are dissociated from one another before forming the cell suspension.

[0081] In other words, the process for obtaining a multicellular skin spheroid of the invention can comprise, prior to step (a): separating an isolated human skin sample into an epidermis layer and a dermis layer; and dissociating the cells from each separated layer, thereby obtaining a first population of cells constitutive of a human epidermis layer and a second population of cells constitutive of a human dermis layer, respectively.

[0082] These steps can be accomplished by conventional means in the art such as a mechanical and / or a chemical treatment. By “ mechanical treatment', it is meant the disruption of connections between associated tissue layers and / or associated cells by a mechanical mean or method, for example, using a scalpel or scissors or a machine such as a homogenizer. By “chemical treatment', it is meant the disruption of connections between associated tissue layers and / or associated cells by a chemical mean or method, for example, using enzymes such as a collagenase, a dispase, trypsin, or any combination thereof. One or more enzymes may be used under different reaction conditions, such as incubation at 37°C in a water bath or at room temperature.

[0083] A particularly preferred protocol for preparing cells constitutive of the human epidermis and dermis layer from a human skin sample is detailed in the experimental part described below, notably in Examples, section 1.2. Briefly, this protocol can be summarized as comprising the sub steps of: i. separating of the isolated human skin sample into an epidermis layer and a dermis layer by dispase digestion; ii. dissociating the cells from the epidermis layer by trypsin digestion, and collecting said cells, thereby obtaining a first population of cells constitutive of an epidermis layer; and iii. dissociating cells from the dermis later by collagenase digestion, and collecting said cells, thereby obtaining the second population of cells constitutive of the dermis layer,

[0084] The skilled person will understand that steps ii. and iii. may be implemented concomitantly to each other, or one after the other, in either order.

[0085] Because the process for producing a multicellular skin spheroid of the invention relies on cells constitutive of human epidermis and dermis layers, the hypodermis layer will be preferably removed from the human skin sample, such as by mechanical treatment and / or chemical treatment.

[0086] Once the cells from each separated layer are dissociated, one can also, if desired, remove dead cells and / or dying cells and / or cellular debris. Removal of dead cells and / or dying cells can be accomplished by any conventional means known in the art, for example using beads and / or antibody methods. It is known, for example, that Annexin V-Biotin binding followed by binding of the biotin to streptavidin magnetic beads can enable separation of apoptotic cells from living cells. Similarly, removal of cell debris may be achieved by any suitable technique in the art, including, for example, filtration.

[0087] As previously explained, the cell suspension obtained in step (a) is then incubated, in a step (b), in a particular growth medium.

[0088] In a preferred embodiment, step (b) is initiated by seeding the cell suspension at an amount ranging from about 70 cells per pL of growth medium to about 2 500 pL cells per pL of growth medium. Cell counting can be achieved by conventional means or methods in the art, such as using a manual counting chamber (e.g. Malassez chamber, hemacytometer), an automated cell counter (e.g. flow cytometry, microscopy image analysis), or an indirect cell counter (e.g. spectrophotometer).

[0089] In a preferred embodiment, the cell suspension obtained in step (a) is incubated, in step (b), under low-adherent or non-adherent culture conditions. Such culture conditions can notably promote the aggregation of the cells, especially in absence of exogenous scaffold.

[0090] When referring to culture conditions, the terms “ low-adherent” or “non-adherent” refer to conditions under which the cells minimally adhere, or do not adhere, to the portion of the culture system or vessel which is in contact with the cell suspension, respectively. The term “low-adherent” can encompass herein, or be synonymous with, “substantially non-adherent” , or “ultra-low adherent” . Low-adherence or non-adherence can be achieved by conventional means known in the art. For instance, at least a portion of the cell culture system or vessel that is in contact with the cell suspension can be coated with a low-adhesive or non-adhesive material such as with a hydrophilic material, and / or the cells can be cultured in the culture system or vessel as floating cells. Non limiting examples of low-adherent or non-adherent culture systems include magnetically rotated spinner flasks or shaken flasks or dishes in which the cells are kept actively suspended in the medium, as well as stationary culture vessels or T- flasks and bottles in which, although the cells are not kept agitated, they are unable to attach firmly to the culture system or vessel.

[0091] As described in the experimental part, at least a portion of the cell culture system or vessel that is in contact with the cell suspension is preferably coated with a hydrophilic material and optionally neutrally charged.

[0092] Also, as described in the experimental part, the cell suspension can advantageously be kept actively suspended in the medium when performing step (b). Any shaker can be used for that purpose, such as, for example, a rotator which agitates the cultures in three dimensions. In other words, the cell suspension is preferably incubated in step (b) under shaking conditions. A constant agitation throughout step (b) will be herein preferred.

[0093] The culture conditions of step (b) require a particular growth medium.

[0094] By “growth medium", “culture medium", or “cell medium", it is meant any medium comprising growth-promoting components, and which is suitable for the maintenance and / or proliferation of the cells to be cultured.

[0095] The growth medium used herein term is an endothelial cell growth medium. In other words, said medium is suited for the maintenance and / or proliferation of endothelial cells. This type of medium is well-known in the art, and is typically an endothelial basal medium that is supplemented with growth-promoting components.

[0096] In a preferred embodiment, the endothelial cell growth medium used in step (b), that is preferably an endothelial basal medium, comprises, or consists essentially of, ascorbic acid, hydrocortisone, and growth factors EGF (Epidermal Growth Factor), FGF (Fibroblast Growth Factor) such as bFGF (also known as FGF2 or FGF beta), IGF (Insulin Growth Factor) such as the Long R3 Insulin-like growth factor (IGF-1 LR3), and VEGF (Vascular Endothelial Growth Factor) such as human VEGF 165. It will be understood that the growth factors used herein are preferably human growth factors. In a more preferred embodiment, the endothelial cell growth medium used in step (b), that is preferably an endothelial basal medium, further comprises heparin, serum such as Fetal Bovine Serum (FBS) or Fetal Calf Serum (FCS), or any combination thereof. The presence of heparin can further optimize self-organization of the cells. It shall also be noted, that when serum is present in the growth medium used herein, the presence of human platelet lysate is not mandatory to produce the multicellular skin spheroid of the invention.

