Skin organoids for biologic discovery

Abstract

A culture system for human dermal and epidermal layers of the skin is provided, which culture reproduces the histomorphology of native human skin. The system is highly reproducible and maintains the cellular diversity of human skin for extended periods of time, both in the types of cells that are present, and in the architecture of the tissue. Features of the culture include self-production of molecules and proteins required for maintenance and maturation of the diverse cellular compartments.

Description

ATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171SKIN ORGANOIDS FOR BIOLOGIC DISCOVERYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 687,029, filed August 26, 2024, benefit of U.S. Provisional Patent Application No. 63 / 686,076, filed August 22, 2024, which applications are incorporated herein by reference in their entirety.BACKGROUND

[0002] The human integumentary system is a complex composition of diverse structures and cell types that represents the first physical barrier and defense against pathogens, chemicals, and other environmental insults. In addition to acting as a physical barrier, human skin possesses a complex immune cell repertoire to further survey and defend against pathogenic insults. With its own microbiome, the keratinocytes of the epithelial layer and immune cells within the epidermis and dermis must communicate and coordinate to ensure appropriately tailored immune responses for productive protection. The importance of this coordination is best evidenced when it goes awry. In addition to being an entry point for an expansive number of well-known and emerging pathogens, human skin is also afflicted by an expansive number of auto-inflammatory conditions.

[0003] While human skin and skin diseases have benefited greatly from mouse and animal models, fundamental biological differences between human and animal models limit their utility. As such, there has been a rapid expansion in the development of ex vivo culture techniques and organoid culture methods aimed at reproducing human skin biology. While currently available systems are capable of recreating critical elements of human skin, none support the growth and maintenance of the native epithelial, stromal, and immune cells resident in human skin.

[0004] The present disclosure provides methods for the generation of 3-dimensional airliquid interface organoid systems that cultures skin en bloc with its endogenous immune, epithelial, and stromal cells.SUMMARY

[0005] A culture system for human dermal and epidermal layers of the skin is provided, which culture reproduces the histomorphology of native human skin. The human skin culture system maintains epidermal proliferation, differentiation, maturation and barrier function, and possesses diverse specialized cell types critical for skin function, including endothelial, fibroblast, stromal and immune cells. The system is highly reproducible and maintains the cellular diversity of human skin for extended periods of time, both in the types of cells that areATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 present, and in the architecture of the tissue. Features of the culture include self-production of molecules and proteins required for maintenance and maturation of the diverse cellular compartments.

[0006] The systems of the disclosure are shown to be responsive to diverse chemical and biological stimuli, responding in stereotypic manner phenocopying known in vivo responses, including without limitation viral infection. Conditions of interest include immune responses, e.g. autoimmune responses, responses associated with transplantation, responses associated with cancer, responses associated with contact allergens, responses to infection and vaccination, and the like. Other conditions of interest include genetic diseases of the skin, responses of skin to chemical, including without limitation, skin irritants, gene editing in a pathway of interest, response of skin to drug therapy, and the like.

[0007] In an embodiment, a method is provided for generating a human skin culture system, the method comprising admixing small blocs of human skin, e.g. a biopsy sample, with a gel forming polymer, layering the admixture over a gel to form an insert, and placing the insert in a well with media outside the well, such that the skin admixture is exposed to liquid media from the bottom and to air from the top. The blocs of skin placed into culture may be referred to as an initiating tissue bloc, where all the cells in the bloc are derived from the same individual source. In an embodiment, all of the human cells present in the culture are derived from the initiating bloc. In an embodiment, the medium is free of exogenous protein factors during the initiation of the culture, e.g. from about 1 to about 4 weeks. Factors such as Rspo family members, Wnt family members, BMP2, TGF-Beta 1 , EGF, EGFR, can be endogenously produced by cells present in the culture itself.

[0008] This air-liquid interface (ALI) method allows culturing of the diverse cells present in skin together as a cohesive 3-dimensional unit that recapitulates the function and the microanatomy of the original tissue, and includes endogenous immune cells. In ALI, adequate oxygenation is achieved by culturing microscopic fragments of tissue embedded in a matrix within a trans-well ("inner dish”) in which direct air exposure is obtained from the top; whilst contact with tissue culture media contained in an "outer dish”; is obtained from the bottom via the trans-well permeable membrane

[0009] Cells present in the culture may comprise, without limitation, macrophages, keratinocytes, T cells, endothelial cells, fibroblasts, melanocytes, mast cells, and neuronal cells that are derived from the initiating tissue bloc and are present in the culture for an extended period of time. Specifically, keratinocytes are shown to have normal histology and differentiation, and in culture have a stratum basalis (basal keratinocytes), stratum spinosum (spinous keratinocytes), and stratum corneum (cornified keratinocytes). In addition to these layers, observed structures may also comprise sweat glands, hair shafts and follicles, and blood vessels. The immune cells in the culture may comprise CD4+T-cells, CD8+T-cells, mastATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 cells, Langerhans cells, macrophages, and y5 T-cells. In some embodiments, each of the cell types listed above are present in the culture system.

[0010] In some embodiments, a method is provided for screening assays to determine the effect of an agent on the skin. In the methods, an initiating tissue bloc is placed in culture. The culture is then subjected to a treatment or stimulus of interest and the response of the cultured cells to the treatment is measured. Various responses can be measured, including without limitation: changes in gene expression, changes in protein levels, changes in histology, changes in the absolute number of specific cell types, changes in the relative number of specific cell types, viability of specific cell types, etc. In such methods, the post-initiation culture system may comprise, for example, an infectious virus, e.g. a pathogenic virus; bacteria, e.g. pathogenic bacteria; exogenous factors; exogenous mammalian cells; drugs; genetic constructs, skin irritants; etc.

[0011] In some embodiments, screening methods are used in precision medicine and diagnostics, where the initiating tissue bloc is obtained from an individual of interest. The response of the culture system to an agent or treatment is used to predict or inform the expected clinical response to the same treatment by the individual of interest. In some embodiments, the individual is treated in accordance with the findings of the screening assay.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIGS. 1 A-1 E: Schematic and initial characterization of an air-liquid interface (ALI) skin organoid maintains native architecture, a, Schematic of technique to generate human skin airliquid interface organoid, b, Air-liquid interface culture setup, consisting of transwell insert within a 6-well dish with media surrounding the outside of insert containing organoids, c, Brightfield transwell images at day 0 and day 28. Inserts highlight fragments with interval change and growth, d, Representative full collagen inserts from ALI culture at day 14, day 28, and day 56, sectioned en face and H&E stained. Insets highlight individual skin organoid fragments, e, H&E stained primary skin and skin organoids.

[0013] FIGS. 2A-2M: Immunohistochemical and immunofluorescent characterization of skin organoid culture, a, Representative immunohistochemical staining of p63 and b, p40 basal keratinocytes on day 14 organoids, c, Representative Immunofluorescence staining of KRT5, KRT15, and Hoechst, and d, KRT1 , KRT5, and Hoechst of day 14 organoids, e, Immunofluorescence staining of KRT5, KRT15, and Hoechst, and f, KRT5, KRT10, Loricrin, and Hoechst on day 28 organoids, g, Immunohistochemical staining of Ki67 on day 28 organoids, h, Immunofluorescence staining of PCNA, KRT5, and Hoechst on a day 28 organoids, i, Immunohistochemical staining of SOX10 melanocytes on day 14 organoids, j, H&E staining of eccrine glands in a day 28 organoid, k, H&E staining of hair follicles in a dayATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-17128 organoid. I, H&E staining of a hair follicle shaft embedded in the dermis, m, Immunohistochemical staining of CD34 endothelial cells on a day 28 organoid.

[0014] FIGS. 3A-3L: Pharmacologic perturbation of human skin organoid cultures, a, Representative histological image of an organoid following treatment + / - 5 M IWP-2 for 14 days, b, Quantification of epidermal thickness N = 4 biological replicates with 4 different organoids each, c, Representative histological image of an organoid following treatment + / - 50nM erlotinib for 14 days, d, quantification of basalis and spinosum thickness N = 5 biological replicates with 4 different organoids each, e, Representative histological image of a skin organoid following treatment + / - 100ng / mL TPA treatment for 28 days, f, Quantification of epidermal thickness N = 3 biological replicates with 4 organoids each, g, Representative immunohistochemistry of SQX10 melanocyte staining of skin organoids following treatment + / - 100 ng / mL TPA for 28 days, h, Quantification of SQX10+ cells per 500 pm of epidermis N = 4 biological replicates with 4 organoids each, i, Representative immunohistochemistry of MelanA production in skin organoids following treatment + / - 100 ng / mL TPA for 28 days, j, Quantification of MelanA per nucleus N= 4 biological replicates with 4 organoids each, k, Representative immunofluorescence staining of yH2AX, keratin 1 (KRT1 ), keratin 5 (KRT5), with Hoechst stain of day 3 skin organoids treated + / - 5pM DMBA for 48 hours. I, Quantification of single base substitution DNA mutations in day 28 ALI organoids following 2 day + / - 5pM DMBA treatment at day 3. For statistical analysis, paired t tests were performed in all summary plots.

[0015] FIGS. 4A-4D: Human skin ALI organoids infected with Mpox. a, Immunofluorescence staining with KRT5, KRT10, Mpox, and Hoechst of organoids 7 days post + / - Mpox infection, b, Representative immunohistochemistry staining for Mpox cross reactive polyclonal antibodies in organoids 7 days post + / - Mpox infection c, Representative H&E staining of organoids 7 days post + / - Mpox infection, d, Quantitative PCR of Mpox genes at day 1 , 3, 5, and 7 on organoids + / - Mpox infection.

[0016] FIGS. 5A-5I: Immune cells and specialized cell types present in skin organoids. Immunohistochemical staining of day 28 organoids for a, CD45RB immune cells b, CD3 T cells c, CD8 T cells d, CD68 macrophages e, Langerin for Langerhans cells and f, CD1 17 mast cells, g, Flow cytometry summary results of immune cells out to day 100. N = 3+ biological replicates h,i, UMAPs generated for single cell data to show cell types present at day 0 (N = 3), day 14 (N = 1 ), and day 28 (N = 3) organoids.

[0017] FIGS. 6A-6E: Human skin ALI organoids contain a responsive immune component, a, Flow cytometry plot of day 9 ALI organoids for CD103 and CD69 tissue resident immune markers pre-gated on live single CD45+ immune cells, b, Flow cytometry plot of day 9 ALI organoids for CD1 1 c and HLA-DR / DP / DQ MHC class II antigen presenting dendritic cells pregated on CD45+ immune cells, c, Flow cytometry plot of CD49f and HLA-DR / DQ / DP MHCATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 class II presenting keratinocytes pre-gated on CD45- cells, d, Flow cytometry plots assessing double positive AIM markers CD40L, 0X40, CD25, and 4-1 BB on CD8+ T cells in ALI organoids treated for 6 days with a T cell stimulating cocktail containing activating CD3, CD28, and CD2 antibodies. The results were summarized and assessed using paired t-test. e, Box and whisker plot of secreted factors in ALI organoids + / - treatment with a T cell stimulating cocktail containing activating CD3, CD28, and CD2 antibodies analyzed by Luminex. N=7.

[0018] FIGS. 7A-7F: Human skin ALI organoids infected with HSV. a, H&E stained ALI organoids 2 days post + / - HSV-1 infection of day 3 organoids, b, Immunofluorescence and immunohistochemistry analysis of HSV-1 infection using two different HSV-1 antibodies 2 days post infection of day 3 ALI organoids, c, Box and whisker plot of secreted factors in ALI organoids 6 days post + / - HSV-1 infection. N=5. d,e,f, Flow cytometry of double positive AIM markers CD40L, 0X40, and CD69 on CD8+ or CD4+ T cells in ALI organoids 6 days post + / - HSV-1 infection. The results were summarized using paired t test analysis, d, CD69 and 0X40. e, CD69 and CD40L, f, 0X40 and CD40L.

[0019] FIG. 8: Dermis only fragment from skin organoid cultures. Full collagen inserts from organoids at day 14, day 28, and day 56, sectioned en face and H&E stained the same inserts as FIG. 1 but insets highlight individual skin organoid fragments that are made of only dermis instead of epidermis and dermis.

[0020] FIG. 9: H&E of blood vessels from human skin organoid culture. Full collagen inserts from organoids at day 28 sectioned en face and H&E stained. Insets highlight blood vessels in the dermis of the skin organoid fragments.

