Systems and methods for skin tissue development and analysis - Patent Application 20070122997

Abstract

The present disclosure relates to skin tissue systems, devices and methods.The system described includes a system comprising a tissue sample; an insert; and a porous layer configured to promote mass transport into the tissue sample; the skin tissue system can support tissue viability for at least one week.The method described includes a method for skin tissue analysis, comprising: contacting a skin tissue array with at least one test agent; after contacting, measuring a plurality of data sets; mapping the plurality of data sets based on scoring panel results; and based on the mapped data set, generating at least one threshold criterion for selecting a therapeutic agent from at least one test agent.

Description

[Technical Field]

[0001] cross reference This application claims priority to U.S. Provisional Patent Application No. 63 / 488,673, filed March 6, 2023, which is incorporated by reference herein in its entirety for all purposes. [Background technology]

[0002] background Animal and human testing is expensive, time-consuming, and carries inherent degree of experimental risk to living organisms.In the field of compound or biologics testing, there is currently no effective test system for skin research.Commercially available methods for skin tissue analysis lack data consistency, because the skin models used to obtain such data lack life span, hindering their ability to provide clinically relevant evaluation items.Therefore, there is a need for a reliable analytical workflow that uses model systems to reduce the costs associated with testing on living organisms, while also providing meaningful tissue life span to provide clinically relevant evaluation items. Summary of the Invention [Means for solving the problem]

[0003] A brief overview This section contains a summary of the claims, in the commonly accepted definition of a comprehensive, usually concise, summary of the claims. It should be understood that these embodiments are merely illustrative, that the embodiments are not limited to operating according to the specific examples shown in the drawings and discussed below, and that other embodiments are possible. The following embodiments are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the ex vivo human skin models, compositions, and methods of use and manufacture described in certain aspects and embodiments herein, and are not intended to limit the scope of the various aspects and embodiments herein.

[0004] In some embodiments, the present disclosure provides an ex vivo skin tissue system, comprising: an insert comprising at least one opening and a wall; a skin tissue sample sealed inside the insert; and a porous layer positioned inside the insert, wherein the porous layer has a first side facing at least one opening of the insert and a second side facing the opposite side of the at least one opening, and the porous layer comprises a structure and arrangement relative to the skin tissue sample, such that the porous layer promotes the mass transport of reagents and / or fluids into the skin tissue sample, and maintains the viability of the skin tissue sample for at least one week.In some embodiments, the ex vivo skin tissue system further comprises an adipose layer positioned inside the insert.In some embodiments, the porous layer is in contact with at least one of the adipose layer and the tissue sample.In some embodiments, the ex vivo skin tissue system further comprises at least one inlet channel and outlet channel positioned laterally relative to the porous layer. In some embodiments, the ex vivo skin tissue system further includes a gel layer in contact with the second side, the gel layer configured to receive one or more of adipose-derived cells, dermal cells, and endothelial cells. In some embodiments, the gel layer includes a gel material. In some embodiments, the gel layer includes an extracellular matrix. In some embodiments, the ex vivo skin tissue system further includes an adhesive film configured for at least one of contacting and sealing the adipose layer. In some embodiments, the ex vivo skin tissue system further includes a cell composition seeded within the porous layer between the first side and the second side. In some embodiments, the ex vivo skin tissue system further includes a gel layer in contact with the second side. In some embodiments, the seeded cells include one or more of endothelial cells, primary cells, iPSCs, keratinocytes, fibroblasts, melanocytes, resident immune cells, circulating immune cells, stem cells, adipocytes, and microorganisms. In some embodiments, the insert includes a first thread along an inner surface of the wall perpendicular to the first side of the porous layer. In some embodiments, the tissue sample is sealed using an adhesive, the adhesive being selected from medical super glue and UV curable glue.In some embodiments, the tissue sample includes at least one of an epidermis layer, a dermis layer, and a subcutaneous tissue layer. In some embodiments, the porous layer includes a porosity gradient. In some embodiments, the porous layer includes a plurality of pores, each of the plurality of pores being gyroid. In some embodiments, the ex vivo skin tissue system further includes at least one standoff along a second side of the porous layer. In some embodiments, an inner surface of a wall perpendicular to the first side of the porous layer includes a plurality of pores. In some embodiments, each of the plurality of pores is gyroid. In some embodiments, the plurality of pores penetrates the wall to a desired thickness. In some embodiments, the ex vivo skin tissue system further includes a growth medium in contact with the tissue sample. In some embodiments, the growth medium is maintained at a temperature between 32 and 37 degrees Celsius. In some embodiments, the growth medium includes at least one test agent. In some embodiments, the ex vivo skin tissue system further comprises at least one test agent contacted with the tissue sample, wherein the contacting is by one or more of topical application, subcutaneous application, systemic application, intradermal administration, infusion, perfusion, and injection.

[0005] In some embodiments, an ex vivo skin tissue system is provided herein, comprising: an insert including at least one opening and a wall; a sealing unit along the inner wall of the insert; a tissue sample positioned inside the insert via the sealing unit; and a porous layer positioned inside the insert, the porous layer configured to facilitate bulk transport of reagents and / or fluids into the tissue sample, the porous layer having a first side facing at least one opening of the insert and a second side facing the opposite side of the at least one opening, wherein the ex vivo skin tissue system is capable of supporting tissue viability for a period of more than one week. In some embodiments, the ex vivo skin tissue system further comprises an adipose layer positioned inside the insert. In some embodiments, the ex vivo skin tissue system further comprises a cell composition seeded in the porous layer between the first side and the second side. In some embodiments, the ex vivo skin tissue system further comprises a gel layer in contact with the second side. In some embodiments, the seeded cells include one or more of endothelial cells, primary cells, iPSCs, keratinocytes, fibroblasts, melanocytes, resident immune cells, circulating immune cells, stem cells, adipocytes, and microorganisms. In some embodiments, the insert includes a first thread along an inner surface of the wall perpendicular to a first side of the porous layer. In some embodiments, the sealing unit includes a compression unit, the compression unit including a second thread along the periphery configured to engage with the first thread. In some embodiments, the compression unit provides either (1) a complete seal or (2) a partial seal of the tissue sample. In some embodiments, the compression unit includes an injection port in fluid communication with the microchannel. In some embodiments, the sealing unit is an adhesive, the adhesive being selected from medical super glue and UV-curable glue. In some embodiments, the porous layer is in contact with at least one of the fat layer and the tissue sample. In some embodiments, the ex vivo skin tissue system further comprises at least one of an inlet channel and an outlet channel positioned laterally relative to the porous layer.In some embodiments, the tissue sample includes at least one of an epidermis layer, a dermis layer, and a subcutaneous tissue layer. In some embodiments, the porous layer includes a porosity gradient. In some embodiments, the porous layer includes a plurality of pores, each of the plurality of pores being gyroid. In some embodiments, the ex vivo skin tissue system further includes at least one standoff along a second side of the porous layer. In some embodiments, an inner surface of a wall perpendicular to the first side of the porous layer includes a plurality of pores. In some embodiments, each of the plurality of pores is gyroid. In some embodiments, the plurality of pores penetrates the wall to a desired thickness. In some embodiments, the ex vivo skin tissue system further includes a gel layer in contact with the second side, the gel layer configured to receive one or more of adipose-derived cells, dermal cells, and endothelial cells. In some embodiments, the gel layer includes a gel material. In some embodiments, the gel layer includes an extracellular matrix. In some embodiments, the ex vivo skin tissue system further comprises an adhesive film configured for at least one of contacting and sealing the fat layer. In some embodiments, the ex vivo skin tissue system further comprises a growth medium in contact with the tissue sample. In some embodiments, the growth medium is maintained at a temperature between 32 and 37 degrees Celsius. In some embodiments, the growth medium comprises at least one test agent. In some embodiments, the ex vivo skin tissue system further comprises at least one test agent in contact with the tissue sample, the contacting being by one or more of topical application, subcutaneous application, systemic application, intradermal administration, infusion, perfusion, and injection.