[0097] Particularly preferred concentrations of the components in the endothelial cell growth medium used in step (b) are as follows: ascorbic acid concentration can range from about 0.1 pg / mL to about 100 pg / mL, preferably is of about 1 pg / mL; and / or

[0098] - EGF concentration can range from about 0.5 ng / mL to about 50 ng / mL, preferably is of about 5 ng / mL; and / or

[0099] - FGF concentration can range from about 0.5 ng / mL to about 50 ng / mL, preferably is of about 10 ng / mL; and / or

[0100] IGF concentration can range from about 2 ng / mL to about 100 ng / mL, preferably is of about 20 ng / mL; and / or

[0101] VEGF concentration can range from about 0.1 ng / mL to about 50 ng / mL, preferably is of about 0.5 ng / mL; and / or optionally, hydrocortisone concentration can range from about 0.02 pg / mL to about 2 pg / mL, preferably is of about 0.2 pg / mL; and / or optionally, heparin concentration can range from about 2 pg / mL to about 200 pg / mL, preferably is of about 22.5 pg / mL; and / or optionally, serum concentration can range from about 0.005 mL / mL to about

[0102] 0.2 mL / mL, preferably is of about 0.02 or about 0.05 mL / mL.

[0103] As described in the experimental section below, a particularly preferred endothelial cell growth medium used in step (b) is EGM2 (Endothelial Cell Growth Medium-2) or EGMMV2 (Microvascular Endothelial Cell Growth Medium MV-2), more preferably is EGM2 as it is supplemented with heparin.

[0104] In a preferred embodiment, the cell suspension is incubated in step (b) for a time period of at least about 5 days to about 10 days, such as at least about 5, 6, 7, 8, 9, 10 days, preferably at least 7 days. This time period allows sufficient cells to aggregate and self-organize in a multicellular skin spheroid. For example, the cell suspension can be incubated in step (b) for a time period ranging from about 5 days to about 10 days, such as for a time period of about 5, 6, 7, 8, 9, 10 days, more preferably of about 7 days, especially for later assembly of a plurality of multicellular skin spheroids.

[0105] In a preferred embodiment, the cell suspension is incubated in step (b) for a time period of at most about 15 days to about 25 days, such as at most about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 days, preferably at most about 20 days. This time period allows a more advanced self-organization of the cells.

[0106] For example, the cell suspension can be incubated in step (b) or a time period ranging from about 15 days to about 25 days, such as a time period of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 days, preferably of about 20 days.

[0107] In another aspect, the present invention pertains to a multicellular human skin spheroid obtainable or obtained by the above-described process. More precisely, the present invention relates to a multicellular human skin spheroid directly obtained by the above-described process. The multicellular human skin spheroid of the present invention is accordingly a scaffold-free multicellular human skin spheroid.

[0108] It shall accordingly be understood that the cells used in step a) of that process are comprised in said spheroid. The Inventors have indeed detected the expression of biological markers specific of key cell types that are naturally present in human epidermis and dermis.

[0109] Thus, in a preferred embodiment, the multicellular human skin spheroid according to the invention comprises, or consists essentially of, a population of cells constitutive of a human epidermis layer and a population of cells constitutive of a human dermis layer. Those populations of cells and their self-organization are as described above.

[0110] In that regard, it is important to note that, contrary to human skin organoids or spheroids disclosed in the art, the multicellular human skin spheroid of the present invention comprises a wide range of skin cell types, including, but not limited to, endothelial cells and immune cells. These endothelial cells can also form a network resembling vascular capillaries in the multicellular skin spheroid.

[0111] Thus, in a particularly preferred embodiment, the multicellular human skin spheroid according to the invention comprises immune cells and endothelial cells, preferably a network of endothelial cells. Because of the population of cells contained in that spheroid, and their self-organization, one would readily understand that the multicellular human skin spheroid of the invention exhibits characteristics that grossly mimic those of natural skin or of the skin tissue sample used, especially when primary cells are used.

[0112] As explained above, the multicellular human skin spheroids according to the invention can be used as a building-block units to engineer a tissue model that recapitulates the complexity and organization of human skin. To do so, the invention provides a further process which assembles these multicellular human skin spheroids into an artificial human skin construct. As demonstrated in the experimental section, this process essentially aims at using an exogenous porous scaffold for hosting the cells of the spheroids, and inducing the formation of a skin organoid in which the cells have undergone a re-organization as well as differentiation / stratification thereby reproducing key hallmarks of human skin. This process is a accordingly a scaffold-based process.

[0113] It is thus another aspect of the present invention to provide a method for the in vitro assembly of multicellular human skin spheroids into an artificial human skin construct, comprising:

[0114] (1) placing a plurality of multicellular human skin spheroids obtained or obtainable according to the above-described process, on or in a porous scaffold; and

[0115] (2) culturing the plurality of multicellular human skin spheroids placed on or in the scaffold in step (1), in presence of an organoid growth medium.

[0116] More precisely, the present method can comprise the following steps:

[0117] (0) obtaining a plurality of multicellular human skin spheroids according to the above-described process;

[0118] (1) placing (or transferring) the plurality of multicellular human skin spheroids obtained in step (0), on or in a porous scaffold; and

[0119] (2) culturing the plurality of multicellular human skin spheroids placed (or transferred) on or in the scaffold in step (2), in presence of an organoid growth medium.

[0120] Preferred embodiments for obtaining multicellular human skin spheroids according to the process of the invention apply to the present method, mutatis mutandis. In order to notably speed-up the present method, one may generate the plurality of multicellular skin spheroids in a relatively short time period. Accordingly, in step (0) representing the process for obtaining the plurality of spheroids, the cell suspension can be preferably incubated in step (b) for a time period of at least about 5 days to about 10 days, such as at least about 5, 6, 7, 8, 9, 10 days, preferably at least about 7 days. For example, the cell suspension can be incubated in step (b) for a time period ranging from about 5 days to about 10 days, such as of about 5, 6, 7, 8, 9, 10 days, preferably of about 7 days.

[0121] The term “plurality” means herein at least two. Thus, by “a plurality of multicellular human skin spheroids”, it is meant at least two multicellular human skin spheroids as described above. In the context of the present invention, the plurality of multicellular human skin spheroids preferably comprises from about 5 to about 50 multicellular human skin spheroids, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 spheroids, more preferably from about 20 to about 40 multicellular human skin spheroids, such as about 20, 25, 35, or 40 spheroids. Even more preferably, the plurality of multicellular human skin spheroids comprises about 30 multicellular skin spheroids.

[0122] This plurality of multicellular human skin spheroids is assembled by the present method to obtain a construct which closely mimics the native skin, referred herein as an artificial human skin construct.