[0021] FIGS. 10A-10I: Representative H&E and immunohistochemistry p63 and Ki67 stained human skin organoid cultures, a, Representative immunohistochemistry of p63 basal keratinocytes in ALI organoids treated + / - 5pM IWP-2 for 14 days, b, Quantification of p63+ cells per 500pm of epidermis N = 4 biological replicates with 4 organoids each, c, Representative immunohistochemistry of Ki67-positive cells in ALI organoids treated + / - 5pM IWP-2 for 14 days, d, Quantification of Ki67-positive cells per 500 pm epidermis. N = 4 biological replicates with 4 organoids each, e, SuperTopFlash Wnt reporter luciferase assay quantification of Wnt secretion in ALI organoids treated + / - 5pM IWP-2 for 14 days, f, Representative immunohistochemistry of p63 basal keratinocytes in ALI organoids treated + / - 50nM erlotinib, g, Quantification of p63+ cells per 500 pM epidermis N = 4 biological replicates with 4 organoids each, h, Representative immunohistochemistry of Ki67-positive cells in ALI organoids treated with + / - 50nM erlotinib for 14 days, i, Quantification of Ki67-positive cells per 500um epidermis N = 4 biological replicates with 4 organoids each. For all summary plots, paired t tests were performed.

[0022] FIGS. 1 1 A-11 F: Mpox infection of human skin organoid cultures at early time points shown by immunofluorescence and quantitative PCR. a, Immunofluorescence staining ofATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171KRT5, KRT10, Mpox, and DAPI, on organoids 3 days post + / - Mpox infection, b, H&E stained organoids 3 days post + / - Mpox infection, c, Immunofluorescence staining of KRT5, KRT10, Mpox, and DAPI, on organoids 5 days post + / - Mpox infection, d, H&E stained organoids 5 days post + / - Mpox infection, e, Immunofluorescence staining of KRT5, KRT10, Mpox, and DAPI, on organoids 14 days post + / - Mpox infection, f, H&E stained organoids 14 days post + / - Mpox infection. For all data N = 4+.

[0023] FIGS. 12A-12I: Human skin organoid cultures self-produce bioactive molecules assessed by scSEQ. a, Violin plots from scSEQ data for primary human skin tissue (blue) or skin ALI organoids at day 14 and 28 combined (red) for IL2, IL7, and IL15. b, Quantification of IL-2, IL-7, and IL-15 secretion by Luminex analysis of organoid conditioned media, c, Violin plots from scSEQ data for CSF1, CSF2, IL6, and KITLG. d, Quantification of M-CSF and GM- CSF secretion by Luminex analysis of organoid conditioned media, e, Violin plots from scSEQ data for RSPO1, RSPO2, and RSPO3. f, Violin plots from scSEQ data for WNT4, WNT5A, and WNT5B. g, Violin plots from scSEQ data for BMP2, TGFB1, EGF, and EGFR. h, Violin plots from scSEQ data for HGF and VEGFA. i, Quantification of EGF, HGF, and VEGF secretion by Luminex analysis of day 14 organoid conditioned media. For violin plots N = 3 for primary human skin and N = 4 biological replicates for organoids. For Luminex experiments N = 3 biological replicates.

[0024] FIGS. 13A-13B: a, mock and HSV-1 infected organoids day 6 infected at day 4. b, 10 day old organoids. 24 hour HSV infected organoids at day 9. 6 day HSV-1 infected at day 4.DETAILED DESCRIPTION

[0025] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0026] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.ATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0028] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0029] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the date of publication provided may be different from the actual publication date, which may need to be independently confirmed.

[0030] As used herein, compounds which are "commercially available" may be obtained from commercial sources including but not limited to Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee Wl, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester NY), Fisher Scientific Co. (Pittsburgh PA), Fisons Chemicals (Leicestershire UK), Frontier Scientific (Logan UT), ICN Biomedicals, Inc. (Costa Mesa CA), Key Organics (Cornwall U.K.), Lancaster Synthesis (Windham NH), Maybridge Chemical Co. Ltd. (Cornwall U.K.), Parish Chemical Co. (Orem UT), Pfaltz & Bauer, Inc. (Waterbury CN), Polyorganix (Houston TX), Pierce Chemical Co. (Rockford IL), Riedel de Haen AG (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland OR), Trans World Chemicals, Inc. (Rockville MD), Wako Chemicals USA, Inc. (Richmond VA), Novabiochem and Argonaut Technology.

[0031] Compounds can also be made by methods known to one of ordinary skill in the art. As used herein, "methods known to one of ordinary skill in the art" may be identified though various reference books and databases. Suitable reference books and treatises that detail the synthesis of reactants useful in the preparation of compounds of the present invention, or provide references to articles that describe the preparation, include for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; S. R. Sandler et al., "OrganicATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H. O. House, "Modern Synthetic Reactions", 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L. Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, New York, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanisms and Structure”, 4th Ed., Wiley-lnterscience, New York, 1992. Specific and analogous reactants may also be identified through the indices of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases (the American Chemical Society, Washington, D.C., may be contacted for more details). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (e.g., those listed above) provide custom synthesis services.

[0032] In the description that follows, a number of terms conventionally used in the field of cell culture are utilized extensively. In order to provide a clear and consistent understanding of the specification and claims, and the scope to be given to such terms, the following definitions are provided.

[0033] The term "cell culture” or "culture" means the maintenance of cells in an artificial, in vitro environment. It is to be understood, however, that the term "cell culture" is a generic term and may be used to encompass the cultivation not only of individual cells, but also of tissues or organs.

[0034] The term “culture system” is used herein to refer to the culture conditions in which the subject explants are grown that promote prolonged tissue expansion with proliferation, multilineage differentiation and recapitulation of cellular and tissue ultrastructure. A culture system also refers to a non-ALI culture in which cells of interest can be expanded, e.g. on feeder layer cells.

[0035] Culture conditions of interest provide an environment permissive for differentiation, in which the complex cell system from a skin bloc will proliferate, differentiate, or mature in vitro. Such conditions may also be referred to as “differentiative conditions”. Features of the environment include the medium in which the cells are cultured, any endogenous growth factors or differentiation-inducing factors that may be present, and a supporting structure (such as a substrate on a solid surface) if present.

[0036] “Gel substrate”, as used herein has the conventional meaning of a semi-solid extracellular matrix. Gel described herein includes without limitations, collagen gel, matrigel, extracellular matrix proteins, fibronectin, collagen in various combinations with one or more of laminin, entactin (nidogen), fibronectin, and heparin sulfate; and human placental extracellular matrix. In some embodiments the matrix comprises collagen, e.g. Type I collagen.ATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171

[0037] An “air-liquid interface” is the interface to which the tissue bloc is exposed to in the cultures described herein. The initiating tissue bloc is mixed with a gel solution which is then poured over a layer of gel formed in a container with a lower semi-permeable support, e.g. a membrane. This container is placed in an outer container that contains the medium such that the gel containing the tissue in not submerged in the medium. The initiating tissue is exposed to air from the top and to liquid medium from the bottom, see for example US Patent no. 9,464,275, herein specifically incorporated by reference.

[0038] By "containei1' is meant a glass, plastic, or metal vessel that can provide an aseptic environment for culturing cells.

[0039] The term “exogenous protein factors” refers to soluble cytokines, chemokines, differentiation factors, growth factors, etc. These factors are commonly included in mammalian cell culture systems. Examples of factors include Rspo family members, Wnt family members, BMP family members, epidermal growth factors, nerve growth factors, TGF, etc., as known in the art. In some embodiments, serum from animals, e.g. fetal bovine serum, is included in the ambit of the term.

[0040] Without being bound by the theory, the cultures of the present disclosure may benefit from a lack of exogenous factors, as it may allow endogenous production and regulation of factors by cells present in the culture system.

[0041] The term “explant’ is used herein to mean a piece of skin tissue and the cells thereof originating from the skin tissue that is cultured in vitro, for example according to the methods of the invention. The tissue from which the explant is derived may be obtained from an individual of interest, i.e. a patient with a disorder of the skin. Methods of interest include patient-specific analysis of skin cell responses.

[0042] The term “organoid' is used herein to mean a 3-dimensional growth of skin tissue in culture that retains characteristics of the skin in vivo, e.g. recapitulation of cellular and tissue ultrastructure, differentiation, immune cell interactions, etc. Organoids for use in the methods disclosed herein are generally cultured from a skin biopsy section. Organoids that find use in the present disclosure, include without limitation, those cultured en bloc in an air liquid interface method.

[0043] Methods are provided for the culture of small amounts of clinical specimens. Samples of interest include human tissue, e.g. skin microbiopsy samples such as needle or fine needle aspirate. Samples may be taken at a single timepoint, or may be taken at multiple timepoints. Samples may be as small as 107cells, 106cells, 105cells, or less; e.g. a biopsy section of from about 0.1 mm2, about 1 mm2, about 10 mm2, etc.

[0044] The air-liquid interface (ALI) method allows the propagation of organoids with all the cellular components and structures of skin. The ALI method utilizes Boyden chambers (cell culture inserts) used for cell migration assays. Cells are embedded in a gel matrix in an upperATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 surface of the cell culture inserts with a porous membrane underneath and cells are directly exposed to oxygen, which substantially increases the oxygen supply to the cells as compared to a submerged organoid method. Cells obtain nutrients and growth factors from the medium placed in the outer dish through diffusion across the porous membrane on the lower surface. Methods of using the ALI method to culture PDOs is known within, for instance, as disclosed by Neal et al. Cell. 2018 Dec 13;175(7):1972-1988.e16 which is incorporated herein in its entirety by reference. The various cells present in the culture are generally derived from the initiating tissue bloc, although in some specific embodiments, after the culture is established, cells of various sorts are added to determine the response of the culture.

[0045] As used herein, the term “immune cell” includes cells that are of hematopoietic origin and that play a role in the immune response. Immune cells include lymphocytes, such as B cells and T cells; natural killer cells; dendritic cells; myeloid cells, such as Langerhans cells, monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0046] The term “T cells” refers to mammalian immune effector cells that may be characterized by expression of CD3 and / or T cell antigen receptor. In some embodiments, the T cells are selected from naive CD8+T cells, cytotoxic CD8+T cells, naive CD4+T cells, helper T cells, e.g. TH1 , TH2, TH9, TH11 , TH22, TFH; regulatory T cells, e.g. TR1 , natural TReg, inducible TReg; memory T cells, e.g. central memory T cells, T stem cell memory cells (TSCM). effector memory T cells, NKT cells, □ □ T cells. In some embodiments, the immune cells comprise a complex mixture of immune cells, as are typically present in the skin.

[0047] The phrase “mammalian cells" means cells originating from mammalian tissue. Typically, in the methods of the invention, pieces of tissue are obtained surgically, e.g. biopsy, needle biopsy, etc. and minced to a size less than about 2 mm3, and may be less than about 1 mm3, or less than about 0.5 mm3. “Mammalian” used herein includes human, equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc. “Mammalian tissue cells” and “primary cells” have been used interchangeably.

[0048] “Ultrastructure”, or histomorphology, refers to the three-dimensional structure of a cell or tissue observed in vivo. For example, the ultrastructure of a cell may be its polarity or its morphology in vivo, while the ultrastructure of a tissue would be the arrangement of different cell types relative to one another within a tissue. In the cultures of the disclosure, for example, structures such as blood vessels, hair follicles, sweat glands, etc. may be observed.

[0049] The term “sample” with respect to a patient encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. For the purposes of initiating cultures of the disclosure, samples comprise a skin bloc comprising at least the dermal and epidermalATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 layers of the skin. The definition also includes samples that have been manipulated in any way after their procurement. A “biological sample” includes a sample obtained from a patient's tissue, e.g., a sample comprising polynucleotides and / or polypeptides that is obtained from a patient’s cell (e.g., a cell lysate or other cell extract comprising polynucleotides and / or polypeptides); and a sample comprising cells from a patient.

[0050] The term “diagnosis” is used herein to refer to the identification of a molecular or pathological state, disease or condition, such as the identification of a disease of the skin.

[0051] The term “prognosis” is used herein to refer to the prediction of the likelihood of a disease progression, including recurrence, spread; drug responsiveness, etc. The term “prediction” is used herein to refer to the act of foretelling or estimating, based on observation, experience, or scientific reasoning. In one example, a physician may predict the likelihood that a patient will respond to a treatment of interest. The present methods allow prediction of whether a patient will be responsive to a therapy of interest.

[0052] As used herein, the terms “treatment,” “treating,” and the like, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of effecting a partial or complete cure for a disease and / or symptoms of the disease. “Treatment,” as used herein, may include treatment of a skin condition in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0053] Treating may refer to any indicia of success in the treatment or amelioration or prevention of disease, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the compounds or agents to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with cancer or other diseases. The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.ATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171Conditions of interest for samples, screening assays and / or treatment

[0054] The skin is the largest organ in the body, covering its entire external surface. The skin has 3 layers — the epidermis, dermis, and hypodermis, which have different anatomical structures and functions. The skin's structure comprises an intricate network that serves as the body's initial barrier against pathogens, ultraviolet (UV) light, chemicals, and mechanical injury. This organ also regulates temperature and the amount of water released into the environment.

[0055] The skin's barrier function makes it susceptible to various inflammatory and infectious conditions. In addition, wound healing, sensory changes, and cosmesis are significant surgical concerns. Understanding the skin's anatomy and function is crucial for managing conditions across all medical fields.