[0006] In some embodiments, a method is provided herein that includes the steps of: providing an insert including a porous layer, the porous layer including a structure for promoting fluid mass transport; placing and sealing a skin tissue sample against a first side of the porous layer along an inner wall of the insert to promote fluid mass transport into the skin tissue sample; culturing the skin tissue sample, thereby creating an ex vivo skin model; and perfusing a reagent through the ex vivo skin model to support the viability of the skin tissue sample for a period of at least one week. In some embodiments, the method further includes depositing an adipose layer on one or more of the first side, the interior region, and the second side of the porous layer. In some embodiments, the porous layer is configured to promote vascularization of the tissue sample. In some embodiments, the method further includes seeding a cell composition into the porous layer between the first side and the second side. In some embodiments, the seeding is performed using one or more of bioprinting, manual or automated liquid handling, and perfusion. In some embodiments, the cell composition comprises at least one of endothelial cells, primary cells, iPSCs, keratinocytes, fibroblasts, melanocytes, resident immune cells, circulating immune cells, stem cells, preadipocytes, and adipocytes. In some embodiments, the resident immune cells comprise at least one of Langerhans cells, dendritic cells, macrophages, mast cells, monocytes, and lymphocytes. In some embodiments, the circulating immune cells comprise at least one of neutrophils, T lymphocytes, B lymphocytes, natural killer cells, monocytes, and dendritic cells. In some embodiments, the reagent comprises at least one of medium, non-essential amino acid solution, insulin, transferrin, selenite, bovine serum albumin, linoleic acid, hydrocortisone, vitamin C, serum, blood substitute, penicillin, and streptomycin. In some embodiments, the method further comprises contacting the second side with a gel layer, the gel layer configured to receive at least one of adipose-derived cells, dermal cells, and endothelial cells. In some aspects, the gel layer comprises a gel material, hi some aspects, the gel layer comprises an extracellular matrix.In some embodiments, the gel layer is bioprinted. In some embodiments, the porous layer is in contact with at least one of an adipose layer and a tissue sample. In some embodiments, the method further includes at least one of an inlet channel and an outlet channel positioned laterally relative to the porous layer. In some embodiments, the tissue sample includes at least one of an epidermal layer, a dermal layer, and a subcutaneous tissue layer. In some embodiments, the porous layer includes a porosity gradient. In some embodiments, the porous layer includes a plurality of pores, each of the plurality of pores being in the shape of one or more of a gyroid, a Schwarz, an x-cell, and an intersection.

[0007] In some embodiments, the method further includes at least one standoff along the second side of the porous layer. In some embodiments, an inner surface of the wall perpendicular to the first side of the porous layer includes a plurality of pores. In some embodiments, each of the plurality of pores is gyroid. In some embodiments, the plurality of pores penetrates the inner wall to a desired thickness. In some embodiments, the method further includes a gel layer in contact with the second side, the gel layer configured to receive at least one of the fat layer and the tissue sample. In some embodiments, the gel layer includes a gel material. In some embodiments, the gel layer includes an extracellular matrix. In some embodiments, the sealing step includes a compression unit, the compression unit including a microchannel. In some embodiments, the compression unit includes an injection port in fluid communication with the microchannel. In some embodiments, the method further includes sealing the fat layer with a gas-permeable film. In some embodiments, an oscillatory pressure is applied to the fluid to facilitate filling and emptying of the fluid. In some embodiments, the method further comprises perfusing a composition through the insert; culturing the tissue sample; applying an environmental condition and / or monitoring the environmental condition; providing at least one sensor for obtaining at least one functional readout; monitoring at least one of a cellular characteristic and a tissue characteristic using the at least one sensor; and scoring at least one of a cellular characteristic and a tissue characteristic. In some embodiments, one or more of culturing the tissue sample and monitoring at least one of a cellular characteristic and a tissue using the at least one sensor are performed for at least two weeks. In some embodiments, the composition is adapted to maintain and / or promote a disease state. In some embodiments, the composition is adapted to promote cell differentiation. In some embodiments, the composition is adapted to promote one or more of promoting tissue complexity, maintaining multiple cell types, and generating cell-cell and / or cell-tissue interactions.In some embodiments, the environmental conditions include at least one of temperature, humidity, gas composition, pollution, and light exposure. In some embodiments, monitoring at least one of the cellular and tissue characteristics includes monitoring at least one of irritation, corrosion, and phototoxicity. In some embodiments, monitoring at least one of the cellular and tissue characteristics includes monitoring at least one of anti-aging, skin moisturization, brightening, discoloration, pigmentation, UV protection, cleansing, permeability, inflammation, anti-inflammatory, antibacterial, wound healing, skin barrier, epidermal thickness, dermal matrix, cellular stress, cellular senescence, sagging, and wrinkles. In some embodiments, each of the at least one sensor includes at least one clinical sensor. In some embodiments, each of the at least one sensor includes a viscoelastic sensor, a moisturization sensor, a pH sensor, an oil content sensor, a barrier function sensor, a pigmentation sensor, and a skin surface sensor. In some embodiments, the scoring step includes calculating the percent change in each characteristic from normal or vehicle conditions. In some aspects, the scoring step comprises characterizing the method used to obtain the feature. In some aspects, an oscillatory pressure is applied to the perfused composition to facilitate filling and emptying of the perfused composition.

[0008] In some embodiments, the techniques described herein relate to a method for skin tissue analysis, the method comprising: receiving a plurality of ex vivo skin tissue systems described herein; mapping a plurality of datasets based on scoring panel results, wherein the scoring panel results are calculated according to the strength and effectiveness of each measurement in the mapped plurality of datasets; and generating at least one threshold criterion for identifying a test agent from at least one test agent based on the mapped plurality of datasets, wherein each of the at least one threshold criterion corresponds to at least one biological pathway. In some embodiments, the skin tissue array comprises one or more of an epidermal tissue layer and a dermal tissue layer. In some embodiments, the skin tissue array comprises one or more tissue layers from a biopsy, a donor, or a graft. In some embodiments, the skin tissue array comprises an adipose layer, the adipose layer being below a porous layer. In some embodiments, the skin tissue array comprises an adipose layer, the adipose layer being within the porous layer. In some embodiments, the plurality of datasets comprises measurements from one or more of the skin surface, histological staining, immunostaining, biochemical assays, clinical sensors, gene expression, and protein expression. In some embodiments, measurements generated from the skin surface include measurements from one or more of dermoscopy, megurometer, cutometer, and photography. In some embodiments, measurements generated from histological staining include measurements from one or more of hematoxylin and eosin (H&E), Masson's trichrome, and Movat pentachrome. In some embodiments, measurements generated from one or more of gene expression and protein expression include one or more of dermal differentiation, epidermal-dermal junction, dermal markers, matrix metalloproteinases (MMPs), MMP inhibitors, regeneration, wrinkles, sagging, inflammation, intrinsic apoptosis, extrinsic apoptosis, anti-apoptosis, immune cell markers, skin elasticity, skin rejuvenation, and antioxidant defense. In some embodiments, measurements generated from clinical sensors include measurements from one or more of moisture, barrier function, sebum, pH, dermoscopy, photography, melanin content, and ultrasound.In some embodiments, at least one threshold criterion is either positively or negatively correlated with the condition. In some embodiments, the condition comprises one or more of anti-aging, skin moisturizing, toxicity, brightening, discoloration, pigmentation, UV protection, cleansing, permeability, inflammation, anti-inflammatory, antibacterial, wound healing, skin barrier, epidermal thickness, dermal matrix, cell stress, cell senescence, sagging and wrinkles. In some embodiments, the skin tissue array is viable for a period longer than one week. In some embodiments, the at least one biological pathway comprises one or more of skin surface, barrier function, epidermal thickness, matrix density, epidermal differentiation, epidermal-dermal junction, dermal papilla thickness and projection, stem cell and regenerative activity, adipogenesis, melanogenesis, sagging pathway, wrinkle pathway, cell senescence, cell stress, inflammation and apoptosis. In some embodiments, the at least one threshold criterion is generated by applying a machine learning algorithm to the mapped multiple datasets. In some embodiments, the machine learning algorithm is further configured to perform one or more of: verifying and generating product claims for skin concerns; conducting broad efficacy and / or toxicity screening of a chemical library for quantitative comparison against product benchmarks; and performing virtual screening of at least one test agent. In some embodiments, the machine learning algorithm is either an unsupervised algorithm or a supervised algorithm. In some embodiments, the porous layer is configured to promote mass transport into each tissue sample in the skin tissue array. In some embodiments, the porous layer comprises a porosity gradient. In some embodiments, the porous layer comprises a plurality of pores, each of the plurality of pores being one or more of a gyroid, a Schwarz, an x-cell, and a cross shape. In some embodiments, the method further comprises contacting the skin tissue array with at least one test agent, wherein the contacting is one or more of topical application, subcutaneous application, systemic application, intradermal administration, infusion, perfusion, and injection.