[0123] The term “human skin construct”, “human skin substitute” or “human skin organoid” can be defined herein as an engineered three-dimensional cellular construct which recapitulates, to a high extent, the cellular composition, architecture, and / or function of native human skin. Like the multicellular spheroids of the invention, this construct, substitute or organoid has essentially a closed or confined shape. This construct, substitute or organoid is herein artificial, in that it is engineered in vitro and is not naturally-occurring.

[0124] The Inventors have indeed observed that the plurality of spheroids of the invention undergo a re-organization as well as a differentiation / stratification process when assembled according to the present method. The resulting construct displays key hallmarks of human skin, including, inter alia, the stratification of keratinocytes and fibroblasts into multiple layers with a polarization of the keratinocytes and interconnection of keratinocytes via desmosomes, the formation of collagen fibers, the presence of melanin pigments and vascular vessel-like structures as well as genesis of sweat gland-like structures. The presence of immune cells, as observed in the initial multicellular human skin spheroids, is also maintained, which is a promising sign of immunocompetency.

[0125] Yet, the resulting construct is not strictly identical to native human skin. Indeed, while a dermis-like layer and an epidermis-like layer can be observed in the artificial human skin construct of the invention, said layers may not adopt a planar organization as in native human skin, due notably to the relatively round shape of the multicellular skin spheroids and to the present method used for generating the construct. Instead, the dermis-like layer and the epidermis-like layer preferably form an inner core-outer shell structure, where the epidermislike layer surrounds the dermis-like layer.

[0126] In other words, the human skin construct obtained or obtainable by the present method is preferably a spherical construct comprising, or consisting essentially of, an outer epidermislike layer and an inner dermis-like layer, where the dermis-like layer is surrounded by the epidermis-like layer.

[0127] Each of the dermis- and epidermis-like layer is herein multilayered, and may be contiguous, substantially contiguous, non-contiguous. This means that the epidermis-like layer may not be contiguous when surrounding the dermis layer, or that the dermis layer may not be contiguous when surrounded by the epidermis-like layer.

[0128] The term “dermis- or epidermis-like layer", “dermis- or epidermis mimic layer" or “dermis- or epidermis simulating layer" designates herein a layer resembling those of the native human dermis or epidermis layer, in terms of architecture, composition, and / or function.

[0129] More precisely, in a preferred embodiment, the epidermis-like layer comprises a basal layer adjacent to the dermis-like layer and / or a suprabasal layer on top of the basal layer.

[0130] In a preferred embodiment, the dermis- or epidermis-like layer comprises a population of cells constitutive of a human dermis layer or a population of cells constitutive of a human epidermis layer, respectively, said population being as described above. Indeed, besides fibroblasts and keratinocytes, the Inventors have confirmed the presence of immune cells and endothelial cells in the layers of the artificial human skin construct.

[0131] In a preferred embodiment, the dermis-like layer and / or the epidermis-like layer, especially the basal layer, comprises one or more proliferating cells, i.e. the cells in that layer are mitotically active. In a preferred embodiment, the dermis-like layer and / or the epidermis-like layer, especially the suprabasal layer, comprises one or more differentiating cells, i.e. the cells in that layer undergo a maturation process.

[0132] In a preferred embodiment, the dermis-like layer and / or the epidermis-like layer, preferably the epidermis-like layer, comprises melanin, i.e. these layers may protect e.g. against UV.

[0133] In a preferred embodiment, the dermis-like layer comprises one or more collagen fibers, i.e. the cells in that layer are metabolically active.

[0134] In a preferred embodiment, the dermis-like layer comprises one or more vascular vessellike structures, i.e. this layer may, under appropriate conditions, supply nutrients and oxygen to the cells of the construct, evacuate waste products away from the construct, and / or promote graft take of the skin construct of the invention in a recipient subject.

[0135] In a particularly preferred embodiment, the dermis-like layer comprises one or more skin appendages, more preferably a sweat gland-like structure, i.e. this layer may enable the excretion of watery substances from the construct and / or be useful should the skin construct of the invention be grafted in a recipient subject.

[0136] All these hallmarks can be detected by conventional means in the art, for instance, by histology and / or microscopy. For example, a human skin construct of the invention, or a portion thereof, can be stained for well-known biological markers by immunohistochemistry (IHC), and the staining visualized by confocal microscopy, as described above. Ki67 can be used as a further biological marker for detecting proliferating cells; appropriate antibodies specific for this marker are known and commercially available. Conventional IHC dies may be used to observe the distribution and organization of the cells: hematoxylin eosin (HE) can help distinguish epidermal cells from dermal cells and detect their stratification, spot melanin or even anatomic features of sweat glands; Masson Trichrome can reveal collagen fibers; and / or Periodic Acid Schiff can uncover mucopolysaccharides / mucin typically present in sweat glands.

[0137] In step (1) of the present method, the plurality of multicellular human skin spheroids is placed on or in a porous scaffold comprising an extracellular matrix. In other words, step (1) consists in either placing the plurality of multicellular human skin spheroids on top of said porous scaffold, or in partially or fully embedding the plurality of multicellular human skin spheroids within said porous scaffold. It will be appreciated that placing the plurality of multicellular human skin spheroids on top of said porous scaffold does not necessarily prevent the plurality of spheroids to later become partially or fully embedded in the scaffold when performing step (2).

[0138] The term “porous scaffold" or “porous tissue scaffold" as used herein refers to a porous three-dimensional matrix or network of polymers, which acts as a microenvironment to which cells are attracted and can adhere. This cell adhesion is essential for the assembly of the cells from the spheroids in the three-dimensional construct provided herein. This way, cells can receive adhesive cues that control their polarity, proliferation, differentiation, and survival. A porous scaffold can be natural, synthetic, or hybrid (i.e. a mixture of synthetic and natural). Natural scaffolds are those whose polymers have a natural origin, such as collagen or elastin, while synthetic scaffolds are those whose polymers are synthetic such as polyamides or polyethylene glycol. In biomedical applications, the use of natural polymers can be advantageous due to their biocompatibility, biodegradability and / or non-toxicity, whereas, synthetic polymers can be hydrophobic and / or possess strong covalent bonds within their matrix, which can improve the mechanical strength, service life and absorbability of the scaffolds. Hybrid scaffolds can combine the advantages of both natural and synthetic scaffolds.

[0139] In the context of the present invention, the porous scaffold may comprise extracellular matrix components, meaning that the scaffold used herein is advantageously a natural or hybrid scaffold. Said scaffolds can indeed be a better fit for human skin tissue engineering. The presence of exogenous extracellular matrix components may however not be necessary in some embodiments of the invention, as demonstrated in the experimental section.