[0056] Diseases of the skin are wide-ranging and span all medical disciplines. These include, for example, infections, immune-associated diseases, cancers, drug reactions, contact dermatitis, genetic conditions, and the like. The culture systems of the present disclosure are suitable for screening of agents that act on these conditions, and as research tools for investigating the etiology and progression of these diseases. In some embodiments, an initiating tissue bloc is obtained from an individual suffering from a skin disease. In some embodiments, an initiating tissue bloc is obtained from an individual free of the disease, where a disease condition is induced in the culture system, for example by introducing an infectious agent, by inducing an immune response, by treating with a contact allergen, etc.

[0057] Skin cancer is the most common type of cancer and commonly develops in sun- exposed areas of skin. Skin cancers can be found on any location of the body but are most commonly diagnosed on the head and neck. The incidence is highest among people who spend a lot of time outdoors for work or recreation and is inversely related to the amount of melanin skin pigmentation; people who have light skin are most susceptible. Skin cancers may also develop years after therapeutic radiation or exposure to carcinogens.

[0058] The most common forms of skin cancer are basal cell carcinoma (approximately 80%), squamous cell carcinoma and melanoma. The less common forms of skin cancer are Paget disease of the breast or extramammary Paget disease, Kaposi sarcoma, merkel cell carcinoma, atypical fibroxanthomas, tumors of the adnexa, cutaneous T-cell lymphoma (mycosis fungoides), and squamous cell carcinoma in situ.

[0059] Squamous cell carcinoma is a malignancy arising from mutated keratinocytes, typically due to UV damage in individuals with type I or II skin types. These individuals typically have light skin, blue or green eyes, and red or blonde hair and burn without tanning. The lesions often appear as scaly, flaky, thick red patches that may bleed. Some squamous cell carcinoma tumors resemble warts. This type of skin cancer can metastasize. Squamous cellATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 carcinoma often arises from actinic keratoses — premalignant lesions with cutaneous horns developing from chronic UV damage.

[0060] Basal cell carcinoma is a malignant neoplasm of the basal layers of the epidermis. Unlike squamous cell carcinoma, it is much less likely to metastasize. This type of skin cancer is more common in sun-exposed areas, often appearing as pearly papules on the face, with telangiectasias and a great tendency to ulcerate.

[0061] Melanoma is a highly invasive malignant melanocyte tumor that is fatal but rarer than skin squamous cell carcinoma and basal cell carcinoma. This neoplasm's high metastatic potential is significantly mediated by lesion depth. Melanoma can be found anywhere on the body and is typically irregularly pigmented but can be amelanotic.

[0062] Langerhans cell histiocytosis is a type of cancer in which Langerhans cells accumulate in the body and form granulomas, often in the bones, causing bone pain. These granulomas can also appear in the skin, producing rashes, erythematous papules, or blisters. Notably, Langerhans cell histiocytosis can affect the pituitary gland, leading to diabetes insipidus, infertility, or other endocrine disorders due to hormone deficiencies. Pancytopenia is a potentially fatal Langerhans cell histiocytosis complication, manifesting with anemia, thrombocytopenia, and leukocytopenia, caused by overcrowding of Langerhans cells in the bone marrow.

[0063] Merkel cell carcinoma is an uncommon cancer of the Merkel cells. This tumor is categorized as a neuroendocrine small cell carcinoma. Clinically, Merkel cell carcinoma often presents as a painless, solitary cutaneous or subcutaneous nodule, sometimes with a cystic appearance. The nodule can be red, pink, violet, blue, or skin-colored. Lesions may ulcerate or have satellite lesions. Merkel cell carcinoma is typically smaller than 20 mm at diagnosis but shows rapid tumor growth over a few months.

[0064] The immune system plays a significant role in many skin disorders. Immune associated diseases of the skin include autoimmune conditions, responses to infection, contact dermatitis, hypersensitivity to an agent, etc.

[0065] Allergic contact dermatitis (ACD) is a type IV, T-cell-mediated, delayed-type hypersensitivity reaction to an environmental allergen that has 2 phases: sensitization to an antigen; and allergic response after reexposure. In the sensitization phase, allergens are captured by Langerhans cells. When activated by innate immunity cascades, these cells migrate to regional lymph nodes, where they process and present the antigen to naive, antigen-specific T cells. When a naive T cell recognizes its antigen via binding to its T-cell receptor, it expands clonally and differentiates into memory / effector T cells. The sensitization phase, which is asymptomatic, may be brief or prolonged. During differentiation, sensitized T cells become able to express cutaneous homing antigens that enable them to migrate fromATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 cutaneous capillaries to the epidermis. When antigen-presenting cells present the antigen to the sensitized T cells, the T cells can expand and trigger an inflammatory reaction at that location (elicitation phase of ACD), resulting in the characteristic symptoms and signs of ACD.

[0066] Multiple allergens can cause ACD. Nickel sulfate is a common contact allergen in most populations. The Toxicodendron species of plant, e.g. poison ivy, poison oak, poison sumac, accounts for a large percentage of ACD, including moderate and severe cases. The offending allergen is urushiol.

[0067] Autoimmune diseases of the skin include, for example, scleroderma, vitiligo, systemic lupus erythematosus, dermatitis herpetiformis; and alopecia areata.

[0068] Scleroderma is a rare connective tissue disorder with an unknown and complex pathogenesis. Scleroderma can be divided into 2 primary forms -localized scleroderma and systemic sclerosis. Systemic sclerosis can be further classified as limited systemic sclerosis (formerly known as CREST syndrome, characterized by calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia) or diffuse systemic sclerosis based on clinical and serological criteria. Localized scleroderma primarily affects the skin and subcutaneous tissue, leading to patches of thickened skin that, on biopsy, reveal dermal fibrosis similar to the histopathological changes seen in the thickened skin in systemic sclerosis. Limited cutaneous systemic sclerosis, previously known as CREST syndrome, is characterized by skin thickening distal to the elbows and knees and / or on the face without trunk involvement. On the other hand, diffuse cutaneous systemic sclerosis involves skin thickening that may affect areas proximal to the elbows, knees, face, and / or trunk. Antinuclear antibodies may be present in more than 90% of cases of systemic sclerosis, and up to 70% of cases exhibit at least one of the more specific autoantibodies (anti-centromere, anti-Scl-70, and anti-RNA polymerase III).

[0069] Systemic lupus erythematosus is a chronic, multisystem, inflammatory disorder of autoimmune etiology, occurring predominantly in young women. Common manifestations may include arthralgias and arthritis, Raynaud syndrome, malar and other rashes, pleuritis or pericarditis, renal or central nervous system involvement, and autoimmune cytopenias. Treatment of severe, ongoing, active disease requires corticosteroids and immunosuppressants. Skin lesions include malar butterfly erythema (flat or raised) that generally spares the nasolabial folds. A variety of other erythematous, firm, maculopapular lesions can occur elsewhere, including exposed areas of the face and neck, upper chest, and elbows. Skin blistering and ulceration are rare, although recurrent ulcers on mucous membranes (particularly the central portion of the hard palate near the junction of the hard and soft palate, the buccal and gum mucosa, and the anterior nasal septum) are common (sometimes called mucosal lupus); findings can sometimes mimic toxic epidermal necrolysis.ATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171

[0070] Pemphigus vulgaris is an autoimmune disease that targets the desmosomes, the intercellular proteins connecting keratinocytes. Desmosome degradation results in acantholysis and the formation of easily ruptured blisters within the epidermis. The disease is characterized by a positive Nikolsky sign, where the epidermis peels away upon rubbing. Bullous pemphigoid is a blistering disease that affects older adults, causing tense subepidermal blisters. The condition is caused by antibodies targeting hemidesmosomes, which connect the epidermis to the dermis at the basement membrane. This condition is not acantholytic and does not show a positive Nikolsky sign.

[0071] Psoriasis is hyperproliferation of epidermal keratinocytes combined with inflammation of the epidermis and dermis. The cause of psoriasis is unclear but involves immune stimulation of epidermal keratinocytes; T cells seem to play a central role. Family history is common, and certain genes and human leukocyte antigens (Cw6, B13, B17) are associated with psoriasis. Genome wide linkage analysis has identified numerous psoriasis susceptibility loci; the PSORS1 locus on chromosome 6p21 plays the greatest role in determining a patient's susceptibility of developing psoriasis. An environmental trigger is thought to evoke an inflammatory response and subsequent hyperproliferation of keratinocytes. Psoriasis manifests most commonly as well-circumscribed, erythematous papules and plaques covered with silvery scales. Multiple factors contribute, including genetics. Common triggers include trauma, infection, and certain medications. Symptoms are usually minimal, but mild to severe itching may occur. Cosmetic implications may be major. Some people also develop psoriatic arthritis. Diagnosis is based on appearance and distribution of lesions. Treatment can include topical treatments (eg, corticosteroids, vitamin D3 analogs, calcineurin inhibitors, tazarotene, roflumilast, tapinarof, emollients, salicylic acid, coal tar, anthralin), phototherapy, and, when severe, systemic medications, such as methotrexate, oral retinoids, cyclosporine, and other immunosuppressants.

[0072] Skin hypersensitivity disorders, also known as hypersensitivity reactions or allergic skin conditions, involve an exaggerated immune response to a substance that is usually harmless. These disorders can manifest in various ways, including rashes, itching, redness, and swelling. Contact dermatitis is inflammation of the skin caused by direct contact with irritants (irritant contact dermatitis) or allergens (allergic contact dermatitis). Symptoms include pruritus and sometimes a burning pain. Skin changes include erythema, scaling, skin swelling, and sometimes blistering and ulceration. The location depends on the site of contact. Diagnosis is by exposure history, examination, and sometimes skin patch testing. Treatment includes topical corticosteroids, antipruritics, and avoidance of irritants and allergens.ATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171

[0073] Atopic Dermatitis (Eczema) is chronic skin condition often associated with a personal or family history of allergies or asthma. It is characterized by dry, itchy, and inflamed skin, typically affecting the face, neck, and folds of the elbows and knees.

[0074] Urticaria (Hives) is characterized by raised, itchy welts that can appear anywhere on the body. These welts are usually red or skin-colored and can vary in size. It is caused by allergens, infections, medications, insect bites, or physical stimuli (e.g., cold, heat, pressure). Acute urticaria lasts for less than six weeks, while chronic urticaria persists longer and may not have a clear trigger. Angioedema is similar to hives but affects deeper layers of the skin, often around the eyes, lips, throat, and sometimes the genitals or hands.

[0075] Photodermatitis is a reaction to sunlight (UV radiation) causing rashes, blisters, or other skin damage. It can be triggered by certain medications, chemicals, or plants that sensitize the skin to sunlight. Symptoms can vary from mild redness to severe blistering and peeling.

[0076] Stevens-Johnson Syndrome (SJS) and T oxic Epidermal Necrolysis (TEN) are severe, potentially life-threatening skin reactions often triggered by medications, infections, or unknown causes; characterized by widespread skin blistering, peeling, and mucous membrane involvement.

[0077] Erythema Multiforme is an acute, self-limited hypersensitivity reaction often triggered by infections, particularly herpes simplex virus (HSV). It presents as target-like lesions, primarily on the hands, feet, and mucous membranes, and can range from mild (minor) to severe (major), the latter involving mucous membranes and potentially progressing to SJS.

[0078] Genetic diseases of the skin include Dystrophic Epidermolysis Bullosa (DEB), which includes three subtypes: recessive DEB, severe generalized (RDEB-sev gen) (formerly called Hallopeau-Siemens type (RDEB-HS); recessive DEB, generalized other (RDEB-O) (formerly called non-Hallopeau-Siemens type (RDEB-non-HS); and dominant DEB (DDEB). In addition to inherited forms of EB, the acquired form of Epidermolysis Bullosa (EBA) involves pathology in type VII collagen, where circulating autoantibodies in patients with EBA recognize epitopes in type VII collagen molecules. In RDEB-sev gen, blisters affecting the whole body may be present in the neonatal period. Oral involvement may lead to mouth blistering, fusion of the tongue to the floor of the mouth, and progressive diminution of the size of the oral cavity. Esophageal erosions can lead to webs and strictures that can cause severe dysphagia. Consequently, severe nutritional deficiency and secondary problems are common. Corneal erosions can lead to scarring and loss of vision. Blistering of the hands and feet followed by scarring fuses the digits into “mitten” hands and feet, a hallmark of this disorder. The lifetime risk of aggressive squamous cell carcinoma is over 90%. In DDEB, blistering is often mild andATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 limited to hands, feet, knees, and elbows, but nonetheless heals with scarring. Dystrophic nails, especially toenails, are common and may be the only manifestation of DDEB.

[0079] Viruses that infect human skin, including HSV-1 and mpox, as provided in the Examples, can cause a variety of conditions, ranging from mild rashes to more severe infections.