[0009] In some embodiments, the techniques described herein relate to a method comprising providing any of the ex vivo skin tissue systems described herein; perfusing a composition through the ex vivo skin tissue system; culturing the ex vivo skin tissue system; performing one or more of applying environmental conditions and monitoring the environmental conditions; providing at least one sensor; monitoring at least one cellular and tissue characteristic using the at least one sensor; and scoring at least one cellular and tissue characteristic. In some embodiments, one or more of the culturing and monitoring steps are performed for at least two weeks. In some embodiments, the composition is adapted to maintain and / or promote a disease state. In some embodiments, the composition is adapted to promote cell differentiation. In some embodiments, the composition is adapted to one or more of promoting tissue complexity, maintaining multiple cell types, and generating cell-cell and / or cell-tissue interactions. In some embodiments, the environmental conditions include at least one of temperature, humidity, gas composition, pollution, and light exposure. In some embodiments, monitoring at least one of the cellular and tissue characteristics includes monitoring at least one of irritation, corrosion, phototoxicity, viability, and metabolic activity. In some embodiments, monitoring at least one of the cellular and tissue characteristics includes monitoring at least one of anti-aging, skin moisturization, brightening, discoloration, pigmentation, UV protection, cleansing, permeability, inflammation, anti-inflammatory, antibacterial, wound healing, skin barrier, epidermal thickness, dermal matrix, cellular stress, cellular senescence, sagging, and wrinkles. In some embodiments, each of the at least one sensor includes a viscoelastic sensor, a moisturization sensor, a pH sensor, an oil content sensor, a barrier function sensor, a pigmentation sensor, and a skin surface sensor. In some embodiments, scoring includes calculating the percent change in each characteristic from normal or vehicle conditions.In some aspects, the scoring step comprises characterizing the method used to obtain the feature. In some aspects, an oscillatory pressure is applied to the perfused composition to facilitate filling and emptying of the perfused composition.

[0010] In some embodiments, the techniques described herein relate to an ex vivo skin tissue system, comprising one or more inserts, each of which comprises a drip reservoir at a proximal end; a base reservoir at a distal end; a via sandwiched between the drip reservoir and the base reservoir; and a tissue sample positioned inside at least one of the drip reservoir and the base reservoir; the via is configured to promote one or more of the following: mass transport into the tissue sample and cell growth from seeded cells, the via has a first side facing the opening of the drip reservoir and a second side facing the opening of the base reservoir, and the ex vivo skin tissue system can support tissue viability for a period longer than one week.In some embodiments, the ex vivo skin tissue system further comprises an adipose layer positioned inside at least one of the drip reservoir and the base reservoir.In some embodiments, the via is in contact with at least one of the adipose layer and the tissue sample. In some embodiments, the tissue sample comprises at least one of an epidermal layer, a dermal layer, and a subcutaneous tissue layer. In some embodiments, the via comprises a porosity gradient. In some embodiments, the via comprises a porous layer, the porous layer comprising a plurality of pores. In some embodiments, each of the plurality of pores has one or more of the following shapes: gyroid, Schwarz, x-cell, and cross. In some embodiments, the ex vivo skin tissue system further comprises a gel layer in contact with the second side, the gel layer configured to receive at least one of adipose-derived cells, dermal cells, and endothelial cells. In some embodiments, the gel layer comprises a synthetic gel material. In some embodiments, the gel layer comprises an extracellular matrix. In some embodiments, the seeded cells comprise one or more of primary cells, iPSCs, keratinocytes, fibroblasts, melanocytes, endothelial cells, resident immune cells, circulating immune cells, stem cells, adipocytes, and microorganisms. In some embodiments, the drip reservoir, base reservoir, and via are dimensioned to have a Bond number < 1. In some embodiments, the via comprises an axially straight interior wall.In some embodiments, the ex vivo skin tissue system further comprises a growth medium in contact with the tissue sample. In some embodiments, the growth medium is maintained at a temperature between 32 and 37 degrees Celsius. In some embodiments, the growth medium comprises at least one test agent. In some embodiments, the ex vivo skin tissue system further comprises at least one test agent in contact with the drip reservoir.

[0011] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0012] [Figure 1-1] 1A-1G are cross-sectional views of an ex vivo skin tissue system upon assembly of a tissue sample and an adipose layer in contact with a porous layer in an insert. FIG. 1A provides a cross-sectional view of an ex vivo skin tissue system that may include one or more of a compression unit, a tissue sample, a porous layer, an adipose layer, and an adhesive film. FIG. 1B provides a cross-sectional view of an ex vivo skin tissue system that may include one or more of an insert, a seeded adipose layer, a 3D-printed porous structure, a gas-permeable film, a tissue biopsy, a humidity control element, a nutrient delivery element, and optional endothelial cell seeding. FIGS. 1C-1G provide representations and photographs of a perfusion device according to one or more embodiments of the present disclosure. FIG. 1C provides a representation of a side view of a perfusion device. FIG. 1D provides a representation of a perfusion device including a reservoir container according to one or more embodiments of the present disclosure. FIG. 1E provides a photograph showing a top view of a perfusion device according to one or more embodiments of the present disclosure. FIG. 1F provides a photograph showing a side view of a perfusion device according to one or more embodiments of the present disclosure. FIG. 1G provides a photograph showing a perfusion apparatus and perfusion controller according to one or more embodiments of the present disclosure. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above.

[0013] [Figure 2-1] 2A-2C are cross-sectional views of an insert. 2A-2B show embodiments in which pores are confined to a porous layer or extend along the inner surface of the insert. 2A shows an embodiment of an ex vivo skin tissue system that may include one or more of a porous structure, a gas-permeable film, and a porous structure along the insert wall. 2B shows an embodiment of an ex vivo skin tissue system that may include one or more of a lip, a porous structure, threading for a compression nut, and one or more standoffs. 2C shows an embodiment in which the insert has a smaller outer diameter. [Figure 2-2] Same as above. [Figure 2-3] Same as above.

[0014] [Figure 3-1] Figures 3A-3B show a workflow for preparing and analyzing an ex vivo skin tissue system. Figure 3A provides a flowchart illustrating the process for collecting data, which includes controlling and / or monitoring relevant biological and environmental conditions. Figure 3B provides a flowchart illustrating a potential system for experimentation. [Figure 3-2] Same as above.

[0015] [Figure 4-1]4A-4J are exemplary embodiments of a porous layer according to aspects of the present disclosure. 4A-4D illustrate exemplary embodiments in which the porous layer comprises a gyroid structure. 4A illustrates an embodiment of an ex vivo skin tissue system. 4B provides a photograph of a skin tissue model insert. 4C provides a photograph showing an alternative view of a skin tissue model insert. 4D provides a photograph showing an alternative view of a skin tissue model insert. 4E-4J illustrate further exemplary embodiments in which the porous layer comprises microneedles or a mesh. 4E provides a photograph of a custom skin tissue model insert with microneedles. 4F provides a photograph of an alternative view of a custom skin tissue model insert with hollow microneedles in fluid communication with perfusable microchannels. 4G provides a photograph of an alternative view of a custom skin tissue model insert with microneedles. 4H provides a photograph of an alternative view of a custom skin tissue model insert with microneedles. 4I provides a photograph of a mesh structure for a skin tissue culture insert. FIG. 4J provides a photograph of an alternative view of the mesh structure for the tissue culture insert. [Figure 4-2] Same as above. [Figure 4-3] Same as above.

[0016] [Figure 5-1] 5A-5B are cross-sectional and perspective views, respectively, of an exemplary embodiment of a compression nut. Figure 5A provides a cross-sectional view of a model of a compression nut for holding a tissue sample, including microchannels. Figure 5B provides a perspective view of a model of a compression nut for holding a tissue sample, including an injection port. [Figure 5-2] Same as above.

[0017] [Figure 6-1]6A-6E show various views of a recirculation system according to some embodiments of the present disclosure. FIG. 6A provides a diagram of a tissue culture modeling system that may include one or more of an insert for holding a tissue sample, an insert cap, a recirculation system, a reservoir cap, a fluid capacitor, and a media reservoir. FIG. 6B provides a diagram of a tissue culture modeling system that may include an integrated check valve. FIG. 6C provides a diagram of a tissue culture modeling system that may include one or more of a fluid inlet, a fluid outlet, and a membrane. FIG. 6D provides a diagram of a model of an ex vivo skin tissue system and media reservoir with a recirculation pump. FIG. 6E provides a diagram of a model of an ex vivo skin tissue system with multiple culture inserts, with an inset of a human skin structure. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above. [Figure 6-5] Same as above.