[0140] The term “extracellular matrix components"" (ECM) refers to a set of natural components or polymers that can provide the support, adherence, movement, and regulation of cells within an environment, and which can form a matrix or network. This set of components or polymers typically comprises extracellular macromolecules that are naturally present in connecting tissue and other tissues of multicellular organisms, such as proteins, peptides, proteoglycans, and the like. In vivo, the extracellular matrix is largely made of glycoproteins and pure proteins, as well as glycosaminoglycans - though its composition may vary between tissue types.

[0141] In a preferred embodiment, the extracellular matrix components, when used in the invention, are extracellular matrix components of the skin.

[0142] Two distinct forms of extracellular matrix can be found in the skin, which differ by their localization and composition: the interstitial dermal matrix (IDM), which is mainly composed of collagens and elastic fibers, cushions the skin from stress and strain, while the basement membrane (BM) separates the keratinocytes from the dermal stroma and is mainly composed of laminins and isoforms of collagen IV. Regardless of their localization, major components of the skin extracellular matrix include collagen (e.g. type I, III, IV, VII, XIV), elastic fibers (elastin), glycosaminoglycans / proteoglycans (hyaluronic acid, chondroitin sulfate, dermatan sulfate, decorin, dermatopontin, lumican), matricellular proteins (osteopontin, SPARC, tenascin-C, Fibulin-5, CCN2) and fibronectin. Extracellular matrix components of the skin can be easily obtained, by conventional methods in the art, such as by decellularization of a skin tissue sample (Mclnnes et al., J. Funct. Biomater. 2022, 13(4), 240); such matrix can be referred as a decellularized extracellular matrix (dECM).

[0143] When used in the present invention, extracellular matrix components are preferably embedded in a hydrogel. In other words, the porous scaffold is preferably in the form of a hydrogel comprising extracellular matrix components. The term “hydrogel” refers herein to a porous scaffold, as defined above, in which polymers are held together by covalent or noncovalent crosslinks, and in which water has been absorbed thereby forming a gel. It shall be understood that the hydrogel typically can undergo a gradual degradation when implementing the present method, and is preferably completely or almost completely degraded when completing said method. In other words, the artificial human skin construct obtained or obtainable by the present method is preferably devoid or essentially devoid of hydrogel (i.e. scaffold-free or essentially scaffold-free). Hydrogels and their methods of preparation are well- known in the art, and will thus not be detailed herein. For further information on methods for preparing hydrogels, one may refer to Ahmed et al. in J Adv Res., 2015; 6(2): 105-21.

[0144] In a more preferred embodiment, the porous scaffold can comprise at least one synthetic polymer, i.e. said scaffold is advantageously a synthetic scaffold. In such case, the presence of extracellular matrix components may not be necessary.

[0145] Examples of synthetic polymers include, without limitation, poly(epsilon- caprolactone) (PCL), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(D-lactic acid) (PDLA), poly(glycolide), poly(glycolic acid) (PGA), polycaprolactone, poly(lactide-co-glycolide) copolymer (PLA / PGA), poly(epsilon- caprolactone-co-glycolide) copolymer (PCL / PGA), poly(epsilon-caprolactone-co-L-lactide) copolymer (PCL / PLLA), polydioxanone (PDO), polygluconate, poly(lactic acid-co-ethylene oxide) copolymer, polyhydroxybutyrate, poly(hydroxpriopionic acid), polyphosphoester, poly(alpha-hydroxy acid), polycarbonates, polyamides, polyacrylamides, polyanhydrides, polyorthoesters and polyurethanes, and also polyethylene glycol) (PEG), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyacrylamides, and any combination thereof.

[0146] As illustrated in the experimental section, a particularly preferred synthetic polymer according to the invention is poly(epsilon-caprolactone) (PCL), especially 3D poly(caprolactone). The Inventors have indeed observed that such polymer promotes a finer organization of the cells in the human skin construct of the invention, and increases the construct mechanical stability, even in the absence of extracellular matrix components.

[0147] In another particularly preferred embodiment, the synthetic polymer as described above may be combined with a natural scaffold, i.e. said scaffold is a hybrid scaffold. For example, the porous scaffold can comprise poly(epsilon- caprolactone) (PCL), especially 3D poly(caprolactone), a hydrogel and extracellular matrix components.

[0148] When the porous scaffold is a hybrid scaffold, one may preferably pour a hydrogel forming solution containing the extracellular matrix components into the network of the synthetic polymer, as described by Kim et al. in Biofabrication., 2017; 9(2):025034.

[0149] In the following step (2), the plurality of multicellular human skin spheroids, that is placed on or in the scaffold, is cultured in presence of an organoid growth medium.

[0150] By “organoid growth medium", “organoid culture medium", “organotypic growth medium", or “organotypic culture medium", it is meant herein a growth medium, as defined above, that is suitable for organoid or organotypic culture. In other words, it refers to a growth medium which favors the assembly and maturation of multiple cell types in a manner that closely replicates the natural tissue architecture of the organ from which the cells are derived, said organ being herein the skin. This medium may also advantageously promote the differentiation and / or the proliferation of the cultured cells.

[0151] A particularly preferred organoid growth medium according to the invention is an epidermalization medium.

[0152] Epidermalization media that are suited for skin organoid culture or HSE (human skin equivalent) engineering are well-known in the art. These media can be used to promote the stratification (epithelialization), organization, and / or morphologic differentiation of keratinocytes into layers resembling those of an epidermis, such as a basal layer and / or a suprabasal layer, and eventually stimulate the proliferation of keratinocytes. This type of medium is also referred in the art as keratinocytes differentiation medium or keratinocyte stratification medium. Non-limiting examples of such media include the SKDM medium (Keratinocyte Defined Medium supplemented with calcium, transferrin, ascorbic acid and 0.1% BSA) developed by Stark et al. (The Journal of Investigative dermatology 1999, 112(5): 681- 691), the Epidermalization I, Epidermalization II and Cornification media (010 medium, supplemented with Hams’s F12 supplements, L-glutamine, adenine, hydrocortisone, T3, transferrin, insulin, PES, FBS, optionally progesterone, optionally calcium chloride) devised by Carlson et al. (Curr Protoc Cell Biol. 2008; CHAPTER: Unit-19.9), or the Keratinocyte Growth Medium, supplemented with KGF, CalCh and ascorbic acid, as used by Roger et al. (J Anat. 2019; 234(4):438-455). Epidermalization media are also commercially available, and include notably the 3dGRO medium (Merck), which is herein particularly preferred.