[0080] Human Papillomavirus (HPV) is a common virus with over 100 different types. It can cause warts on different parts of the body, including the hands, feet (plantar warts), and genitals (genital warts). HPV is spread through direct skin-to-skin contact, including sexual contact.

[0081] Herpes Simplex Virus (HSV), HSV-1 primarily causes oral herpes, leading to cold sores around the mouth; and HSV-2 primarily causes genital herpes. It is spread through direct contact with an infected person’s skin, saliva, or sexual contact.

[0082] Varicella-Zoster Virus (VZV) causes chickenpox and shingles. It spreads through respiratory droplets or direct contact with the rash.

[0083] Measles Virus causes measles, and a red, blotchy rash that starts on the face and spreads downward, accompanied by high fever, cough, runny nose, and red eyes. Like Rubella virus, it spreads through respiratory droplets.

[0084] Smallpox (Variola) causes a high fever, fatigue, and a characteristic rash that develops into pus-filled sores. It can be spread through respiratory droplets or direct contact with sores.

[0085] Bacterial skin infections can be classified as skin and soft-tissue infections (SSTI) and acute bacterial skin and skin structure infections (ABSSSI). SSTI include carbuncles, ecthyma, erythrasma, folliculitis, furuncles, impetigo, lymphadenitis, and minor cutaneous abscesses. The primary pathogens in SSTI are Streptococcus and Staphylococcus species, including methicillin-resistant Staphylococcus aureus (MRSA). MRSA is a common pathogen in the United States. However, the proportion of cases attributed to MRSA differs substantially elsewhere in the world. Because MRSA can be resistant to multiple antibiotics, recommended antibiotics for bacterial skin and soft-tissue infections depend largely on local prevalence and resistance patterns of MRSA. Scalded skin syndrome arises from the effects of the exfoliative toxin released by Staphylococcal aureus. The condition manifests as generalized skin exfoliation with a positive Nikolsky sign, a severely burned (intensely red) appearance, and fever.

[0086] Various drug reactions manifest in the skin, including erythema multiforme and the syndromes of drug reaction with eosinophilia and systemic symptoms, Stevens-Johnson, andATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 toxic epidermal necrolysis. These conditions are often associated with certain medications, including sulfa-containing drugs, nonsteroidal anti-inflammatory drugs, and antiepileptics.Methods

[0087] A culture system for human dermal and epidermal layers of the skin is provided, which culture reproduces the histomorphology of native human skin. The human skin culture system maintains epidermal proliferation, differentiation, maturation and barrier function, and possesses diverse specialized cell types critical for skin function, including endothelial, fibroblast, stromal and immune cells. The system is highly reproducible and maintains the cellular diversity of human skin for extended periods of time, both in the types of cells that are present, and in the architecture of the tissue. Features of the culture include self-production of molecules and proteins required for maintenance and maturation of the diverse cellular compartments.

[0088] Skin tissue comprising at least dermal and epidermal layers is obtained as a bloc, i.e. comprising the normal diversity of cells and structures present in situ, and may be obtained by any convenient method, e.g. by biopsy, and is typically obtained as aseptically as possible. Upon removal, tissue is immersed in ice-cold buffered solution, e.g. PBS, Ham’s F12, MEM, culture medium, etc. The skin tissue may be from any mammalian species, e.g. human, equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc., usually from a human. Pieces of tissue may be minced to a size less than about 3 mm3, and may be less than about 2 mm3, or less than about 1 mm3.

[0089] The minced tissue is mixed with a gel substrate, e.g. a collagen gel solution, e.g. Cellmatrix type l-A collagen; a matrigel solution, etc. Subsequently, the tissue-containing gel substrate is layered over a layer of gel (a “foundation layer”) in a container with a lower semi- permeable support, e.g. a membrane, supporting the foundation gel layer, and the tissuecontaining gel substrate is allowed to solidify. This container is placed into an outer container containing a suitable medium. In an embodiment, the medium is free of exogenous protein factors during the initiation of the culture, e.g. from about 1 to about 4 weeks. Factors such as Rspo family members, Wnt family members, BMP2, TGF-Beta 1 , EGF, EGFR, may be endogenously produced by cells present in the culture itself. Following the initiation period, additional factors are optionally added, e.g. in order to screen potential therapies.

[0090] The arrangement described above allows nutrients to travel from the bottom, through the membrane and the foundation gel layer to the gel layer containing the tissue. The level of the medium is maintained such that the top part of the gel, i.e. the gel layer containing the explants, is not submerged in liquid but is exposed to air. Thus the tissue is grown in a gel with an air-liquid interface. A description of an example of an air-liquid interface culture system is provided in Ootani et al. in Nat Med. 2009 Jun;15(6):701 -6, the disclosure of which isATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 incorporated herein in its entirety by reference. The air-liquid interface organoid cultures can be moved into other formats such as multi-wells for screening.

[0091] This air-liquid interface (ALI) method allows culturing of the diverse cells present in skin together as a cohesive 3-dimensional unit that recapitulates the function and the microanatomy of the original tissue, and includes, inter alia, endogenous immune cells. In ALI, adequate oxygenation is achieved by culturing microscopic fragments of tissue embedded in a matrix within a trans-well ("inner dish”) in which direct air exposure is obtained from the top; whilst contact with tissue culture media contained in an "outer dish”; is obtained from the bottom via the trans-well permeable membrane.

[0092] The skin cultures can be maintained for an extended period of time, for example up to 15 days, up to 21 days, up to 28 days, up to 6 weeks, up to 8 weeks, or more. Reference may be made to the “initiation” period of culture, from about 1 week, about 2 weeks, up to about 4 weeks.

[0093] In an embodiment, all of the human cells present in the culture after the initiation period are derived from the initiating bloc. Cells present in the culture may comprise, without limitation, macrophages, keratinocytes, T cells, endothelial cells, fibroblasts, melanocytes, mast cells, and neuronal cells derived from the initiating tissue bloc. In an embodiment, each of keratinocytes, endothelial cells, fibroblasts, melanocytes, and immune cells derived from the initiating tissue bloc are present in the culture for an extended period of time, e.g. at least about 2 weeks, at least about 4 weeks, or more. Immune cells may comprise, for example, CD4+T-cells, CD8+T-cells, mast cells, Langerhans cells, macrophages, and gd T-cells. In an embodiment, following the initiation period, a specific cell type of interest may be introduced into the culture for screening or other purposes.

[0094] Keratinocytes in the culture are shown to have normal histology and differentiation, and have a stratum basalis (basal keratinocytes), stratum spinosum (spinous keratinocytes), and stratum corneum (cornified keratinocytes). In addition to these layers, observed structures may also comprise sweat glands, hair shafts and follicles, and blood vessels. In some embodiments, each of the structures listed above are present in the culture system following the initiation period.Screening Assays

[0095] The culture systems of the disclosure find use in screening assays, to determine the effect of a candidate agent or therapeutic regimen on the skin. Skin conditions are disclosed herein, and the culture system may be initiated with skin from an individual with a disease of interest. Alternatively, the culture system may be initiated with skin from a normal individual, and a disease condition is induced, e.g. by infection, introduction of autoimmune cells, introduction of cancer cells, introduction of a skin irritant, and the like. In other embodiments,ATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 healthy skin is tested for sensitivity to an agent of interest. An initial screening, particularly a high-throughput screening, may utilize a panel of tissue conditions.

[0096] The effect of the treatment on the cells is determined by measuring or observing changes in parameters of interest. Parameters include, for example, changes in gene expression, changes in protein levels, changes in histology, changes in the absolute number of specific cell types, changes in the relative number of specific cell types, viability of specific cell types, and the like.

[0097] Candidate agents or therapeutic interventions may be added in solution, or readily soluble form, to the medium of cells in culture. The agents may be added in a flow-through system, as a stream, intermittent or continuous, or alternatively, adding a bolus of the compound, singly or incrementally, to an otherwise static solution. In a flow-through system, two fluids are used, where one is a physiologically neutral solution, and the other is the same solution with the test compound added. The first fluid is passed over the cells, followed by the second. In a single solution method, a bolus of the test compound is added to the volume of medium surrounding the cells. The overall concentrations of the components of the culture medium should not change significantly with the addition of the bolus, or between the two solutions in a flow through method.

[0098] Preferred agent formulations do not include additional components, such as preservatives, that may have a significant effect on the overall formulation. Thus preferred formulations consist essentially of a biologically active compound and a physiologically acceptable carrier, e.g. water, ethanol, DMSO, etc. However, if a compound is liquid without a solvent, the formulation may consist essentially of the compound itself.

[0099] A plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations. As known in the art, determining the effective concentration of an agent typically uses a range of concentrations resulting from 1 :10, or other log scale, dilutions. The concentrations may be further refined with a second series of dilutions, if necessary. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the phenotype.

[0100] Various methods can be utilized for quantifying the presence of the selected parameters. For measuring the amount of a molecule that is present, a convenient method is to label a molecule with a detectable moiety, which may be fluorescent, luminescent, radioactive, enzymatically active, etc., particularly a molecule specific for binding to the parameter with high affinity. Fluorescent moieties are readily available for labeling virtually any biomolecule, structure, or cell type. Immunofluorescent moieties can be directed to bind not only to specific proteins but also specific conformations, cleavage products, or site modifications like phosphorylation. Individual peptides and proteins can be engineered toATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 autofluoresce, e.g. by expressing them as green fluorescent protein chimeras inside cells (for a review see Jones et al. (1999) Trends Biotechnol. 17(12):477-81 ).

[0101] The use of high affinity antibody binding and / or structural linkage during labeling provides dramatically reduced nonspecific backgrounds, leading to clean signals that are easily detected. Such extremely high levels of specificity enable the simultaneous use of several different fluorescent labels, where each preferably emits at a unique color. Fluorescence technologies have matured to the point where an abundance of useful dyes are now commercially available. These are available from many sources, including Sigma Chemical Company (St. Louis Mo.) and Molecular Probes (Handbook of Fluorescent Probes and Research Chemicals, Seventh Edition, Molecular Probes, Eugene Oreg.). Other fluorescent sensors have been designed to report on biological activities or environmental changes, e.g. pH, calcium concentration, electrical potential, proximity to other probes, etc. Methods of interest include calcium flux, nucleotide incorporation, quantitative PAGE (proteomics), etc.

[0102] Multiple fluorescent labels can be used on the same sample and individually detected quantitatively, permitting measurement of multiple cellular responses simultaneously. Many quantitative techniques have been developed to harness the unique properties of fluorescence including: direct fluorescence measurements, fluorescence resonance energy transfer (FRET), fluorescence polarization or anisotropy (FP), time resolved fluorescence (TRF), fluorescence lifetime measurements (FLM), fluorescence correlation spectroscopy (FCS), and fluorescence photobleaching recovery (FPR) (Handbook of Fluorescent Probes and Research Chemicals, Seventh Edition, Molecular Probes, Eugene Oreg.).

[0103] Depending upon the label chosen, parameters may be measured using other than fluorescent labels, using such immunoassay techniques as radioimmunoassay (RIA) or enzyme linked immunosorbance assay (ELISA), homogeneous enzyme immunoassays, and related non-enzymatic techniques. These techniques utilize specific antibodies as reporter molecules, which are particularly useful due to their high degree of specificity for attaching to a single molecular target. U.S. Pat. No. 4,568,649 describes ligand detection systems, which employ scintillation counting. These techniques are particularly useful for protein or modified protein parameters or epitopes, or carbohydrate determinants. Cell readouts for proteins and other cell determinants can be obtained using fluorescent or otherwise tagged reporter molecules. Cell based ELISA or related non-enzymatic or fluorescence-based methods enable measurement of cell surface parameters and secreted parameters. Capture ELISA and related non-enzymatic methods usually employ two specific antibodies or reporter molecules and are useful for measuring parameters in solution. Flow cytometry methods are useful for measuring cell surface and intracellular parameters, as well as shape change and granularity and for analyses of beads used as antibody- or probe-linked reagents. ReadoutsATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 from such assays may be the mean fluorescence associated with individual fluorescent antibody-detected cell surface molecules or cytokines, or the average fluorescence intensity, the median fluorescence intensity, the variance in fluorescence intensity, or some relationship among these.

[0104] Flow cytometry, e.g. FACS or mass cytometry, may be used to quantitate parameters such as the presence of cell surface proteins or conformational or posttranslational modification thereof; intracellular or secreted protein, where permeabilization allows antibody (or probe) access, and the like. The tissue present in the culture may be dissociated for this purpose. Flow cytometry methods are known in the art, and described in the following: Flow Cytometry and Cell Storing (Springer Lab Manual), Radbruch, Ed., Springer Verlag, 2000; Ormerod, Flow Cytometry, Springer Verlag, 1999; Flow Cytometry Protocols (Methods in Molecular Biology, No 91 ), Jaroszeski and Heller, Eds., Humana Press, 1998; Current Protocols in Cytometry, Robinson et al., eds, John Wiley & Sons, New York, N.Y., 2000. The readouts of selected parameters are capable of being read simultaneously, or in sequence during a single analysis, as for example through the use of fluorescent antibodies to cell surface molecules. As an example, these can be tagged with different fluorochromes, fluorescent bead, tags, mass cytometry labels, etc., allowing analysis of up to 4 or more fluorescent colors simultaneously. Both single cell multiparameter and multicell multiparameter multiplex assays, where input cell types are identified and parameters are read by quantitative imaging and fluorescence and confocal microscopy are used in the art.