[0018] [Figure 7-1] 7A-7G are perspective and cross-sectional views, respectively, of a high-throughput cartridge (HTC) system. FIG. 7A provides a perspective view of a skin tissue modeling system compatible with an SBS microtiter plate. FIG. 7B provides a cross-sectional view of a skin tissue modeling system that may include one or more of an insert, a medium reservoir, and a lid. FIGs. 7C-7F show further embodiments of an HTC system according to some aspects of the present disclosure. FIG. 7C provides a diagram of a skin tissue modeling system that may include one or more of a drip reservoir, a via, and a basal reservoir. FIG. 7D provides a diagram of a model of a skin tissue modeling system. FIG. 7E provides a diagram of a model of a skin tissue culture modeling system. FIG. 7F provides a diagram of a model of a skin tissue culture modeling system. FIG. 7G provides a close-up view of a single insert in an HTC system according to some aspects of the present disclosure. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above. [Figure 7-5] Same as above. [Figure 7-6] Same as above.

[0019] [Figure 8-1] 8A-8C are diagrams illustrating a data extraction workflow and diagrams describing workflows for data extraction, data mapping, and assertion quantification, respectively, according to one or more aspects of the present disclosure. [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 8-4] Same as above. [Figure 8-5] Same as above.

[0020] [Figure 9] FIG. 9 is a block diagram of a system in which some embodiments may operate.

[0021] [Figure 10] FIG. 10 is a block diagram of a computing device in which some embodiments may operate.

[0022] [Figure 11-1] Figures 11A-11F are a series of photomicrographs of tissue samples subjected to test conditions with different stains applied. Figures 11A-11B provide photomicrographs using hematoxylin-eosin staining. Figures 11C-11D provide photomicrographs using Masson's trichrome staining. Figures 11E-11F provide photomicrographs using Movat pentachrome staining. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above. [Figure 11-5] Same as above. [Figure 11-6] Same as above.

[0023] [Figure 12-1]12A-12F are gene expression maps for several different study conditions according to one or more embodiments of the present disclosure. FIG. 12A provides a gene expression map for epidermal differentiation and the epidermal-dermal junction. FIG. 12B provides a gene expression map for dermal markers and MMPs. FIG. 12C provides a gene expression map for MMP inhibitors and regeneration. FIG. 12D provides a gene expression map for wrinkle-related and sagging-related genes. FIG. 12E provides a gene expression map for inflammation and apoptosis (intrinsic). FIG. 12F provides a gene expression map for apoptosis (extrinsic) and anti-apoptotic genes. [Figure 12-2] Same as above. [Figure 12-3] Same as above. [Figure 12-4] Same as above. [Figure 12-5] Same as above. [Figure 12-6] Same as above. [Figure 12-7] Same as above. [Figure 12-8] Same as above. [Figure 12-9] Same as above. [Figure 12-10] Same as above. [Figure 12-11] Same as above. [Figure 12-12] Same as above.

[0024] [Figure 13] 13A-13B are micrographs of stained tissue samples according to one or more embodiments of the present disclosure. Figure 13A provides a histologically stained skin tissue sample on day 0 of culture. Figure 13B provides a histologically stained skin tissue sample after 3 weeks of culture.

[0025] [Figure 14-1] FIG. 14 is a gene expression map showing expression data for genes involved in skin development and function across several samples, according to one or more embodiments of the present disclosure. [Figure 14-2] Same as above.

[0026] [Figure 15] 15A-15G are photomicrographs of histologically stained skin tissue samples according to one or more embodiments of the present disclosure. FIG. 15A provides a photomicrograph showing a histologically stained skin tissue culture using medium mix 1 from Table 6. FIG. 15B provides a photomicrograph showing a histologically stained skin tissue culture using medium mix 3 from Table 6. FIG. 15C provides a photomicrograph showing a histologically stained skin tissue culture using medium mix 6 from Table 6. FIG. 15D provides a photomicrograph showing a histologically stained skin tissue culture using medium mix 8 from Table 6. FIG. 15E provides a photomicrograph showing a histologically stained skin tissue culture using medium mix 9 from Table 6. FIG. 15F provides a photomicrograph showing a histologically stained skin tissue culture using medium mix 11 from Table 6. FIG. 15G provides a photomicrograph showing a histologically stained skin tissue culture using medium mix 12 from Table 6.

[0027] [Figure 16] 16A-16B are photomicrographs showing histologically stained skin tissue samples according to one or more embodiments of the present disclosure. Figure 13A provides a photomicrograph showing histologically stained skin tissue cultures after day 0 of culture using medium mix 12 of Table 6. Figure 16B provides a photomicrograph showing histologically stained skin tissue cultures after 4 weeks of tissue culture using medium mix 12 of Table 6.

[0028] [Figure 17]17A-17C are photographs of the skin surface of skin tissue cultures after 6 weeks of culture according to one or more embodiments of the present disclosure. Figure 17A provides a photograph of the skin tissue surface after 6 weeks of culture in medium mix 12 of Table 6. Figure 17B provides a photograph of the skin tissue culture surface after 6 weeks of culture in medium mix 12 of Table 6 with 5% human platelet lysate. Figure 17C provides a photograph of the human skin tissue culture surface after 6 weeks of culture in medium mix 12 of Table 6 with 5% human platelet lysate and 10 nanograms / milliliter of EGF and 10 nanograms / milliliter of bFGF.

[0029] [Figure 18-1] 18A-18B are photographs and photomicrographs of skin biopsies sealed in tissue culture inserts according to one or more embodiments of the present disclosure. Figure 18A provides a photograph of three 20 mm sealed biopsies. Figure 18B provides a photomicrograph of an H&E stained cultured skin tissue biopsy adhered to a skin tissue culture insert. [Figure 18-2] Same as above.

[0030] [Figure 19-1] 19A-19B are graphs of TEWL measurements for both sealed and unsealed biopsies in tissue inserts, according to one or more embodiments of the present disclosure. Figure 19A provides a graph of TEWL time course measurements for a skin biopsy contained in a tissue culture insert without sealing. Figure 19B provides a graph of TEWL time course measurements for a skin biopsy sealed in a tissue culture insert. [Figure 19-2] Same as above.

[0031] [Figure 20] FIG. 20 provides a graph of TEWL measurements over a 6-day time course in skin tissue biopsies in sealed tissue culture inserts according to one or more embodiments of the present disclosure.

[0032] [Figure 21-1] Figure 21A provides a representation of the incorporation of raw multimodal data into a data map, according to one or more embodiments of the present disclosure. Figures 21B-21C provide representative examples of data maps corresponding to compounds of interest, according to one or more embodiments of the present disclosure. [Figure 21-2] Same as above. [Figure 21-3] Same as above. [Figure 21-4] Same as above.

[0033] [Figure 22] 22A-22B provide photographs and micrographs of a 3D-printed gyroid support structure according to one or more embodiments of the present disclosure. Figure 22A provides a photograph of a 3D-printed gyroid support structure with an endothelial cell network stained with the CD31 endothelial cell marker. Figure 22B provides a micrograph showing a detailed view of a 3D-printed gyroid support structure with an endothelial cell network stained with the CD31 endothelial cell marker.

[0034] [Figure 23]Figures 23A-23H provide micrographs of cellular networks after 14 days of tissue culture and CD31 staining in CELLnTEC-Promocell media mix, according to one or more embodiments of the present disclosure. Figure 23A provides a micrograph showing cells stained with ObaGel™ ECM. Figure 23B provides a micrograph showing cells stained with ObaGel™ Original. Figure 23C provides a micrograph showing cells cultured with fibrinogen (2 mg / mL), aprotinin (1 μg / mL), Geltrex™ (40 μg / mL), and thrombin* (0.5 U / mL). Figure 23D provides a micrograph showing cells cultured with fibrinogen (2 mg / mL), aprotinin (5 μg / mL), Geltrex™ (40 μg / mL), and thrombin* (0.5 U / mL). Figure 23E provides a micrograph showing cells cultured with 2D coating: collagen IV (400 μg / mL), FN (100 μg / mL), and Geltrex (50 μg / mL). Figure 23F provides a micrograph showing cells cultured with 2D coating: collagen IV (200 μg / mL), FN (50 μg / mL), and Geltrex™ (50 μg / mL). Figure 23G provides a micrograph showing cells cultured with 2D coating: collagen I (100 μg / mL), collagen IV (200 μg / mL), FN (50 μg / mL), Geltrex™ (50 μg / mL). Figure 23H provides a micrograph showing the cells-only control group.