[0153] In a preferred embodiment, the epidermalization medium used in the present method can comprise (if not already present) ascorbic acid, VEGF, M-CSF (Macrophage colonystimulating factor), or any combination thereof. The presence of any of those components can indeed further optimize the process of the present invention, notably by promoting the viability and / or the growth of cells other than keratinocytes within the artificial human skin construct. VEGF and M-CSF can indeed respectively assist in the maintenance of endothelial cells and macrophages, while ascorbic acid can stimulate the secretion of collagen by fibroblasts.

[0154] Particularly preferred concentrations of these components in the epidermalization medium used in step (2) are as follows: ascorbic acid concentration can range from about Ipg / mL to about 250 pg / mL; and / or

[0155] VEGF concentration can range from about 1 ng / mL to about 50 ng / mL; and / or

[0156] - M-CSF concentration can range from about 2 ng / mL to about 100 ng / mL.

[0157] A particularly preferred epidermalization medium used in step (2) is the 3dGRO medium, which is further supplemented with ascorbic acid, VEGF, M-CSF, or any combination thereof, as described above.

[0158] It will be appreciated that the organoid growth medium can be renewed during step (2), i.e. replaced with fresh an organoid growth medium, as often as needed.

[0159] The organotypic organization, notably the epidermalization process, is herein optimal when the plurality of skin spheroids of the invention is exposed in step (2) to an air-liquid interface. Accordingly, in a preferred embodiment, the plurality of multicellular human skin spheroids is cultured in step (2) at an air-liquid interface. In other words, the plurality of spheroids is exposed to air from the top and to the culture medium from the bottom. It will be understood that the liquid or culture medium is herein the organoid growth medium, as described above.

[0160] Means and methods for culturing cells at an air-liquid interface are well-known in the art, and will not be detailed herein. Briefly, cells (herein, the plurality of spheroids) can be deposited in a culture system or vessel, which is either suspended or supported to allow the culture medium (herein, the organoid growth medium) to access the underside of the culture, while raising the cells (herein, the plurality of spheroids) to the air-liquid. For further details of culture methods at an air-liquid interface, one may refer to Prunieras et al. (J Invest Dermatol. 1983; 81(1 Suppl):28s-33s), Fauzi et al. (Polymers (Basel), 2020;12(12):2784), or Chen et al. (Reprod Domest Anim. 2019; 54 Suppl 3:38-45).

[0161] The Inventors have further observed that the assembly of the spheroids into a human skin construct is efficient when the plurality of spheroids is cultured in step (2) under conditions that favor minimal adherence of the cells of the spheroids to the culture system or vessel.

[0162] Thus, in a preferred embodiment, the plurality of multicellular human skin spheroids is cultured in step (2) under low-adherent or non-adherent culture conditions. Such conditions are as described above.

[0163] The method for the in vitro assembly of skin spheroids as proposed by the present invention can provide a skin construct which closely mimics native human skin in a very short time period typically less than a month.

[0164] In a preferred embodiment, the plurality of multicellular human skin spheroids is cultured in step (2) for a time period of at least about 10 days to about 30 days, such as at least about 10, 15, 20, 25, 30 days, preferably for a time period of at least about 15 days to about 25 days, such as at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 days, more preferably for a time period of at least about 20 days. This time period allows an optimal assembly of the plurality of multicellular skin spheroids into a multicellular human skin spheroid system.

[0165] For example, the plurality of multicellular human skin spheroids is cultured in step (2) for a time period ranging from about 10 to about 30 days, such as about 10, 15, 20, 25, 30 days, preferably for a time period ranging from about 15 days to about 25 days, such as about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 days, more preferably for a time period of about 20 days. In another aspect, the present invention pertains to an artificial human skin construct obtainable or obtained by the above-described method. More precisely, the present invention relates to an artificial human skin construct directly obtained by the above-described process.

[0166] Preferred embodiments related to this human skin construct are as described above.

[0167] It is especially important to note that, contrary to artificial human skin models disclosed in the art, the human skin construct of the present invention comprises immune cells, vascular- vessel like structures and skin appendages resembling sweat glands.

[0168] Thus, in a particularly preferred embodiment, the artificial human skin construct according to the invention comprises immune cells, one or more vascular vessel-like structures and one or more skin appendages, especially sweat gland-like structures.

[0169] Further aspects of the present invention relate to uses of the multicellular human skin spheroid or artificial human skin construct as described herein.

[0170] These may indeed serve as model systems for in vivo skin tissue. As such it can be used to study skin development (e.g., keratogenesis, etc), determine the possible ill effects of substances (e.g., cosmetics, medical treatments etc), or for screening for the cosmetic or therapeutic effects of candidate substances. The invention can thus pertain to the use of a multicellular human skin spheroid or artificial human skin construct, as described above, in in vitro assays. The in vitro assays may be toxicology assays, physiology assays, or screening assays.

[0171] The multicellular human skin spheroid or artificial human construct of the invention may also be used for therapeutic purposes, notably to repair the skin, i.e. for treating any loss, damage or impairment of the skin such as bums or wounds. As such, the invention can be directed to a multicellular human skin spheroid or artificial human skin construct, as described above, for use as a medicament. The invention can preferably relate to a multicellular human skin spheroid or artificial human skin construct, as described above, for use in the treatment of skin loss, damage or impairment. More precisely, the present invention can pertain to the use of the multicellular human skin spheroid or artificial human skin construct of the invention, for manufacturing a medicament to treat skin loss, damage or impairment. The invention can also be drawn to a method for treating skin loss, damage or impairment comprising the step of administering an effective amount of the multicellular human skin spheroid or artificial human skin construct, to a subject in need thereof. The term “treating, “treatment” or “treat” as used herein encompasses, among other, preventing, ameliorating, inhibiting, or curing a deficiency, dysfunction, disease, or other deleterious process, including those that interfere and / or result from therapy.

[0172] Pathological loss, damage or impairment of skin can result either from a disease or disorder, and / or from a trauma or injury to the skin. Typical examples of pathological skin loss, damage or impairment include, without limitation, skin redness or soreness, irritated skin, blisters, wounds (such as open wounds, closed wounds, acute wounds, or chronic wounds), bums, abscess, bedsores and / or skin ulcer.

[0173] An “effective amount” means herein an amount that is sufficient to achieve the effect for which it is indicated, herein the treatment of skin loss, impairment or damage. It is within the skill of the person in the art to determine such amount.