[0105] The quantitation of nucleic acids, especially messenger RNAs, is also of interest as a parameter. These can be measured by hybridization techniques that depend on the sequence of nucleic acid nucleotides. Techniques include polymerase chain reaction methods as well as gene array techniques. See Current Protocols in Molecular Biology, Ausubel et al., eds, John Wiley & Sons, New York, N.Y., 2000; Freeman et al. (1999) Biotechniques 26(1 ):112- 225; Kawamoto et al. (1999) Genome Res 9(12):1305-12; and Chen et al. (1998) Genomics 51 (3):313-24, for examples.

[0106] Changes in morphology and histology may utilize traditional methods, e.g. staining of formalin-fixed and paraffin-embedded (FFPE) tissues. Alternatively, microscopy techniques with optical sectioning capability enable slide-free imaging of thick resection specimens, greatly simplifying the procedures associated with tissue sectioning. Scanning-based depth- resolved approaches include confocal microscopy, photoacoustic microscopy (PAM), multiphoton microscopy (MPM), stimulated Raman scattering (SRS), second harmonic generation (SHG), and their spectral multiplexing. Wide-field depth-resolved techniques, include microscopy with ultraviolet surface excitation (MUSE), light-sheet microscopy, and structured illumination microscopy (SIM). Machine learning algorithms may find use in the analysis of histology.ATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171

[0107] Candidate agents for testing are biologically active agents that encompass numerous chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, etc. An important aspect of the invention is to evaluate candidate drugs, select therapeutic antibodies and protein-based therapeutics, with preferred biological response functions. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, frequently at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

[0108] Included are pharmacologically active drugs, genetically active molecules, etc. Compounds of interest include chemotherapeutic agents, anti-inflammatory agents, hormones or hormone antagonists, ion channel modifiers, and neuroactive agents. Exemplary of pharmaceutical agents suitable for this invention are those described in, "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth edition, under the sections: Drugs Acting at Synaptic and Neuroeffector Junctional Sites; Drugs Acting on the Central Nervous System; Autacoids: Drug Therapy of Inflammation; Water, Salts and Ions; Drugs Affecting Renal Function and Electrolyte Metabolism; Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function; Drugs Affecting Uterine Motility; Chemotherapy of Parasitic Infections; Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Used for Immunosuppression; Drugs Acting on Blood-Forming organs; Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology, all incorporated herein by reference. Also included are toxins, and biological and chemical warfare agents, for example see Somani, S. M. (Ed.), "Chemical Warfare Agents," Academic Press, New York, 1992).

[0109] Test compounds include all of the classes of molecules described above, and may further comprise samples of unknown content. Of interest are complex mixtures of naturally occurring compounds derived from natural sources such as plants. While many samples will comprise compounds in solution, solid samples that can be dissolved in a suitable solvent may also be assayed. Samples of interest include environmental samples, e.g. ground water, sea water, mining waste, etc.; biological samples, e.g. lysates prepared from crops, tissue samples, etc.; manufacturing samples, e.g. time course during preparation of pharmaceuticals; as well as libraries of compounds prepared for analysis; and the like.ATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171Samples of interest include compounds being assessed for potential therapeutic value, i.e. drug candidates.

[0110] Compounds, including candidate agents, are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

[0111] Agents are screened for activity by adding the agent to at least one and usually a plurality of cultures, usually in conjunction with assay combinations lacking the agent. The change in parameter readout in response to the agent is measured, desirably normalized, and the result may then be evaluated by comparison to reference results. The reference results may include readouts in the presence and absence of the agents, in the absence or presence of disease initiating factors, comparing normal and diseased tissues, etc.

[0112] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. It is also understood that the terminology used herein is for the purposes of describing particular embodiments

[0113] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0114] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the appended claims.ATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171EXPERIMENTAL

[0115] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.Example 1

[0116] Human skin is characterized by dynamically interacting epithelial, immune, stromal, and adnexal components. In particular, the immune-mediated inflammation that underlies diverse infectious, autoimmune, neoplastic, and drug-mediated cutaneous pathologies has raised the need for human in vitro skin culture systems that retain tissue-resident immune cells. Here, we successfully developed human skin organoids where intact fragments of skin can be grown for months in an air-liquid interface (ALI) and preserve a full range of native epidermal and dermal elements without artificial reconstitution. The epidermal components include keratinocytes that mature from proliferative basal cells to stratum corneum, hair follicles, melanocytes, while dermal fibroblasts, endothelium, and eccrine glands are also represented. The skin organoids accurately recapitulated known adverse cutaneous toxicities of therapeutics such as erlotinib, and mutagens and tumor promoters, with Mpox infection in epithelial, and to a lesser extent non epithelial, compartments. Notably, ALI human skin organoids contained diverse tissue-resident immune subsets including T lymphocytes, myeloid cells, mast cells and antigen-presenting Langerhans cells. Further, ALI skin organoids were robustly infected by HSV-1 , to which pre-existing immunity is widespread, which induced TCR signaling markers in organoid resident T cells. Collectively, the successful and robust organoid culture of human adult skin, holistically co-preserving endogenous immune, epidermal and dermal components, provides a versatile platform for studies of cutaneous biology, pathogenesis, and therapy.RESULTS

[0117] Air-liquid interface culture produces stable, stereotypic human skin cultures. Tools to study human skin biology have traditionally relied on keratinocyte cultures, engineeredATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 induced pluripotent stem cell organoids, mouse models, or a combination of these techniques. However, each of these systems suffers from limitations such as a lack of known cell types, lengthy and costly culture techniques, and known differences between human and model organism biology. Further, none of these methods contain an autologous functional tissueresident immune repertoire, known to be critically important to skin biology and homeostasis. As such there remains a need for a robust, reliable, cost-effective culture technique that captures, maintains, and facilitates the growth of the full diversity of cell types present in adult human skin. To address this need, we generated a novel adult human skin culture system using an air-liquid interface culture method.

[0118] Human skin samples obtained under Stanford Institutional Review Board approval from already planned surgical operations or post-mortem donations were trimmed of adipose tissue (step 1 ), the remaining dermis and epidermis mechanically minced with scissors (step 2), and the resulting fragments plated into an ALI culture setup (step 3, Fig. 1 a). Tissue fragments are embedded within a collagen matrix, spread within a trans-well insert, and the insert placed within a culture well. The inserts are exposed to air on one side and media on the other side, generating an air-liquid interface (Fig. 1 b). Brightfield images of the trans-wells at the time of plating (day 0) and 28 days later demonstrated obvious morphologic changes of tissue fragments (Fig. 1 c). These changes are suggestive of tissue growth and remodeling, consistent with an active culture. Hematoxylin and eosin (H&E) stained sections of formalin- fixed paraffin-embedded (FFPE) collagen inserts at day 14, 28, and 56 expectedly showed a mixture of fragments with dermis only (FIG. 8) and combined dermis and epidermis (insets, Fig. 1d).

[0119] Epidermis containing fragments exhibited two dominant histologic morphologies - full circumferential growth, with epidermis encasing a dermal fragment (day 28 & 56, blue and black inset, Fig. 1 d) or a dermal fragment with an epidermal cap (blue inset, day 14, Fig. 1 d). All epidermis containing fragments showed consistent and stereotypic histologic evidence of keratinocyte maturation and differentiation, composed of a stratum basalis, stratum spinosum, and stratum corneum, compatible with native adult human skin. Scattered fragments also demonstrated a granular layer, consistent with physical barrier maintenance. These cultures are readily produced from any source of adult human skin and, by H&E, faithfully reproduce the histologic features of native human skin out to a time point of at least 56 days of continuous culture. Human skin ALI cultures show molecular hallmarks of epidermal maturation and contain diverse cell types.

[0120] Given the prototypic appearance of the human skin ALI cultures on H&E-stained sections, we next sought to examine whether the cultured epidermis maintained selective expression of keratinocyte specific transcription factors and cytokeratins throughout the histologic maturation profile. Immunohistochemical (IHC) stains for p63 and the associatedATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 isoform p40, two proteins indicative of squamous differentiation, demonstrated a strong signal with the stratum basalis, congruent with the known staining pattern in native human skin (Fig. 2a & 2b).

[0121] Further, cytokeratin expression is well-known to change throughout the differentiation process of the epidermis. We assessed cytokeratin expression within the epidermis by immunofluorescence (IF) on FFPE sections. The epidermis of ALI cultured human skin showed robust expression of cytokeratin-5 (KRT5) within basal and suprabasal keratinocytes, highlighting rete ridges, while cytokeratin-10 (KRT 10) was predominantly expressed within the stratum corneum (Fig. 2c). Cytokeratin-1 (KRT1 ) expression mirrored that of KRT10, showing restricted expression to the stratum corneum and the superficial most layer of the stratum spinosum (Fig. 2d). Cytokeratin-15 (KRT15) was restricted to expression within basal cells, a subset of the cells expressing KRT5 (Fig. 1 e).

[0122] Finally, loricrin, a protein critical for epidermal barrier maintenance, showed expression at the junction between KRT5, which highlights the stratum basalis and spinosum, and KRT10, which shows restricted expression within the stratum corneum (Fig. 2f). We next sought to determine whether the keratinocytes within the culture were actively proliferating. To assess whether the keratinocytes of the epidermis were proliferating we performed an IHC stain for Ki-67, which showed staining within the basal cells of the epidermis (Fig. 2g). To further validate proliferation of the keratinocytes, we performed IF for proliferating cell nuclear antigen (PCNA). PCNA immunostaining was similarly restricted to the basal most KRT5- positive keratinocytes (Fig. 2h). Collectively, ALI-cultured human skin faithfully reproduces the histologic, cytokeratin expression profile, and specific barrier protein expression known to be present in native human skin with a proliferating basal layer.

[0123] While keratinocytes of the epidermis are a major component of the human integumentary system, there are additional specialized structures and cell types that contribute to its function. H&E stained sections of human skin ALI cultures demonstrate the presence of sweat glands (Fig. 2i) as well as hair shafts and follicles (Fig. 2j & 2k) within the dermis. Blood vessels are identified histologically (FIG. 9) and confirmed by IHC staining for the vascular endothelial marker CD34 (Fig. 2I). Further, melanocytes which provide pigmentation to the skin, are readily identified by IHC staining for the transcription factor SOX10 (Fig. 2m). Collectively, human skin ALI cultures show maintenance of epidermal proliferation, differentiation, maturation and barrier function and possess diverse specialized cell types critical for skin function.

[0124] Immune and overall cellular diversity is preserved within human skin ALI cultures. While there have been an expansive number of methods to generate in vitro human skin models, none to date capture the immunologic diversity present in native skin. Methods to reconstitute human skin equivalents with immune cells often only include a single immune cellATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 type, are limited in duration, are tailored to answer specific questions, or require destruction of the native architecture, relying on digestive procedures. Therefore, a robust and readily accessible adult human skin culture method containing the full complement of immune cells would represent a remarkable advancement for the field. As the expected epidermal, dermal, and other specialized cell types were present within the adult human skin ALI cultures, we hypothesized that immune cells would similarly be present within the same cultures without the need for media optimization, additives, or other extensive tissue engineering practices.

[0125] To begin an assessment of the immune cell content of the cultures, we performed IHC staining on FFPE sections of human skin ALI cultures after 14 or 28 days of culture. Immunohistochemical staining demonstrated a robust population of CD45RB-positive cells distributed throughout the dermis consistent with the presence of immune cells (Fig. 3a). Small lymphocytes were highlighted by IHC staining of CD3, which showed T-cells predominantly as clusters centered around the superficial dermal vasculature (Fig. 3b). A subset of the CD3- positive T-cells also demonstrate CD8 positivity, consistent with CD8 T-cells (Fig. 3c). Macrophages, highlighted by IHC staining of CD68, were distributed throughout the dermis with no predilection for a particular niche (Fig. 3d). Langerhans cells, the specialized epidermal resident antigen presenting cell, are present throughout the epidermis (Fig. 3e). Finally, mast cells, the major tissue-resident myeloid cell of human skin, are scattered throughout the dermis, highlighted by IHC staining for CD117 (Fig. 3f).