[0035] [Figure 24]Figures 24A-24H provide micrographs of cells after endothelial growth medium pretreatment (8 days) followed by CELLnTEC-Promocell medium mix (6 days) and CD31 staining, according to one or more embodiments of the present disclosure. Figure 24A provides a micrograph showing cells stained with ObaGel™ ECM. Figure 24B provides a micrograph showing cells stained with ObaGel™ Original. Figure 24C provides a micrograph showing cells cultured with fibrinogen (2 mg / mL), aprotinin (1 μg / mL), Geltrex™ (40 μg / mL), and thrombin* (0.5 U / mL). Figure 24D provides a micrograph showing cells cultured with fibrinogen (2 mg / mL), aprotinin (5 μg / mL), Geltrex™ (40 μg / mL), and thrombin* (0.5 U / mL). Figure 24E provides a micrograph showing cells cultured with 2D coating: collagen IV (400 μg / mL), FN (100 μg / mL), and Geltrex (50 μg / mL). Figure 24F provides a micrograph showing cells cultured with 2D coating: collagen IV (200 μg / mL), FN (50 μg / mL), and Geltrex™ (50 μg / mL). Figure 24G provides a micrograph showing cells cultured with 2D coating: collagen I (100 μg / mL), collagen IV (200 μg / mL), FN (50 μg / mL), Geltrex™ (50 μg / mL). Figure 24H provides a micrograph showing the cells-only control group.

[0036] [Figure 25] FIG. 25 provides a photomicrograph showing adipose-derived stem cells stained for F-actin using media mix 12 of Table 6, according to one or more embodiments of the present disclosure.

[0037] [Figure 26]26A-26C are representations showing skin tissue subjected to clinically relevant changes according to one or more embodiments of the present disclosure. Fig. 26A provides a representation showing topical chemical treatment of skin tissue. Fig. 26B provides a representation showing skin tissue subjected to contact with a heated metal rod. Fig. 26C provides a representation showing skin tissue subjected to UV treatment.

[0038] [Figure 27] FIG. 27 is a representation of a treatment schedule for replicating dynamic changes in skin across various types of clinically relevant stimuli, according to one or more embodiments of the present disclosure.

[0039] [Figure 28] 28A-28D are photomicrographs showing histologically stained skin tissue samples according to one or more embodiments of the present disclosure. Figure 28A provides a photomicrograph showing untreated skin tissue. Figure 28B provides a photomicrograph showing SDS-challenged skin tissue. Figure 28C provides a photomicrograph showing burned skin tissue. Figure 28D provides a photomicrograph showing skin tissue subjected to UV irradiation.

[0040] [Figure 29-1] 29A-29C are graphs showing histopathological scoring in different groups of cells subjected to clinically relevant stimuli according to one or more embodiments of the present disclosure. Figure 29A provides a graph showing epidermal damage in skin tissue culture samples subjected to UV radiation, SDS, a heated metal rod, and a sample without treatment. Figure 29B provides a graph showing eczematous epidermis in skin tissue culture samples subjected to UV radiation, SDS, a heated metal rod, and a sample without treatment. Figure 29C provides a graph showing epidermal thickness in skin tissue culture samples subjected to UV radiation, SDS, a heated metal rod, and a sample without treatment. [Figure 29-2] Same as above. [Figure 29-3] Same as above.

[0041] [Figure 30]30A-30D are photomicrographs of histologically stained skin tissue samples according to one or more embodiments of the present disclosure. FIG. 30A provides a photomicrograph showing an untreated skin tissue culture. FIG. 30B provides a photomicrograph showing a skin tissue culture subjected to SDS stimulation. FIG. 30C provides a photomicrograph showing a skin tissue culture subjected to burn wounding. FIG. 30D provides a photomicrograph showing a skin tissue culture subjected to UV radiation.

[0042] [Figure 31-1] 31A-31B are graphs showing histopathological scoring of collagen deposition and lactate dehydrogenase (LDH) release, respectively, in skin tissue culture samples subjected to UV radiation, SDS, a heated metal rod, and a sample without treatment, according to one or more embodiments of the present disclosure. [Figure 31-2] Same as above.

[0043] [Figure 32-1] 32A-32B are expression heat maps showing immune responses of skin tissue cultures in response to clinically relevant stimuli according to one or more embodiments of the present disclosure. [Figure 32-2] Same as above.

[0044] [Figure 33-1] 33A-33C are graphs showing inflammatory responses of skin in response to clinically relevant stimuli according to one or more embodiments of the present disclosure. Figure 33A provides a graph showing IL-1β concentrations (pg / mL) in baseline (untreated), SDS-treated, burned, and UV-treated skin tissue culture samples. Figure 33B provides a graph showing IL-6 concentrations (pg / mL) in baseline (untreated), SDS-treated, burned, and UV-treated skin tissue culture samples. Figure 33C provides a graph showing IL-8 concentrations (pg / mL) in baseline (untreated), SDS-treated, burned, and UV-treated skin tissue culture samples. [Figure 33-2] Same as above. [Figure 33-3] Same as above.

[0045] [Figure 34-1] 34A-34C are representations, photomicrographs, and graphs of intradermal injection in skin tissue culture according to one or more embodiments of the present disclosure. FIG. 34A provides a representation of a skin tissue culture sample subjected to intradermal injection. FIG. 34B provides a photomicrograph of a stained intradermal injected skin tissue culture sample. FIG. 34C provides a graph of IL-1β concentrations (pg / mL) in samples treated with saline, LPS 1 μg / mL, LPS 10 μg / mL, and LPS 10 μg / mL together with TNF-α. [Figure 34-2] Same as above.

[0046] [Figure 35-1] 35A-35B are expression heat maps of immune cell profiling of two different donors associated with related compounds, according to one or more embodiments of the present disclosure. [Figure 35-2] Same as above.

[0047] [Figure 36-1] 36A-36B are expression heat maps and graphs of superoxide dismutase (SOD) activity showing antioxidant defense responses, according to one or more embodiments of the present disclosure. Figure 36A provides an expression heat map showing antioxidant-related gene expression associated with treatment with several skin care ingredients. Figure 36B provides a graph showing SOD activity from several UV-treated samples. [Figure 36-2] Same as above.

[0048] [Figure 37] FIG. 37 is an exemplary embodiment of a diagram of a perfusion controller configuration for an ex vivo skin tissue system in accordance with one or more aspects of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0049] Detailed Description Described herein are systems and techniques for compound or biologic screening with increased tissue complexity and lifespan.The present disclosure generally relates to an ex vivo skin tissue model, also referred to herein as an ex vivo skin tissue model, and a support system therefor, such that the ex vivo skin tissue model can promote and maintain advanced features for a wide range of analytical results, for example, efficacy and / or toxicity testing, such as enhanced lifespan, skin-like tissue structure, functionality and immunocompetence.In some embodiments, the methods described herein include analyzing skin tissue during one or more of tissue growth and development, or response to a compound or biologic of interest.In some embodiments, the method of testing a composition includes selecting a therapeutic agent.In some embodiments, the method of testing a composition uses, for example, big data and diverse datasets generated from an ex vivo tissue support system and processed using machine learning algorithms as described herein, to verify and generate evidence-based skin care product claims for skin concerns, to conduct extensive efficacy and / or toxicity screening of chemical libraries for potential quantitative comparison to product benchmarks, and to train artificial intelligence (AI) models to perform virtual screening. In some aspects, the present disclosure provides an ex vivo tissue skin model system comprising: an occlusive unit; skin tissue; and a porous structure configured to promote mass transport into and interaction between one or more of a skin tissue biopsy, an endothelial layer, and an adipose layer.

[0050] Provided herein are (1) insert and cartridge devices; (2) workflows and systems for engineered ex vivo skin tissue analysis; (3) porous layers for enhanced perfusion; (4) samples, cells, and tissues for incorporation into such innovations; (5) high-content model systems; (6) high-throughput model systems; (7) methods for intelligent data analysis; (8) methods for data monitoring and scoring; and (9) computer-implemented systems and methods.