[0174] It should further be understood that “a subject in need thereof refers more particularly to a subject in need of skin repair. In the context of the present invention, said subject is preferably a human.

[0175] The present invention will be better understood in the light of the following detailed experiments. Nevertheless, the skilled artisan will appreciate that the present examples are not limitative and that various modifications, substitutions, omissions, and changes may be made without departing from the scope of the invention.

[0176] EXAMPLES

[0177] 1. MATERIALS AND METHODS

[0178] 1.1. Samples

[0179] Skin samples were obtained from abdominal dermolipectomies from adult human subjects.

[0180] 1.2. Isolation of cells from the epidermis layer and the dermis layer

[0181] The subcutaneous adipose tissue was mechanically removed from the abdominal dermolipectomy. The resulting skin sample, free of fat layer, was then cut into small fragments. In order to separate the dermis layer from the epidermis layer, the skin fragments were firstly incubated in dispase II solution (Roche) at 1.5 U / mL; forceps were then used to complete this separation. The isolated epidermis layer was subsequently digested in trypsin-EDTA solution (0.05%), while the isolated dermis layer was digested in a solution of collagenase at 0.8 U / mL. After counting on a Malassez cell, the composition of cell suspensions obtained after digestion of the epidermis and the dermis was analyzed by flow cytometry. Each cell population was characterized by a combination of well-known specific cellular markers of keratinocytes, melanocytes, fibroblasts, endothelial cells, Langerhans cells, monocytes / macrophages, dendritic cells, neutrophils, eosinophils, lymphocytes T, and lymphocytes B.

[0182] 1.3. Skin spheroids production

[0183] Dissociated dermal cells and epidermal cells were mixed according to a ratio of 1 epidermal cell to 9 dermal cells (1 : 9). The ratio 1 :5 was also tested.

[0184] This mixture was seeded in Endothelial Cell Growth Medium 2 (EGM2) or Endothelial Cell Growth Medium MV 2 (EGM-MV2) (Promocell) at 50,000 cells in an ultra-low-adherence plate, with shaking. According to Promocell, the EGM2 medium is an endothelial cell growth medium supplemented with the recombinant human growth factors EGF (5 ng / ml), basic FGF (10 ng / ml), IGF (long R3 IGF) (20 ng / ml) and VEGF (0.5 ng / ml), ascorbic acid (1 pg / ml), heparin (22.5 pg / ml), hydrocortisone (0.2 pg / ml), and fetal calf serum (FCS) (0.02 ml / ml). The EGM-MV2 medium has a similar composition to EMG2, except that it does not contain heparin and has a slightly higher concentration of FCS (0.05 ml / ml). Overall, both media are low in serum.

[0185] As an alternative to the EGM2 or EGM-MV2 medium, the aMEM medium supplemented with serum (FCS, 0.2 ml / ml) was also assessed. The aMEM medium is a modification of the Minimum Essential Medium (MEM) which contains non-essential amino acids, sodium pyruvate, lipoic acid, B12 vitamin, biotin and ascorbic acid. The aMEM medium is devoid of proteins, lipids and growth factors.

[0186] The medium was changed when needed, and spheroids were either (i) maintained in culture for about 3 weeks or (ii) used after 7 days of culture for assembly.

[0187] The general process for producing skin spheroids is summarized on Figure 1A. 1.4. Skin spheroids assembly

[0188] In a first configuration, a non-adherent culture insert was coated with Skin dECM hydrogel for cell culture (Sigma) following the supplier's recommendations - the dECM used therein is extracellular matrix stemming from decellularized pig skin. In a second configuration, the Skin dECM hydrogel was preloaded into the pores of a 3D PolyCaproLactone (PCL) scaffold (Sigma) previously placed on a non-adherent culture insert. In a third configuration, a porous 3D PCL scaffold was placed alone on the non-adherent culture insert. Finally, tests were also carried out in the absence of any scaffold.

[0189] About 30 spheroids that were cultured for 7 days as described in above section 1.3 were placed on the insert. The insert with the spheroids was then placed at an air-liquid interface, where the liquid was the skin differentiation medium from Merck named 3dGRO™ (Merck, Cat # SCM310) (supplemented with VEGF, ascorbic acid, and MCSF). The medium was renewed when needed, and cultured spheroids were analyzed after 2 to 3 weeks of culture. The experiment was conducted either with a non-adherent culture insert (PTFE), or with an adherent culture insert.

[0190] The general process for assembling skin spheroids is summarized on Figure 1A.

[0191] 1.5. Immunofluorescence analysis

[0192] Cultured spheroids, individually or assembled, were fixed in formalin 10% overnight at room temperature. The fixed spheroids were incubated in citrate buffer, then permeabilized (PBS + 0.2% triton XI 00 solution), followed by an incubation in a blocking buffer (PBS + 0.2% Triton XI 00 + 2% Normal Goat Serum). The treated spheroids were then incubated with various primary antibodies, as well as with secondary antibodies coupled to AlexaFluor (488, 546, 594, 647), in order to characterize the type of cells contained therein: the primary anti-human cytokeratin 14 antibody was used to detect the presence of basal keratinocytes; the anti -human cytokeratin 10 antibody to detect suprabasal keratinocytes; a pan-cytokeratin antibody was used to detect keratinocytes regardless of their differentiation state; the anti-human CD31 antibody to detect endothelial cells; the antibodies directed against human CD68, CD45 and / or CDl lc to detect immune cells (more precisely, CD68 for macrophages, and CD11c for dendritic cells - CD45 being a generic marker for immune cells). DAPI staining was also performed to detect the nuclei and assess the overall morphology of the spheroids. The labelled spheroids were finally imaged using the LSM 780 confocal microscope (Zeiss).

[0193] 1.6. Histological analysis

[0194] Cultured spheroids, individually or assembled, were fixed in formalin 10% overnight at room temperature. The fixed spheroids were incubated in ethanol, embedded in agarose and incubated in formalin. The resulting spheroids were then paraffin embedded, sectioned, then subjected to various types of staining, which included inter alia', a H&E (hematoxylin-eosin) coloration for overall nuclei (purplish blue) and cytoplasm / ECM (pink color) detection but also to check for the potential presence of melanin (yellow / brown color) and sweat glands; Masson's trichrome staining to detect collagen fibers, periodic acid Schiff staining to detect epidermal-dermal junctions; immunostaining with various antibodies. The anti-human vimentin antibody was used to detect dermal compartment; the anti-human CD45 antibody to detect immune cells; the antibodies anti-CDl lc and anti-CD68 to detect more specifically dendritic cells and macrophages, respectively; the anti-human Ki67 antibody to detect mitotic / dividing cells; the antibodies directed against human CD31 to detect endothelial cells.