[0126] To further confirm the presence of immune cells, we performed flow cytometry analysis of the human skin ALI cultures. Cultures harvested after 14, 28, 55, 80, or 100 days of continuous culture showed three major populations. The first population, defined as CD45- CD49L, increased in proportion over time in culture, starting at approximately 45% of live cells at day 14 and increasing to just over 80% of live cells at day 100 (Fig. 3g, “Other”). While it is difficult to assign cell type identity to this negative staining population, we hypothesized they were likely dermal fibroblasts that proliferated within and between the tissue fragments. The second population, defined as CD45- CD49f+, delineated a robust population of basal or progenitor-type keratinocytes. These basal keratinocytes comprise nearly 40% of all live cells at 14 days of culture, decreasing to around 5% at 100 days of culture (Fig. 3g, “CD49f”). This proportional decrease in keratinocytes is not likely due to cell loss, but rather by dilutional expansion of the “other” population. The third and final dominant population of cells was defined as CD45+, immune cells. Immune cells comprised approximately 10% of all live cells at day 14 of culture, and in a similar fashion to the keratinocytes, their proportion decreased throughout extended culture times (Fig. 3g, “Immune”). This immune cell population was further divided into CD4+ T-cells, CD8+ T-cells, mast cells, Langerhans cells, macrophages, and gd T-cells all being detected with no selective loss of any of the unique immune cells through 100 days of continuous culture (Fig. 3g and FIG. 10a). While the presence of immuneATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 cells within the human skin ALI cultures marks a significant extension of the work done to date of generating faithful in vitro models of human skin, we wanted to immediately test whether the immune cells within the culture were responsive to stimuli. To do this, we treated human skin ALI cultures with an antibody cocktail of anti-CD3, anti-CD28, and anti-CD2, well-known to activate T-cells in a TCR dependent manner causing upregulation of T cell activation induced markers - CD40L, 0X40, CD25, and 41 -BB36. As assessed by flow cytometry, every specimen tested showed robust induction of T-cell activation-induced markers upon treatment with the antibody cocktail for 7 or 14 days (FIG. 10b).

[0127] Given the presence of epithelial, immune, and specialized cell types detected by IHC, IF, and flow cytometry, we next sought to determine the cellular composition of the human skin ALI cultures at single cell resolution by single cell RNA sequencing (scSEQ). We performed scSEQ on day 14 and day 28 human skin ALI cultures in addition to the original uncultured skin specimen that initiated the cultures (Day 0) for specimens from three separate donors (Fig. 3h). Following single cell dissociation, capture, library preparation, and sequencing, we integrated the 7 samples successfully without batch effects (FIG. 10c). Using unsupervised clustering by Seurat, we characterized eight populations: macrophages, keratinocytes, T cells, endothelial cells, fibroblasts, melanocytes, mast cells, and neuronal cells (FIG. 10d). We defined each cluster by using categorical markers for each cell type which displayed distinct expression profiles (FIG. 10e).

[0128] We sought to determine differences between uncultured native human skin (day 0) and ALI cultured human skin after 14 or 28 days of continuous culture. Remarkably, we found that the populations present at day 0 persisted at day 14 and were unchanged after 28 days of continuous culture (Fig 3h). We next examined the proportions of each cell type present in the uncultured skin origin specimen versus the human skin ALI cultures. As hypothesized from the flow cytometry data, we found a relative expansion in the number of fibroblasts and a corresponding proportional decrease in other cell types (FIG. 10f). Notably, every detectable cell type present within native uncultured skin specimens is present after at least 28 days of continuous culture as assessed by scSEQ.

[0129] Human skin ALI cultures are responsive to diverse stimuli. Given the diversity of cell types present within the human skin ALI cultures, and that they are initiated and maintained in a simplistic media without growth factor additives, we hypothesized that the cells within the culture produced many factors needed to support these different cell types. Indeed, interrogation of the single cell sequencing data for factors that support the growth, maintenance, differentiation, and maturation of keratinocytes revealed robust expression of Rspo family members, Wnt family members, BMP2, TGF-Beta 1 , EGF, and EGFR (FIG. 1 1 a, b, & c). Further, given the persistence of immune cells, we examined the scSEQ data to determine whether the cultures self-produced factors known to support immune cell viabilityATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 and proliferation. Indeed, the cultures produced known factors critical for T-cell maintenance - IL2, predominantly from T-cells themselves; IL7, predominantly from fibroblasts; and IL15, predominantly from fibroblasts with contributions from several other cell types (FIG. 1 1 d). Further, robust expression of CSF1 (macrophage colony stimulating factor) and CSF2 (granulocyte-macrophages colony stimulating factor) known to support macrophages and granulocytes as well as IL6 and KITLG, factors that support mast cells, were detected (FIG. 1 1 e). Remarkably, all produced factors showed good agreement between primary tissue (blue) and ALI cultured tissue (red) in both the amount and the cellular compartment from which they were expressed (FIG. 1 1 a - 4e).

[0130] This scSEQ data provides evidence that the human skin ALI culture system selfproduces a myriad of signaling molecules critical for the maintenance of the diverse cell types found within the culture. We hypothesized that pharmacologic perturbation of these factors and pathways would alter the histomorphology or cell numbers within the human skin ALI cultures. To begin, we utilized IWP- 2, an inhibitor of the acyltransferase Porcupine, known to be critical in the maturation and secretion of biologically active Wnt protein. Treatment of human skin ALI cultures with 5pM IWP-2 resulted in a significantly reduced epidermal thickness (Fig. 4a &). Quantification of the Ki-67 positive nuclei in a defined length of epidermis revealed a significantly reduced Ki-67 positive number in the IWP-2 treated organoids (Fig. 4d).

[0131] In addition to Wnt, EGF and EGFR signaling are known to be important for the balance of proliferation, differentiation, and lineage selection in the epidermis. This is best evidenced by the high rate of skin toxicity reactions in patients receiving EGFR inhibitor therapy. While the effects of loss of EGFR signaling lead to skin toxicity reactions through multiple mechanisms, we hypothesized that similar to their effect on the epidermis in patients, inhibition of EGFR signaling in the human skin ALI cultures would alter the histomorphology of the epidermis. Indeed, treatment of human skin ALI cultures with 50 nM erlotinib resulted in a distinct morphology on H&E sections with a significant increase in the combined thickness of the stratum basalis and spinosum in cultures treated with erlotinib (Fig. 4e & 4f). This was accompanied by a significant increase in the number of p63-positive cells in a defined length of epidermis and no significant change in the number Ki-67 cells in a defined length of epidermis (Fig. 4g & 4h).

[0132] Finally, given that the human skin ALI cultures showed significant responses to diverse stimuli within multiple cellular compartments we wanted to further examine whether more specialized cell types were similarly responsive. Melanocytes represent a significant specialized cell type within the epidermis, providing pigment to keratinocytes and rarely undergoing cell division. In vitro, the proliferation and melanogenesis of melanocytes can be augmented by addition of 12-Otetradecanoylphorbol- 13-acetate (TPA). To examine whetherATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 melanocytes present within human skin ALI cultures remained responsive to stimuli, we treated the skin organoids with TPA and examined the effect on melanocyte numbers, as assessed by IHC staining for SOX-10 and expression of the melanocytic marker Melan-A. Treatment with TPA significantly increased the number of melanocytes within a defined segment of the epidermis (Fig. 4i & 4j) and significantly increased the amount of Melan-A expression within a defined segment of epidermis (Fig. 4k & 4I).

[0133] Human skin ALI organoids reveal dynamics of Mpox infection. Human skin is the site for an expansive number of microbial infections. The human skin ALI cultures represent a promising model system to interrogate human skin and pathogen interactions in the presence of a full tissue-resident immune repertoire. Further, in the context of the recent COVID-19 pandemic, tools that can aid in the evaluation of active and potential pandemic pathogens are critical to developing effective strategies for biosafety and biocontainment. Given the recent outbreak of Mpox in 2022, we hypothesized that the human skin ALI cultures would be susceptible to infection by this smoldering microbial threat. Incubation of human skin ALI cultures with Mpox resulted in active infection, demonstrated by Mpox staining only in Mpox treated cultures. Mpox infection of human skin ALI cultures showed delayed IF positivity, with no detection of Mpox virus by IF at three days post-infection and beginning at five days postinfection (FIG. 12a & 5c). Mpox co-localized at five- and seven-days post-infection with the basal-most KRT5-positive keratinocytes of the epidermis with additional scattered positive cells within the dermis, sparing the KRT10-positive epidermis (Fig. 5a and FIG. 12c). Culture of Mpox infected skin ALI cultures to 14 days post-infection again showed robust infection with Mpox detected by IF throughout the full thickness of the KRT5-positive layer, again sparing the KRT10-positive layer (Fig 5b). Hematoxylin and eosin stained FFPE sections showed viral cytopathologic changes within the epidermis beginning at five days postinfection (FIG. 12b & 5d). These changes coalesced in areas scattered throughout the human skin ALI cultures to blister-type histologic patterns with loss of cell-cell junctions between large groups of keratinocytes, consistent with the known phenotype seen in patients (Fig. 5c & 5d and FIG. 12b & 5d). These histomorphologic changes were absent from mock treated organoids (Fig. 5c & 5d and FIG. 12b & 5d). Given these encouraging results, we aimed to utilize the human skin ALI culture system to generate the first ever single cell sequencing data set of active Mpox infection.

[0134] Human skin ALI organoids contain functional memory T cells responsive to HSV infection. While flow cytometery and scRNA-seq demonstrated the presence of endogenous immune cells within the human skin ALI organoids (Fig. 5, FIG. 12), we wanted to further confirm the presence of a tissue resident T lymphocyte population. Flow cytometry highlighted a subset of CD45+ immune cells co-expressing CD69 and CD103 (Fig. 6a), consistent with a tissue-resident phenotype. The presence of antigen presenting cells was additionallyATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 confirmed by flow cytometric analysis, demonstrating HLA class II expression in both CD45+ CD1 1c+ dendritic cells (Fig. 6b) as well as on a subset of CD45- CD49f+ keratinocytes (Fig. 6c). While the presence of such tissue-resident immune cells within the skin ALI organoid cultures marks a significant extension upon prior in vitro skin models, we desired to confirm the responsiveness of the endogenous T cells to external stimuli. We first treated the human ALI skin organoids with a cocktail of antibodies targeting CD3, CD28, and CD2, which are well established to activate T cells by TCR stimulation and co-stimulatory signals, with secondary induction of activation-induced markers (AIM) such as CD40L, 0X40, CD25, and 41 -BB. As assessed by flow cytometry, ALI skin organoids uniformly showed robust induction of T-cell activation-induced markers upon treatment with the antibody cocktail for 6 days (Fig. 6d). This was accompanied by the production of numerous cytokines and chemokines, as measured by Luminex (Fig. 6e).

[0135] Human skin is commonly infected by diverse pathogens, which in turn evoke local immune responses that oppose initial skin infection, and respond to repeat pathogen challenge. While human skin ALI organoids were readily infected with Mpox (Fig, 4, FIG. 1 1 ), the relatively recent emergence of this pathogen and lack of recent public health immunization initiatives renders pre-existing mpox immunity comparatively rare in many cohorts. Accordingly, it was unlikely that memory T cells against mpox would be present in random human donors or their corresponding ALI skin organoids. In contrast, human populations manifest pervasive seropositivity to herpes simplex 1 virus (HSV-1) infection, consistent with prior overt, subclinical or asymptomatic exposure. Roughly 50% of the total U.S population is seropositive for HSV-1 antibodies, which increases with age and reaches >70% in some cohorts. Consistent with this broad contact, HSV-specific tissue resident memory T cells have been described following infection and clearance of HSV. We thus sought to demonstrate primary HSV-1 infection and secondary adaptive tissue-resident T-cell responses within the context of human skin ALI organoids. Accordingly, HSV-1 readily infected skin organoids with characteristic histologic changes, including nuclear margination, nuclear molding, multinucleation, and ballooning degeneration of keratinocytes (Fig. 7a), in agreement with the histopathologic features of HSV infection from clinical biopsies. We directly confirmed skin organoid HSV-1 infection by immunofluorescence with a polyclonal antibody to HSV-1 (pHSV- 1 ) (Fig. 7b) and a monoclonal antibody to ICP0 (FIG. 13a), and by immunohistochemistry with a monoclonal antibody against ICP8 (Fig. 7b). HSV-1 infection induced marginal phenotypic consequences at 24 hours predominantly infecting edge keratinocytes not protected by the stratum corneum, which progressed to infect nearly all keratinocytes by day 6 eliciting significant histologic changes (FIG. 13b)

[0136] Having established robust viral infection, we then explored immunologic responses to HSV-1 infection in skin organoids. Luminex analysis of organoid supernatants revealed thatATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171HSV-1 infection resulted in changes in homeostatic cytokine production, increasing inflammatory cytokine production such as GMCSF, IL-1 a, IL-1 RA, IL-18, CCL3, TNFa, IL-23, and IL28a, and decreasing cytokine production of CCL1 1 , IL-7, IL-9, CCL5, and VEGF (Fig. 7c), likely due to the destruction of cells producing them. We further wanted to assess whether the tissue-resident T cells preserved within ALI skin organoids could mount activation responses to HSV-1 . After establishing human skin organoids in ALI culture for 3 days, HSV- 1 was added and flow cytometric analysis performed after day 6 of infection to examine upregulation of AIM markers on CD4+ and CD8+ T-cells. Infection of human skin ALI organoids with HSV-1 resulted in a significant increase in the number of CD4+ and CD8+ T- cells that exhibited double-positivity for AIM activation marker pairs CD69+OX40+, CD69+CD40L+ or OX40+CD40L+. (Fig. 7d-f).