[0051] In some embodiments described herein, ex vivo tissue support systems are curated in a specific manner, and estimates of the efficacy of the systems and illustrative active ingredients when tested with the systems are generated based on analyzing the estimates using various standardized techniques. While laboratory-based experiments have continued to be used to evaluate the efficacy of active ingredients from developers on matrices such as skin tissue, the lack of scalable, physiologically relevant skin models on the market has forced developers to utilize testing platforms that are limited in their ability to screen libraries and evaluate mechanistic insights.

[0052] I. Insert and Cartridge Devices for Engineered Ex Vivo Skin Tissue Models In various aspects, the present disclosure provides an ex vivo skin tissue system that can be used as a platform for maintaining complex tissue models that can be monitored in response to exposure to various test agents over an extended time frame compared to contemporary testing platforms. Figures 1A-1B show an ex vivo skin tissue system according to an exemplary embodiment of the present disclosure. Figure 1A shows an exemplary embodiment in which an adipose layer is deposited on the second side of the porous layer, while Figure 1B shows an exemplary embodiment in which an adipose layer is deposited on the first side of the porous layer.

[0053] In some aspects, ex vivo skin tissue system 1 includes insert 10, which includes a compartment for skin tissue growth containing at least one skin tissue sample 11, porous layer 12, and optionally, adipose tissue layer 13, compression unit 14, and film 15. In certain embodiments, insert 10 may be referred to as a chamber or housing. In some aspects, at least one skin tissue sample 11 includes at least one skin tissue layer. In some aspects, the at least one skin tissue layer includes at least one of an epidermal tissue layer, a dermal tissue layer, and a subcutaneous tissue layer. In some aspects, at least one skin tissue system 11 includes one or more of a stratum corneum, a granular cell layer, a spinous cell layer, a basal cell layer, a sebaceous gland, an erector pili muscle, a sweat gland, a nerve, a hair follicle, collagen and elastin fibers or connective tissue, a blood vessel (e.g., an artery and / or a vein), and adipose (e.g., adipose) tissue. In some aspects, the porous layer 12 includes a structure with sufficient openings for vascularization from the adipose tissue layer into the epidermal and / or dermal tissue layers. In some embodiments, the skin tissue system 11 is viable for a period of more than one week. As used herein, viability may be considered when the percentage of tissue and / or cultured cells is assessed as at least 80% viable. Viability may also be considered when there is less than a 20% change in function, such as cell proliferation, differentiation, migration, and cell-cell, cell-matrix, and immune cell interactions, compared to a control skin tissue sample on day 0. In some embodiments, the skin tissue system 11 is viable for a period of at least two weeks. In some aspects, the ex vivo skin tissue system 1 includes a cell composition seeded within the porous layer 12 between the first and second sides. In some embodiments, the seeded cell composition comprises at least one of primary cells, iPSCs, endothelial cells (e.g., dermal microvascular endothelial cells - HDMECs), dermal cells (e.g., fibroblasts), epidermal cells (e.g., keratinocytes and melanocytes), resident immune cells, circulating immune cells, stem cells, preadipocytes, and adipocytes.In some embodiments, the resident immune cells comprise at least one of Langerhans cells, Merkel cells, dendritic cells, macrophages, mast cells, monocytes, and lymphocytes. In some embodiments, the circulating immune cells comprise at least one of neutrophils, T lymphocytes, B lymphocytes, natural killer cells, monocytes, and dendritic cells.

[0054] The porous layer 12 may provide a primary benefit of increasing mass transport into the tissue biopsy material through its multiple pores. In some embodiments, the insert 10 includes a first thread 161 along the inner surface of the wall 16 perpendicular to the first side of the porous layer 12. In some embodiments, the first thread 161 has a pitch ranging from 1 mm to 0.5 mm. In some embodiments, the compression unit 14 includes a second thread 141 along the periphery configured to mate with the first thread 161. The compression unit 14 is dimensioned to secure the skin tissue sample 11 within the insert 10 at a desired applied pressure based on the height to which the compression unit 14 is tightened within the insert 10. Insufficient pressure, e.g., a compression unit 14 positioned away from the porous layer 12, will loosen the tissue from the insert 10, while excessive pressure, e.g., a compression unit 14 positioned closer to the porous layer 12, will induce torque on the tissue during rotation, causing the tissue to bulge in the center and reduce tightness.

[0055] In some embodiments, insert 10 includes at least one of inlet channels 17a, b and outlet channels 18a, b positioned laterally relative to porous layer 12 such that perfusion of fluid through each of inlet channels 17a, b and outlet channels 18a, b causes fluid to flow through porous layer 12. In some embodiments, fluid is applied via a pressure pump and / or fluidic capacitor. In some embodiments, an oscillating pressure is applied to the fluid. This promotes filling and emptying of fluid in ex vivo skin tissue system 1, such as filling and emptying of vasculature in the tissue sample. In some embodiments, this is achieved via an air to fluid pressure pumping system. In some embodiments, this is achieved by varying hydrostatic pressure in ex vivo skin tissue system 1 through dynamic positioning of one or more of inlet channels 17a, b and outlet channels 18a, b and / or reservoirs connected thereto.

[0056] In some embodiments, insert 10 includes a top inlet 19 and a top outlet 20 for humidity control. In some embodiments, insert 10 includes a lip 101 along the outer edge of the open side of insert 10. In some embodiments, the outer surface of insert 10 includes indentations for mounting one or more O-rings, the one or more O-rings configured to seal insert 10 within a cartridge perfusion system.

[0057] 1C-1D show exemplary embodiments of an ex vivo skin tissue system 1 adapted to establish fluid communication with a perfusion solution. FIG. 1C shows the ex vivo skin tissue system 1 including a compartment for a biopsy of a skin tissue sample 11, a porous layer 12, a flow path 21 including an inlet 17a and an outlet 18a in the porous layer 12, a recirculation path 22 including an inlet 17b and an outlet 18b below the porous layer 12, and fittings 23, 24 for at least a first reservoir 25 and a second reservoir 26. FIG. 1D shows the ex vivo skin tissue system 1 including extensions of the flow path 21 and the recirculation path 22 into a reservoir container, such as a conical tube. In some embodiments, the flow path 21 and the recirculation path 22 further include one or more check valves 27 for controlling flow. FIGS. 1E-1G are photographic images of the embodiment of FIGS. 1C-1D , specifically, top, side, and perspective views, respectively, connected to a perfusion controller 30. In some embodiments, the perfusion controller 30 includes a user interface (UI) configured to send a pressure set point and duration to an air pump engaged with at least one of the first reservoir 25 and the second reservoir 26. The air pump may be configured to provide a vacuum or positive pressure to either the first reservoir 25 or the second reservoir 26, thereby causing fluid to flow through the ex vivo skin tissue system 1. In some embodiments, the perfusion controller 30 is configured to control one or more of a fluid perfusion rate, a fluid perfusion duration, a recirculation rate, and a recirculation frequency. In some embodiments, the perfusion controller 30 is configured to control, for example, an air perfusion rate and duration at the epidermal side of the tissue sample 11 and / or a positive pressure to stretch or distend the tissue sample 11.

[0058] 2A-2C show cross-sectional views of an insert 10 according to an exemplary embodiment of the present disclosure. FIG. 2A shows an embodiment in which multiple pores extend entirely along the inner wall 16 of the insert 10, while FIG. 2B shows an embodiment in which multiple pores extend partially along the inner wall 16 of the insert 10. The multiple pores along the inner wall 16 provide additional control over humidity within the insert 10. In some embodiments, the insert 10 is in a format that can be placed in an SBS microtiter plate, similar to a Transwell® insert. In some embodiments, the insert 10 is configured to be inserted into a cartridge, which can accommodate multiple inserts.

[0059] In some embodiments, insert 10 includes at least one standoff 28 along the second side of porous layer 12. Standoff 28 may allow medium to enter insert 10 from below porous layer 12, eliminating the need for inlet and outlet channels for perfusion. Standoff 28 may include one or more cylindrical stands below insert 10, as shown in FIG. 2B. FIG. 2C shows an embodiment in which insert 10 has an outer diameter of 10 mm. In some embodiments, insert 10 has an outer diameter in the range of 4 mm to 30 mm. In some embodiments, each insert includes a lip 101 along the perimeter of its top surface.