[0195] 2. RESULTS

[0196] 2.1. Isolation of cells from the epidermis layer and of the dermis layer

[0197] Flow cytometry analysis of native cells from the skin biopsy identified: in the epidermis layer, the presence of keratinocytes, melanocytes and immune cells, the latter including Langerhans cells, lymphocytes T, eosinophils, as well as neutrophils; in the dermis layer, the presence of fibroblasts, endothelial cells and immune cells, the latter including monocytes and macrophages, dendritic cells, neutrophils, eosinophils, lymphocytes T, as well as lymphocytes B.

[0198] The percentage of each cell type from each layer is illustrated on Figure IB.

[0199] 2.2. Skin spheroids production

[0200] Native cells isolated from the epidermis and dermis layers, and mixed at a 1 : 9 ratio, were able to aggregate / self-assemble into spheric structures directly after tissue digestion, when cultured for up to 20 days using either the EGM2 medium in the ultra-low adherence plate (Figure 2A). Similar results were obtained in the EGMMV2 medium, albeit with a less effective cellular organization (data not shown).

[0201] Immunofluorescence analysis after 7 days or about 3 weeks of culture shows that in addition to this aggregation, the spheroids of the invention were able to self-organize: a spontaneous separation of cells constitutive of the dermis and the epidermis can indeed be observed (a hallmark of a skin-like structure), as apparent from the regionalized labeling of cytokeratins that are typically found only in epidermis (Figures 2B1 and 2C1). The epidermis appeared to become structured with cells displaying different degrees of differentiation, as shown by the positive staining for cytokeratins 14 and 10 (Figures 2B1 and 2C1). Cytokeratin 14 is indeed expressed by the basal and most immature cells of the epidermis, while cytokeratin 10 is a marker of epidermic suprabasal cells engaged in differentiation. The positive labelling for CD31 shows not only the presence of endothelial cells in the spheroids after 7 days culture and their maintenance after 3 weeks, but also their alignment and organization in an endothelial network which was more advanced after 3 weeks (Figures 2B2 and 2C2). Myeloid immune cells were also present and maintained in the spheroids as shown by the positive immunostaining for macrophages (CD68) and dendritic cells (CD11c) (Figures 2B3, 2B4 and 2C3). Of note, cells mixed at ratios 1 : 5 also gave rise to spheroids that self-organized, albeit with less effective cellular organization than with the 1 : 9 ratio (data not shown). Overall, the lower the number of cells isolated from the epidermal layer by comparison to cells isolated from the dermal layer, the better the organization of the spheroids. Taken together, these results demonstrate that a wide panel of human skin cells are not only maintained but also organized in the single spheroids, after 7 days of culture.

[0202] By contrast, when cells (1 : 9 ratio) were cultured in the supplemented aMEM medium, the spheric cell aggregates did not grow steadily in size over time, but rather appeared to shrunk through compaction and became ultimately necrotic (data not shown). Immunofluorescence analysis of these cell aggregates showed a positive labelling for cytokeratins, CD31, CD45 and CD68 (data not shown). However, the organization of the cells within the aggregates did not display a dermis / epidermis separation nor an endothelial network like in the present invention (data not shown). 2.3. Skin spheroids assembly

[0203] In a first configuration, about 30 individual spheroids, initially formed using the 1 : 9 ratio and the EGM2 medium, successfully merged with one another, when cultured at the air-liquid interface in a non-adherent insert coated with hydrogel and ECM (Figure 3A to 3C).

[0204] Histological analysis was performed after successive inclusions in agarose and paraffin, and sections of the assemblies. Hematoxylin eosin (HE) staining shows that, after about 14 to 21 days of culture at the air-liquid interface, marks of the assembly between spheroids were still present. This is apparent from e.g. Figure 3B, where 3 independent spherical areas of dermal cells (dense areas in dark purple) intermittently surrounded by epidermal cells (light pink) could still be distinguished. HE staining also shows an organization of the epidermis with several superimposed layers of cells (Figure 3D-E). Interestingly, this staining further shows that epithelial positioning bordering the periphery of the assembly was associated with typical epithelial differentiation: a basal layer was apparent in the deeper section of the epidermis, while a Malpighian differentiation of large cells was observed towards the surface (Figure 3E). Melanin pigmented in brown was also detected (arrows) (Figure 3E).

[0205] Immunostaining with the vimentin antibody colors the dermal cells in dark brown; this staining shows here the self-organization of the cells of the spheroids, with a rather neat separation between the dermal and epidermal layers (Figure 3F), the dermal layer being essentially in the center of the spheroid assembly, intermittently surrounded by the epidermis layer (Figure 3C). The emergence of vascular structures was observed thanks to the anti-CD31 staining of endothelial cells bordering lacunas (Figure 3H). Immunostaining with the ki67 antibody detected the presence of proliferating cells, even after 14 to 21 days of culture at the air-liquid interface (arrows) (Figures 3G and 31). This proliferation was more pronounced at the base of the epithelium (Figure 3G) - which is the usual localization of proliferative cells in a natural skin epidermis - and at the border of the vascular lacunas (Figure 31). The later observation confirms the formation of vascular vessel-like structures. The presence of immune cells was confirmed (Figure 3J). Abundant mature collagen fibers were detected by Masson Trichrome staining (Figure 3K), highlighting the activity of fibroblasts, the main components of the dermal compartment. These collagen fibers were sometimes distinctly fibrillar, bordering multiple mesenchymal lobules. Even more surprisingly, sweat glands were observed, showing the cellular richness and complexity of the spheroid assembly of the invention (Figure 3L). Finally, the presence of dendritic cells and macrophages was detected thanks to anti-CDl lc (Figure 3M) and anti-CD68 (Figure 3N) staining, confirming the presence of myeloid immune cells in the spheroid assembly.

[0206] In a second configuration, when skin dECM hydrogel was mixed with a 3D PCL scaffold (Figure 4A), the obtained spheroid assemblies were finely organized. The epidermis was neatly separated from the dermis with a polarization of the epidermis (Figure 4B). The presence of numerous proliferating cells (Figure 4C), collagen fibers (Figure 4E), dendritic cells (Figure 4G), macrophages (Figure 4H), gaps bordered by endothelial cells (Figure 4D), , as well as of a sweat gland were also detected (Figure 4F). By comparison to the first configuration, the presence of 3D PCL appeared to increase the mechanic stability of the spheroid assemblies (data not shown).