[0137] We present here a first-in-class adult human skin culture system that faithfully reproduces the histomorphology of native human skin (Fig. 1 d), contains the specialized cell types of the dermis and epidermis (Fig. 2i-m), and, importantly, contains the full complement of skin resident immune cells (Fig. 3a-g). These findings signify a significant advancement for in vitro human skin models containing immune cells. While some models can support a single immune cell type, none of the prior models can support all of the cell types present in human skin while simultaneously preserving immune cells and native architecture. The culture system is markedly robust with all cellular compartments present in native skin represented after at least 28 days of continuous cultures as assessed by single cell sequencing (Fig. 3h). It is independent of many of the variables associated with culture initiation with successful cultures derived from multiple source types (fresh from surgery or organ donation post-mortem), at two geographic locations (Stanford University and Washington State University), and by numerous lab personnel. The robust, reliable nature of the system likely derives from the self-production of molecules and proteins required for maintenance and maturation of the diverse cellular compartments present within the cultures (FIG. 1 1 ). It is suspected that this self-production allows for self-regulated titration of critical bioactive molecules to homeostatic levels required to maintain the cellular, histologic, and immunophenotypic features of near-native skin, as disruption of these autonomous pathways produces aberrant culture results (Fig. 4).

[0138] Beyond preserving the components of native skin, the system is responsive to diverse stimuli. The T-cells within the culture show robust activation in response to a stimulating antibody cocktail (FIG. 10b), highlighting the capacity for antibodies to diffuse into the perivascular spaces within the dermis and engage T-cells. Given the prominent emergence of antibody therapeutics, this paves the way for utilizing the system to interrogate the activities of prospective immunomodulatory antibody therapeutics. Similarly, the system is responsive to multiple chemicals across multiple cell types (Fig. 4a-h). As small molecules represent aATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 mainstay of therapeutic intervention both in and outside of the skin, these initial studies pave the way not only for using the system for discovery of therapies, but also as a robust platform for screening dermal toxicity, one of the major and most common sites for drug toxicity effects.

[0139] In the context of the recent COVID-19 pandemic, tools that can rapidly generate molecular knowledge on emergent pathogens are critically important. To examine whether the skin ALI organoids can be utilized as a tool for investigating host-pathogen interactions and to examine whether the tissue resident immune cells within the skin organoids can mount a specific response, we infected the skin organoids with the common human pathogen HSV-1 (Fig. 5). Infection with HSV-1 yielded characteristic histologic changes within infected cells of the epidermis. The tissue resident T-cells showed robust TCR activation evidenced by an upregulation of T-cell activation induced markers by flow cytometry. Further, inflammatory cytokines likely produced by multiple responsive cell populations were detected in conditioned culture media (Fig. 5j).

[0140] Finally, utilizing the 2022 clade lib Mpox from the recent worldwide outbreak, we employed the adult human skin ALI organoids to generate active Mpox infection (Fig. 6). Infection of the skin ALI organoids with Mpox yielded an infection with the hallmarks of Mpox pathology - keratinocyte necrosis, loss of basal cell integrity, and blistering-type lesions. Further, we utilize the skin organoids to generate the first ever single cell sequencing dataset of active Mpox infection in human skin. This scSEQ dataset demonstrates that Mpox infects not only keratinocytes but is promiscuous, infecting all major cell types within the organoids. Further, while pox viruses are known to inhibit class I MHC protein expression on infected cells REF, here we report for the first time the ability of Mpox to down-regulate transcription of MHC class I molecules. While there is significant precedent for viruses to block MHC presentation by a variety of post-translational mechanisms, only a limited number of viruses, notably HIV, have been demonstrated to alter transcription from the MHC locus. Thus Mpox is in a rare group of pathogenic viruses, altering transcription from the MHC locus to evade immune recognition.

[0141] With approximately one in four Americans seeking dermatologic care, conditions that afflict human skin represent a major healthcare burden. While there have been significant efforts and progress to generate in vitro systems to address the pathologies of human skin, none to date have provided a system that adequately captures the complexity and dynamics of the human integumentary system. Here, we provide a human skin culture system that fills that need and provides a platform to address pathologic conditions of the skin, ranging from autoimmunity to infectious etiologies. The human skin ALI culture system is useful for a wide range of studies in infectious disease, auto- and allo-immunity, and human skin tissue-resident immunobiology, among many more.ATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171METHODS

[0142] Human tissue procurement. Human skin samples obtained under Stanford Institutional Review Board approval from already planned surgical operations or from postmortem donations.

[0143] Skin AU culture. Discarded skin from surgery was obtained and maintained at 4°C. Skin was trimmed of adipose tissue. Skin was then minced into 2x2 mm pieces and washed with DMEM + Normacin. 1 mL of collagen matrix (Cultrex Rat Collagen I (R&D, #3443-100- 01 ) was added to a 30mm inner transwell, 0.4 mm pore size (Sigma, PICM03050). Once solidified, collagen matrix and 2mm minced pieces of skin were mixed and 1 mL of this combination was plated on top of the previously solidified collagen matrix. Once the second layer solidified, 1 .5 mL of media (described below in Skin ALI culture media composition) was added to the outside of each insert in a standard 6-well tissue culture plate (Corning, #353046).

[0144] Brightfield imaging. Skin ALI culture was imaged in transwell using a Keyence BZX710 microscope. The images were stitched using BZ-Wide viewer software.

[0145] Skin AU culture media composition. Media is composed of advanced DMEM / F12 (ThermoFisher Scientific 12634010), neurobasal medium (Gibco # 21103049), 1x GlutaMax (Gibco # 35050061 ) 0.5 x B-27 minus vitamin A (Gibco # 12587010), 0.5 x N2 (Gibco # 17502048) supplement, 0.1 mM 2-mercaptoethanol (Gibco #21985023) and 100 ug / mL normocin (Invivogen # ant-nr-05).

[0146] Single cell skin dissociation. To dissociate skin organoids in single cell suspension, two steps were performed. The first removed the collagen using collagenase type IV (Worthington Biochemical #LS004212). Collagenase was resuspended with 5 mL PBS, and then 500 mL was added to a 15 mL tube with the insert. The mixture was incubated at 37°C for 30 minutes, with intermittent mixing and then washed with wash media composed of 500 mL DMEM, 100 mL FBS, 1 mL normacin (Invivogen # ant-nr-05), and 5mL Antibiotic- Antimycotic (ThermoFischer Scientific #15240062). The second step followed the protocol of Whole Skin Dissociation Kit (Miltenyi Biotec #130-101 -540) to digest into single cell.

[0147] Flow cytometry. Following dissociation of skin organoids to single cells, cells were washed with PBS and resuspended in PBS containing .55mg / mL BD Horizon Fixable Viability Stain 780 (BD Biosciences #565388) for 10 mins on ice. Cells were quenched with FACS buffer (PBS containing 2% FBS, 500mm EDTA) and resuspended in staining solution comprised of 1 / 3 Brilliant Staining Buffer (BD Biosciences #563794), 2 / 3 FACS buffer, 1 :100 FC block (BD Biosciences #564220), and 1 :100 of each antibody (Supplemental Table 1 ) for 1 .5 hours on ice. Cells were then washed 3x and assessed using a BD FACSAria Fusion flow cytometer. Compensation was performed using OneComp eBeads (ThermoFisher #01 -1 11 1 - 41 ) incubated with individual corresponding antibodies. Cells stained with Live / Dead only wereATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171 used during compensation for the viability stains. Data was analyzed using FlowJo v10.10 software.

[0148] AIM assay. For the activation induced marker (AIM) flow cytometry panel, the following antibodies were used in the antibody staining solution: BUV395-CD45 (BD Biosciences #563792), BV510-CD3 (BioLegend #344827), PE / Cy5-CD8 (BioLegend #301010), Alexa Fluor 700-CD4 (BioLegend #317426) or BB515-CD4 (BD Biosciences #564419), Alexa Fluor 647-CD25 (BioLegend #302618), BV605-CD69 (BioLegend #310937), and PE-CD103 (BioLegend #350206). 1 :1000 Fixable Near IR (780) Viability stain (ThermoFisher Scientific #L34992) was included with the antibody staining solution instead of a pretreatment with Fixable Viability Stain 780. Following staining and washing with FACS buffer, cells were fixed with BD Cytofix (BD Biosciences #554655) for 20 minutes at room temperature. Cells were washed with FACS buffer followed by 1 x permeabilization buffer (ThermoFisher Scientific #00-8333-56) and then stained with PE / Cy7-4-1 BB (BioLegend #309818), BV711 -0X40 (BioLegend #350030), and BV421 -CD154 (BioLegend #310824) antibodies in 1 x permeabilization buffer for 1 .5 hours on ice. Cells were washed with permeabilization buffer twice and twice with FACS buffer followed by assessment and analysis on FlowJo and Prism.

[0149] Pre-processing of scRNA-seq data. Raw sequencing fastqs were processed using CellRanger version v.6.1.1 and human transcriptome GRCh38-2020 as the reference. Mpox fastq files used a custom reference composed of the human transcriptome GRCh38-2020 and the Monkeypox virus isolate MPXV_USA_2022_MA001 , complete genome.

[0150] Single cell sequencing. The scRNA-Seq data of primary and ALI culture skin was preprocessed, clustered, and visualized using Seurat. We removed cells with greater than 200 but less than 5000 genes detected and less than 20% mitochondrial genes detected. We integrated the 7 samples using the default Seurat lntegrateData() function. We annotated the clusters using canonical markers: Mast cells (KIT, TPSAB1 , CPA3, FCER1 G), Keratinocytes (SBSN, KRT5, KRT15, KRTDAP), Endothelial cells (CD93, VWF, PECAM1 , CDH5), Fibroblasts (LUM, PDGFRA, DCN, COL1 A2), Melanocytes (MLANA, PMEL, TYRP1 , DCT), T cells (CD3D, CD3E, CD3G, PTPRC), Macrophages (CD74, HLA-DRA, LYZ, CD68), and Neuronal cells (NRNX1 , S100B, SOX10, PLP1 ).

[0151] Tissue processing. For immunofluorescence, samples were fixed in 10% formalin (Fischer Chemical #SF100-4) for 24 hours, then switched to 70% ethanol (Fischer Bioreagents #BP8203). After routine processing, blocks were cut at 5 micrometer thick sections onto charged glass slides and either stained with hematoxylin and eosin or reserved as unstained sections for immunofluorescence.

[0152] Immunofluorescence. Slides were baked overnight at 60oC. Slides were then placed in xylene (Honeywell #534056) 2X for 5 minutes, 100% ethanol 1 X for 5 minutes, 95% ethanolATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-1711 X for 5 minutes, 70% ethanol 1X for 5 minutes, and H2O 2x for 2 minutes. Slides were then permeabilized with 0.2% Triton X-100 in PBS for 1 hour. Slides were steamed in an antigen unmasking solution (Sigma Aldrich #C9999) for 20 minutes. Slides were then blocked using 10% Donkey serum (Jackson ImmunoResearch #017-000-121 ) diluted in PBS for 1 hour at room temperature. Slides were incubated in primary antibodies diluted in diluent (Epredia #TA- 125-ADQ) and incubated overnight in a humidified chamber. Slides were next washed with PBST 3X for 10 minutes. Secondary antibodies and nuclear stain were added at a dilution of 1 :100 for 2 hours. Slides were washed with PBST 5x 10 minutes each, then in H2O 1 X. Mounting solution (Vector Laboratories #1-1-5501 ) and coverslip (Corning #2975-245) were then placed over the slide.

[0153] KI67 quantification. 500 mM lines were drawn across the epidermis. The entire epidermis was then selected using the closed rectangular selector. The number of Ki67 positive cells was then parsed by the automated processing within QuPath using Analyze -> Cell detection -> positive cell detection. The line and then full area were used because if the area alone had been used, it would have resulted in biased since the erlotinib-treated organoids had a thicker basalis and spinosum. This way of measurement was followed for all other Ki67 quantified organoids. 4 measurements per biological sample were taken.

[0154] p63 quantification. p63 quantification was done in the same method as the Ki67 quantification. 4 measurements per biological sample were taken.

[0155] SOX10 quantification. SOX10 quantification was done in the same method as the Ki67 quantification. 4 measurements per biological sample were taken.

[0156] IWP. 5 mm IWP-2 (STEMCELL Technologies #72124) was added to culture media for 1 month before fixation and processing for downstream analysis.

[0157] TPA. 100 ng / mL TPA (Fisher Scientific #AAJ63916MCR) was added to culture media for 1 month before fixation and processing for downstream analysis.