[0060] II. Workflow and System for Engineered Ex Vivo Skin Tissue Analysis In various aspects, the present disclosure provides methods for preparing and analyzing ex vivo skin tissue systems 1 with enhanced longevity. In vivo testing platforms are important for understanding complex biological processes, but are not easily reproducible and can be expensive when pursuing large data sets. Alternatively, in vitro testing platforms can be performed at significantly lower cost and in a more controlled environment when similar data volumes are sought, but are often less reflective of individualized outcomes. While both types of platforms are useful, ex vivo testing is arguably a better surrogate for in vivo human skin by often allowing normal skin barrier function and support for layers important for various desired skin tests, such as the epidermis (e.g., stratum corneum, lucidum, granular layer, spinous layer, basal layer, Langerhans cells, etc.), dermis (papilla and reticular), subcutaneous tissue (fat / adipose cells, blood vessels, bursae, etc.), and various skin appendages (e.g., sweat glands and hair follicles). However, tissue and cell viability rapidly declines after resection, and most contemporary ex vivo platforms lack the supportive capacity to ensure functionality, e.g., mechanical stability, immunocompetence, metabolic activity, and vascularization.

[0061] In contrast, the ex vivo tissue support system described herein is designed to address this unmet need. The ex vivo tissue support system is shaped to support a tissue sample, such as a tissue sample from a biopsy punch, and is structured to facilitate perfusion through the biopsy material, which better mimics tissue support in a living human. Furthermore, the ex vivo skin tissue system 1 described herein can be used as a platform to collect data that contributes to the DAW described herein by providing potential molecular, genomic, and / or proteomic profiles of skin tissue samples resulting from test agents, such as test agents in skin care products. The following process workflow can enable consistency and reliability in preparing the ex vivo skin tissue system 1.

[0062] Referring to FIG. 3A , an exemplary process workflow 300 for preparing a model system consistent with the present disclosure is provided. In some embodiments, the methods described herein include steps for preparing multiple model systems. In some embodiments, the methods described herein are as follows: step 301 includes receiving a tissue sample; step 302 includes preparing a biopsy from the tissue sample; step 303 includes assembling at least one of tissue and cells within the insert 10; step 304 includes setting / controlling environmental conditions; step 305 includes initiating and maintaining perfusion through the insert 10; step 306 includes culturing at least one of tissue and cells and / or monitoring at least one of tissue and cell conditions and environmental conditions; and step 307 includes collecting data for downstream analysis. In some embodiments, biopsy preparation is performed manually or using a hydraulic / pneumatic press. In some embodiments, the method includes monitoring the ex vivo skin tissue system 1, checking the tolerance of cell and tissue conditions, and the environmental conditions for monitoring include humidity, dryness, temperature, gas control, contamination, perfusion rate, UV, etc. In some embodiments, the clinical measurements of the cells and tissue include measurements of one or more of viability, vascularization, aging, viscoelasticity, differentiation, maturation, and overall complexity. In some embodiments, the environmental conditions include one or more of humidity / dryness, temperature, gas (e.g., CO2, O2, etc.) exposure and control, contamination, and light (e.g., ultraviolet (UV)) exposure. Environmental conditions are further discussed below with reference to Table 1.

[0063] FIG. 3B provides an overview of a method for assembling the ex vivo skin tissue system 1. In some embodiments, the methods described herein include a process workflow 400 including the following steps: Step 401 includes preparing an insert 10 having a porous layer 12. In some embodiments, the porous layer 12 has a first side and a second side, the first side facing one opening of the insert 10 and the second side facing the opposite direction of the opening of the insert 10. Step 402 includes positioning an epidermal and / or dermal biopsy within the insert 10. Step 403 includes optionally applying a sealing unit within the inner wall 16 of the insert 10, the inner wall 16 being adjacent to and perpendicular to the first side of the porous layer 12. The sealing unit enables sealing of the tissue sample within the insert 10. In some embodiments, the sealing unit includes a compression unit 14 having threads for mating with the inner wall 16 of the insert 10. In some embodiments, the sealing unit is an adhesive. In some embodiments, the adhesive is selected from medical super glue and UV-curable glue. Step 404 includes optionally positioning or seeding an endothelial cell layer within the insert 10, for example, within the porous structure of the insert 10. Step 404 may further include optionally positioning an adipose-derived cell layer within or beneath the porous structure of the insert 10. Step 405 includes optionally repeating steps 401-404 to assemble multiple inserts into a cartridge. Step 406 includes optionally sealing the bottom of each insert 10 with a permeable film. Step 406 includes initiating an experiment, embodiments of which are further described in detail herein.

[0064] In some embodiments, the methods described herein further include coating the insert 10 and the porous layer 12 with a gel layer. In some embodiments, the methods described herein further include contacting the second side with a gel layer, the gel layer configured to receive the seeded fat layer. In some embodiments, the gel layer comprises a synthetic gel material. In some embodiments, the gel layer comprises an extracellular matrix (ECM).

[0065] In some embodiments, monitoring at least one of the tissue and cellular conditions and the environmental condition includes the use of at least one sensor. In some embodiments, each of the at least one sensor includes a viscoelastic sensor, a wettability sensor, a pH sensor, an oil content sensor, a barrier function sensor, a pigmentation sensor, and other skin surface sensors. In some embodiments, the at least one sensor includes a corneometer, a transepidermal water loss (TEWL) sensor, a melanin sensor, and an ultrasound sensor. In some embodiments, sensors usable for detecting such signals may include, for example, optical sensors (e.g., imaging devices for detecting fluorescent or optical signals having various wavelengths and frequencies), potential sensors, surface plasmon resonance (SPR) sensors, interferometric sensors, or any other type of sensor suitable for detecting calorimetric, potentiometric, amperometric, optical, or piezoelectric signals.

[0066] In some embodiments, the medium comprises a composition adapted to maintain and / or promote a disease state. In some embodiments, the disease state can be maintained if the tissue sample contains diseased tissue, so that its phenotype is maintained as a control throughout the culture. In some embodiments, the disease state comprises at least one of inflammation, acne, dermatitis, psoriasis, rosacea, and eczema, hyperpigmentation, discoloration, and sun damage. In some embodiments, the disease state comprises at least one of fungal or bacterial infection, such as tinea pedis (athlete's foot), tinea cruris (jock itch), tinea corporis (tinea), cellulitis, erysipelas, and erythrasma. In some embodiments, the medium comprises a composition adapted to promote cell differentiation. In some embodiments, the medium comprises a composition adapted to promote tissue complexity. In some embodiments, the medium comprises components and supplements, including at least one of growth factors, vitamins, hormones, trace elements, and other micronutrients to support cell types for extended periods of time. In some embodiments, the medium comprises a composition to promote microbiota growth.

[0067] Once skin tissue has developed, maintained, and been properly validated using measurements of its progression within the system, condition and effect data of various test agents can be collected and summed for contribution within the DAW.

[0068] III. Porous Layer for Enhanced Perfusion In various embodiments, the present disclosure provides a porous layer 12 that can improve the development and longevity of skin tissue samples during the course of an experiment when collecting data for a DAW. In particular, the porous layer 12 described herein can facilitate multiple benefits, such as increasing adhesion between cells and / or tissue and the insert 10, maintaining the barrier function of the tissue sample, increasing the mass transport of one or more of perfused fluid, nutrients, waste products, and / or oxygen to or from the tissue sample, and promoting vascularization of the tissue sample within the ex vivo tissue support system. The porous layer 12 can further provide a platform for direct seeding of cells during perfusion through the ex vivo tissue support system.