[0207] In a third configuration, when the 3D PCL scaffold was used alone (i.e. without Skin ECM hydrogel), the obtained spheroids assemblies were quite similar to those obtained in the second configuration. A neat separation was indeed observed between the dermis and the epidermis, as well as a stratification of the epidermis (Figure 5A). The later showed a distinct basal layer and the presence of keratin lamellae in the upper layer (stratum corneum). The assembly showed great cellular richness with the presence of a developing sweat gland (Figure 5B), proliferating cells (Figure 5C), collagen fibers (Figure 5D), macrophages (Figure 5E) and dendritic cells (Figures 5F) as well as developing vascular lacunae (Figure 5G). The sole presence of 3D PCL appeared to be sufficient to increase the mechanic stability of the spheroid assemblies (data not shown), similarly to the second configuration.

[0208] By contrast, when the spheroids of the invention were cultured in an adherent insert coated with skin dECM hydrogel, the epidermal cells did not structure correctly and the fibroblasts escaped from the assembly to cover the adherent insert (data not shown).

[0209] Likewise, when the spheroids of the invention were cultured in a non-adherent insert not coated with skin dECM hydrogel and / or 3D PCL, the spheroids merged with each other, but lacked a proper cellular organization. There was no polarization of the epidermis and no secretion of collagen. The merged spheroids started to shrink and to became randomly necrotic (data not shown). At last, when the spheroids of the invention were cultured in a non-adherent insert coated with a synthetic hydrogel (lacking skin dECM), the spheroids did not merge (data not shown).

[0210] 3. CONCLUSION

[0211] Histological and immunofluorescence analysis shows that the individual and assembled spheroids contained numerous organized constituents of human skin, especially the presence of keratinocytes with polarization of the epidermis and keratinization, the presence of dermal fibroblasts, the establishment of an epidermis / dermis interface, as well as the presence of melanin, an endothelial network, immune cells, a sweat gland, cell-secreted extracellular matrix and proliferating cells after only 25 days of culture (starting from production of spheroids until their complete assembly).

Claims

CLAIMS1. A process for the in vitro obtention of a multicellular human skin spheroid, comprising:(a) mixing in suspension a first population of cells constitutive of a human epidermis layer with a second population of cells constitutive of a human dermis layer, in a ratio ranging from about 1 : 4 to about 1 : 14 between the number of cells in the first population and the number of cells in the second population, the first population of cells comprising keratinocytes, melanocytes, and immune cells of the epidermis and the second population of cells comprising fibroblasts, endothelial cells, and immune cells of the dermis; and(b) incubating the cell suspension obtained in step (a) in an endothelial cell growth medium.

2. The process according to claim 1, wherein the immune cells of the epidermis comprise Langerhans cells, neutrophils, eosinophils and lymphocytes and / or the immune cells of the dermis comprise dendritic cells, macrophages, monocytes, neutrophils, eosinophils and lymphocytes .

3. The process according to claim 1 or 2, wherein the first population of cells and / or the second population of cells are primary cells, preferably adult primary cells.

4. The process according to any one of claims 1 to 3, comprising, prior to step (a): separating an isolated human skin sample into an epidermis layer and a dermis layer; and dissociating the cells from each separated layer, thereby obtaining a first population of cells constitutive of a human epidermis layer and a second population of cells constitutive of a human dermis layer, respectively.

5. The process according to any one of the preceding claims, wherein, in step (a), the ratio between the number of cells of the first population and the number of cells of the second population is about 1 : 9.

6. The process according to any one of the preceding claims, wherein, in step (b), the cell suspension is incubated under low-adherent or non-adherent culture conditions.

7. The process according any one of the preceding claims, wherein the endothelial cell culture medium used in step (b) comprises ascorbic acid, hydrocortisone, and growth factors EGF (Epidermal Growth Factor), FGF (Fibroblast Growth Factor), IGF (Insulin Growth Factor) and VEGF (Vascular Endothelial Growth Factor); and optionally heparin, serum, or any combination thereof.

8. The process according to any one of the preceding claims, wherein the cell suspension is incubated in step (b) for a time period of at least about 5 days to about 10 days, preferably at least about 7 days.

9. The process according to any one of the preceding claims, wherein the cell suspension is incubated in step (b) for a time period of at most about 15 days to about 25 days, preferably at most about 20 days.

10. A multicellular human skin spheroid, obtainable or obtained by the process as defined in any one of the preceding claims.

11. The spheroid according to claim 10, comprising a network of endothelial cells.

12. A method for the in vitro assembly of multicellular human skin spheroids into an artificial human skin construct, comprising:(1) placing a plurality of multicellular human skin spheroids as defined in claim 10 or 11, on or in a porous scaffold; and(2) culturing the plurality of multicellular human skin spheroids placed on or in the scaffold in step (1), in presence of an organoid growth medium.

13. The method according to claim 12, comprising, prior to step (1):(0) obtaining a plurality of multicellular human skin spheroids according to the process defined in any one of claims 1 to 9, preferably according to the process defined in claim 8.

14. The method according to claim 12 or 13, wherein the porous scaffold is in form of : a hydrogel comprising extracellular matrix components, a poly(epsilon-caprolactone) (PCL) scaffold, or a combination thereof.

15. The method according to any one of claims 12 to 14, wherein the organoid growth medium is an epidermalization medium.

16. The method according to any one of claims 12 to 15, wherein, in step (2), the plurality of multicellular human skin spheroids is cultured at an air-liquid interface and / or under low-adherent or non-adherent culture conditions.

17. The method according to any one of claims 12 to 16, wherein, in step (2), the plurality of multicellular human skin spheroids is cultured for a time period of at least about 10 days to about 30 days.

18. An artificial human skin construct, obtainable or obtained by the method as defined in any one of claims 12 to 17.

19. The artificial human skin construct according to claim 18, comprising immune cells, one or more vascular vessel-like structures and one or more skin appendages, especially sweat gland-like structures.

20. Use of a multicellular human skin spheroid as defined in claim 10 or 11, or of an artificial human skin construct as defined in claim 18 or 19, in in vitro assays.

21. A multicellular human skin spheroid as defined in claim 10 or 11, or an artificial human skin construct as defined in claim 18 or 19, for use as a medicament.

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