[0158] Erlotinib. 50nM Erlotinib (MedChemExpress #HY-50896) was added to the culture media for 2 weeks before fixation and processing for downstream analysis.

[0159] Statistics and reproducibility. All experiments were repeated at minimum three times.REFERENCES

[0160] Proksch, E., Brandner, J. M. & Jensen, J.-M. The skin: an indispensable barrier. Experimental Dermatology 17 , 1063-1072 (2008).

[0161] Naik, S. One Size Does Not Fit All: Diversifying Immune Function in the Skin. The Journal of Immunology 208, 227-234 (2022).

[0162] Kabashima, K., Honda, T., Ginhoux, F. & Egawa, G. The immunological anatomy of the skin. Nat Rev Immunol 19, 19-30 (2019).ATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171

[0163] Byrd, A. L, Belkaid, Y. & Segre, J. A. The human skin microbiome. Nat Rev Microbiol 16, 143-155 (2018).

[0164] Kobayashi, T., Naik, S. & Nagao, K. Choreographing Immunity in the Skin Epithelial Barrier. Immunity 50, 552-565 (2019).

[0165] Arvin, A. M. Varicella-zoster virus. Clinical Microbiology Reviews 9, 361-381 (1996).

[0166] Moffarah, A. S., Al Mohajer, M., Hurwitz, B. L. & Armstrong, D. G. Skin and Soft TissueInfections. Microbiology Spectrum 4, 10.1128 / microbiolspec.dmih2-0014-2015 (2016).

[0167] Kabuga, A. I. & El Zowalaty, M. E. A review of the monkeypox virus and a recent outbreak of skin rash disease in Nigeria. Journal of Medical Virology 9 , 533-540 (2019).

[0168] Seiringer, P., Garzorz-Stark, N. & Eyerich, K. T-Cell-Mediated Autoimmunity: Mechanisms and Future Directions. Journal of Investigative Dermatology 142, 804-810 (2022).

[0169] Schunkert, E. M., Shah, P. N. & Divito, S. J. Skin Resident Memory T Cells May Play Critical Role in Delayed-Type Drug Hypersensitivity Reactions. Front. Immunol. 12, (2021).

[0170] Su, D., Shen, M., Li, X. & Sun, L. Roles of yd T cells in the pathogenesis of autoimmune diseases. Journal of Immunology Research 2013, e985753 (2013).

[0171] Gudjonsson, J. E., Kabashima, K. & Eyerich, K. Mechanisms of skin autoimmunity: Cellular and soluble immune components of the skin. Journal of Allergy and Clinical Immunology 146, 8-16 (2020).

[0172] Zomer, H. D. & Trentin, A. G. Skin wound healing in humans and mice: Challenges in translational research. Journal of Dermatological Science 90, 3-12 (2018).

[0173] Mestas, J. & Hughes, C. C. W. Of Mice and Not Men: Differences between Mouse and Human Immunology. The Journal of Immunology 172, 2731-2738 (2004).

[0174] Agarwal, Y. et al. Development of humanized mouse and rat models with fullthickness human skin and autologous immune cells. Sci Rep 10, 14598 (2020).

[0175] Hong, Z.-X. et al. Bioengineered skin organoids: from development to applications. Mil Med Res 10, 40 (2023).

[0176] Mewes, K. R. et al. Catch-up validation study of an in vitro skin irritation test method based on an open source reconstructed epidermis (phase I). Toxicology in Vitro 36, 238-253 (2016).

[0177] De Vuyst, E. et al. Reconstruction of Normal and Pathological Human Epidermis on Polycarbonate Filter, in Epidermal Cells: Methods and Protocols (ed. Turksen, K.) 191-201 (Springer, New York, NY, 2014). doi:10.1007 / 7651_2013_40.

[0178] Rosdy, M. & Clauss, L.-C. Terminal Epidermal Differentiation of Human Keratinocytes Grown in Chemically Defined Medium on Inert Filter Substrates at the Air-Liquid Interface. Journal of Investigative Dermatology 95, 409-414 (1990).ATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171

[0179] Lee, J. et al. Hair-bearing human skin generated entirely from pluripotent stem cells. Nature 582, 399-404 (2020).

[0180] Abaci, H. E. et al. Tissue engineering of human hair follicles using a biomimetic developmental approach. Nat Commen d, 5301 (2018).

[0181] Itoh, M. et al. Generation of 3D Skin Equivalents Fully Reconstituted from Human Induced Pluripotent Stem Cells (iPSCs). PLOS ONE 8, e77673 (2013).

[0182] Yang, R. et al. Generation of folliculogenic human epithelial stem cells from induced pluripotent stem cells. Nat Commun 5, 3071 (2014).

[0183] Li, X., Ootani, A. & Kuo, C. An Air-Liquid Interface Culture System for 3D Organoid Culture of Diverse Primary Gastrointestinal Tissues, in Gastrointestinal Physiology and Diseases: Methods and Protocols (ed. Ivanov, A. I.) 33-40 (Springer, New York, NY, 2016). doi :10.1007 / 978-1 -4939-3603-8_4.

[0184] Li, X. et al. Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture. Nat Med 20, 769-777 (2014).

[0185] Neal, J. T. etal. Organoid Modeling of the tumor Immune Microenvironment. Cell 175, 1972-1988. e16 (2018).

[0186] Steurer, S. et al. p63 expression in human tumors and normal tissues: a tissue microarray study on 10,200 tumors. Biomark Res 9, 7 (2021 ).

[0187] Van Muijen, G. N. P., Warnaar, S. O. & Ponec, M. Differentiation-related changes of cytokeratin expression in cultured keratinocytes and in fetal, newborn, and adult epidermis. Experimental Cell Research 171 , 331-345 (1987).

[0188] Ishitsuka, Y. & Roop, D. R. Loricrin: Past, Present, and Future. Int J Mol Sci 21 , 2271 (2020).

[0189] Bergers, L. I. J. C. etal. Immune-competent human skin disease models. Drug Discov Today 21 , 1479-1488 (2016).

[0190] Mulder, P. P. G. et al. Monocytes and T cells incorporated in full skin equivalents to study innate or adaptive immune reactions after burn injury. Front. Immunol. 14, (2023).

[0191] Attiogbe, E. etal. An in vitro autologous, vascularized, and immunocompetent Tissue Engineered Skin model obtained by the self-assembled approach. Acta Biomaterialia 168, 361-371 (2023).

[0192] Kaplan, D. H. Ontogeny and function of epidermal murine Langerhans cells. Nat Immunol 18, 1068-1075 (2017).

[0193] Benyon, R. C. The human skin mast cell. Clinical & Experimental Allergy 19, 375-387 (1989).

[0194] Li, A., Simmons, P. J. & Kaur, P. Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype. Proceedings of the National Academy of Sciences 95, 3902-3907 (1998).ATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171

[0195] Poloni, C. et al. T-cell activation-induced marker assays in health and disease. Immunology & Cell Biology 101 , 491-503 (2023).

[0196] Chen, B. et al. Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer. Nat Chem Biol 5, 100-107 (2009).

[0197] Lim, X. et al. Interfollicular Epidermal Stem Cells Self-Renew via Autocrine Wnt Signaling. Science 342, 1226-1230 (2013).

[0198] Choi, Y. S. et al. Distinct Functions for Wnt / |3-Catenin in Hair Follicle Stem Cell Proliferation and Survival and Interfollicular Epidermal Homeostasis. Cell Stem Cell 13, 720- 733 (2013).

[0199] Robert, C. et al. Cutaneous side-effects of kinase inhibitors and blocking antibodies. The Lancet Oncology 6, 491-500 (2005).

[0200] Lacouture, M. E. Mechanisms of cutaneous toxicities to EGFR inhibitors. Nat Rev Cancer Q, 803-812 (2006).

[0201] Jimbow, K., Roth, S. I., Fitzpatrick, T. B. & Szabo, G. Mitotic activity in non-neoplastic melanocytes in vivo as determined by histochemical, autoradiographic, and electron microscope studies. J Cell Biol 66, 663-670 (1975).

[0202] Arita, Y., O’Driscoll, K. R. & Weinstein, I. B. Growth of human melanocyte cultures supported by 12-O-tetradecanoylphorbol-13-acetate is mediated through protein kinase C activation. Cancer Res 52, 4514-4521 (1992).

[0203] Aly, R. Microbial Infections of Skin and Nails, in Medical Microbiology (ed. Baron, S.) (University of Texas Medical Branch at Galveston, Galveston (TX), 1996).

[0204] Rutjes, S. A., Vennis, I. M., Wagner, E., Maisaia, V. & Peintner, L. Biosafety and biosecurity challenges during the COVID-19 pandemic and beyond. Front Bioeng Biotechnol 11 , 1117316 (2023).

[0205] Harapan, H. et al. Monkeypox: A Comprehensive Review. Viruses 14, 2155 (2022).

[0206] Stagles, M. J., Watson, A. A., Boyd, J. F., More, I. A. R. & McSeveney, D. The histopathology and electron microscopy of a human monkeypox lesion. Transactions of The Royal Society of Tropical Medicine and Hygiene 79, 192-202 (1985).

[0207] Bayer-Garner, I. B. Monkeypox virus: histologic, immunohistochemical and electronmicroscopic findings. Journal of Cutaneous Pathology 32, 28-34 (2005).

[0208] Chames, P., Van Regenmortel, M., Weiss, E. & Baty, D. Therapeutic antibodies: successes, limitations and hopes for the future. Br J Pharmacol 157, 220-233 (2009).

[0209] Hardwick, R. N. et al. Drug-induced skin toxicity: gaps in preclinical testing cascade as opportunities for complex in vitro models and assays. Lab Chip 20, 199-214 (2020).

[0210] Thornhill, J. P. et al. Monkeypox Virus Infection in Humans across 16 Countries - April-June 2022. N Engl J Med 387, 679-691 (2022).ATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171

[0211] Lim, H. W. et al. The burden of skin disease in the United States. Journal of the American Academy of Dermatology 76, 958-972. e2 (2017).

[0212] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

ATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-171What is claimed is:1 . A method for the culture of mammalian skin, the method comprising: culturing an initiating skin tissue bloc comprising epidermal and dermal layers of the skin and skin-resident endothelial, fibroblast, stromal and immune cells, in a gel with an airliquid interface and contact with medium, for an initiation period of time sufficient to maintain epidermal proliferation, differentiation, maturation and barrier function; wherein the culture reproduces the histomorphology of native mammalian skin.

2. The method of claim 1 , wherein the skin tissue bloc is human skin.

3. The method of claim 1 or claim 2, wherein the medium is free of exogenous protein factors during the initiation of the culture.

4. The method of any of claims 1 -3, wherein the initiation period is from about 1 to about 4 weeks.

5. The method of any of the preceding claims, wherein all of the mammalian cells present in the culture after the initiation period are derived from the initiating skin tissue bloc.

6. The method of any of the preceding claims, wherein the culture comprises each of macrophages, keratinocytes, T cells, endothelial cells, fibroblasts, melanocytes, mast cells, and neuronal cells derived from the initiating tissue bloc.

7. The method of any of the preceding claims, wherein the immune cells comprise CD4+T-cells, CD8+T-cells, mast cells, Langerhans cells, macrophages, and gd T-cells.

8. The method of any of the preceding claims, wherein the culture comprises each of a stratum basalis, stratum spinosum, and stratum corneum.

9. The method of any of the previous claims, wherein the culture comprises structures: sweat glands, hair shafts and follicles, and blood vessels.

10. The method of any of the preceding claims, wherein the culture comprises an exogenous disease-inducing agent.1 1 . The method of claim 10, wherein the disease inducing agent is a pathogen, autoimmune-inducing lymphocyte, gene, cancer cell or skin irritant.ATTORNEY DOCKET NAME: STAN-2204WOCLIENT REFERENCE: S22-17112. The method of claim 1 1 , wherein the pathogen is a virus.

13. The method of any of the previous claims, wherein the initiating skin tissue bloc is obtained from an individual with a skin condition of interest.

14. The method of claim 13, wherein the condition of interest is one of an infection, an immune-associated disease, cancers, a drug reaction, contact dermatitis, or genetic disease.

15. A mammalian skin culture obtained by the method according to any of claims 1 - 14.

16. A method of screening a candidate agent for an effect on skin, the method comprising: contacting a mammalian skin culture of claim 15 with the candidate agent; and determining the effect of the agent on skin cells in the culture.

17. The method of claim 16, wherein the effect is one or more of a change in gene expression, a change in protein levels, a change in histology, a change in the absolute number of specific cell types, a change in the relative number of specific cell types, a change in viability of specific cell types.

18. The method of claim 16 or 17, wherein the agent is a candidate for causing disease.

19. The method of claim 18, wherein the agent is a pathogen, autoimmune-inducing lymphocyte, genetic construct, cancer cell or skin irritant.

20. The method of claim 16 or claim 17, wherein the agent is a therapeutic agent.

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