[0069] 4A-4D illustrate exemplary embodiments of porous layer 12 according to aspects of the present disclosure. FIG. 4A shows a cross-sectional view of a 3D rendering of insert 10 having porous layer 12. In some embodiments, the inner surface of wall 16 perpendicular to the first side of porous layer 12 includes a plurality of pores. In some embodiments, each of the plurality of pores is a gyroid. In some embodiments, the plurality of pores penetrates wall 16 to a desired thickness. In some embodiments, each of the plurality of pores is a microchannel. In some embodiments, the gyroid includes a 1 mm unit cell. Gyroids are commonly known in the art as structures lacking straight lines and including triply periodic minimal surfaces. Gyroids are further known to be characterized by zero mean curvature accompanied by local area minimization. In some embodiments, the gyroid is a gyroid infill. Gyroid infill occurs when a gyroid pattern is used as the internal geometry of a structure. The gyroid promotes filling of the porous layer 12 and creates a pressure gradient for vascular sprouting into hypoxic regions of the ex vivo tissue support system, such as for anastomosis or angioplasty. The porous layer 12 is configured to increase mass transport by approximately 70-fold compared to typical track-etched membranes, which can further increase during perfusion. In some embodiments, the porosity of the porous layer 12 ranges from 40% to 90%. In some embodiments, the porosity of the porous layer 12 is 45%, as shown in FIG. 4B. In some embodiments, the porosity of the porous layer 12 is 60%, as shown in FIG. 4C. In some embodiments, at least one of the insert 10 and the porous layer 12 comprises a porosity gradient. FIG. 4D provides a top view of the porous layer 12 with a porosity gradient according to aspects of the present disclosure. In some embodiments, the porosity gradient comprises a plurality of pores. In some embodiments, the porosity gradient comprises a lower density of pores in the center of the porous layer 12 compared to the outer boundary of the porous layer 12. In some embodiments, the porous layer 12 comprises microneedles. In some embodiments, the porous layer 12 comprises porous or hollow microneedles.

[0070] 4E-4H, in some embodiments, the porous layer 12 comprises microneedles, as shown in FIG. 4E. In some embodiments, the microneedles are connected by a series of microchannels, as shown in FIG. 4F, providing hollow microneedles in fluid communication with the perfusable microchannels. In some embodiments, the microneedles are hollow, as shown in FIG. 4G. In some embodiments, the microneedles are solid, as shown in FIG. 4H. In some embodiments, the porous layer 12 comprises a mesh structure, as shown in FIGS. 4I-4J.

[0071] IV. Samples, Cells and Tissues In various aspects, the present disclosure provides insert cartridges and devices for ex vivo skin tissue systems that are adaptable to various tissues and cell types, which can be arranged in a manner that optimizes the tissue model for accuracy and reliability of measurements obtained. For example, tissues and cell types can be selected and refined to promote skin functionality, cell-cell interactions, regenerative capacity, immunocompetence, metabolic activity, mechanical properties, and vascularization, among other important characteristics of the model tissue platform. In some embodiments, the methods described herein include contacting a tissue sample onto a first side of a porous layer. In some embodiments, the tissue sample is reengineered skin tissue. In some embodiments, the tissue sample is natural skin tissue. In some embodiments, the tissue sample is synthetic skin tissue. In some embodiments, the source of the tissue sample can be a human, a non-human primate, or a non-human mammal, including, without limitation, a dog, cat, sheep, horse, pig, rabbit, mouse, rat, goat, llama, duck, chicken, or turkey. In some embodiments, the skin tissue is a biopsy, for example, from a biopsy punch. In some embodiments, the skin tissue is reconstructed three-dimensional (3D) skin having a single or multiple cell types. In some embodiments, the skin tissue includes at least one of the epidermis, dermis, and subcutaneous tissue. In some embodiments, the skin tissue includes cells and ECM components from the vasculature and subcutaneous tissue. In some embodiments, the skin tissue includes one or more of hair follicles, nails, sweat glands, and sebaceous glands. In some embodiments, the skin tissue has a diameter of 20 mm. In some embodiments, the skin tissue has a specific skin type, such as dry, oily, sensitive, and / or a combination thereof. In some embodiments, the skin tissue has a specific skin condition, such as acne, eczema, psoriasis, dermatitis, rosacea, hyperpigmentation, discoloration, sun damage, and / or a combination thereof. In some embodiments, the skin tissue is derived from individuals varying in one or more of age, ethnicity, and gender. In some embodiments, the skin tissue may vary by body region.

[0072] In some embodiments, the methods described herein include seeding cells onto at least one of a first side of the porous layer, an interior region of the porous layer, and a second side of the porous layer. In some embodiments, the seeding is performed using bioprinting, which may include, for example, 3D printing methods. In some embodiments, the seeding is performed using perfusion. In some embodiments, the seeding is performed manually by a user. In some embodiments, the seeded cells are endothelial cells. In some embodiments, the seeded cells are primary cells or immortalized cells. In some embodiments, the seeded cells are induced pluripotent stem cells (iPSCs). In some embodiments, the seeded cells are primary cells from a donor different from the donor of the tissue sample. In some embodiments, the seeded cells are isolated cells from the same donor as the donor in the tissue sample.

[0073] In some embodiments, 3D printing is performed using stereolithography. In some embodiments, bioprinting is performed using digital light processing (DLP) printing. In the case of 3D printing, cells are seeded onto the structure after printing. In the case of bioprinting, cells can be incorporated into the porous structure or can be seeded later. One difference between 3D printing and bioprinting is the materials used. For example, bioprinting allows for the production of porous structures using gel materials such as collagen, gelatin, hyaluronic acid, PEG, etc. These materials are likely to be more biocompatible than plastics produced using 3D printing.

[0074] In some embodiments, the methods described herein include depositing an adipose layer on one or more of the first side of the porous layer, the interior region of the porous layer, and the second side of the porous layer. In some embodiments, the adipose layer comprises one or more of adipose-derived stem cells, adipocytes, and preadipocytes. In some embodiments, the adipose layer comprises one or more of cells isolated from fragmented tissue or adipose tissue. In some embodiments, the adipose layer comprises reconstructed adipose tissue, the reconstructed adipose tissue constructed via one or more of (1) bioprinting and 3D culturing of adipose-derived cells, and then (2) integrating the adipose layer with a skin tissue layer, the skin tissue comprising the epidermis, dermis, and subcutaneous tissue. In some embodiments, the adipose layer is obtained from the same donor as the tissue sample. The methods described herein further include sealing the adipose layer with a gas-permeable film. In some embodiments, the film is an adhesive film. In some embodiments, the gas-permeable film is medical-grade. In some embodiments, the gas-permeable film is a polyurethane adhesive. In some embodiments, the gas permeable film is a polyurethane acrylic adhesive. In some embodiments, the gas permeable film is Saniderm®. In some embodiments, an adipose layer may be added within the porous layer. In some embodiments, an adipose layer may be added below the porous layer. In such embodiments, the adipose layer may be held in place due to its negative buoyancy. The methods described herein further include culturing at least one of the tissue sample and the adipose layer. In some embodiments, the culturing is for a period of at least one week. In some embodiments, the culturing is for a period of at least two weeks.

[0075] In some embodiments, the methods described herein further include seeding a cell composition into the porous layer between the first and second sides. In some embodiments, the seeded cell composition comprises at least one of endothelial cells, primary cells, iPSCs, keratinocytes, fibroblasts, melanocytes, resident immune cells, circulating immune cells, stem cells, preadipocytes, and adipocytes. In some embodiments, the resident immune cells comprise at least one of Langerhans cells, dendritic cells, macrophages, mast cells, monocytes, and lymphocytes. In some embodiments, the circulating immune cells comprise at least one of neutrophils, T lymphocytes, B lymphocytes, natural killer cells, monocytes, and dendritic cells. In some embodiments, the stem cells comprise at least one of adipose-derived stem cells, epidermal stem cells, and dermal stem cells. In some embodiments, the stem cells are isolated from a tissue sample. In some embodiments, the stem cells are configured to provide regenerative capacity to the skin sample. The regenerative capacity of stem cells is beneficial for further extending the lifespan and health of the tissue sample. In some embodiments, the seeded cell composition comprises one or more of 200,000 to about 2,000,000 adipose-derived stem cells per mL and 500,000 to about 10,000,000 dermal endothelial cells per mL. In some embodiments, the cells may be from a resident microbiome maintained on or on the surface of tissue, e.g., from donor skin tissue. In some embodiments, cells from the resident microbiome may be microorganisms from the skin microbiota. In some embodiments, probiotics may be used as single agents or co-treatment agents in ex vivo skin model systems. In some embodiments, the microbiome may be sourced depending on the cell type, donor age, skin region, etc. In some embodiments, microorganisms from the skin microbiota may be evaluated as potential therapeutic agents.

[0076] In some embodiments, the culturing step comprises perfusing a reagent through the porous structure, the reagent comprising at least one of Williams E medium, Glutamax, non-essential amino acid solution, ITS (insulin, transferrin, selenium, BSA, and linoleic acid), hydrocortisone, vitamin C, epinephrine, serum, blood substitute, penicillin, and streptomycin.

[0077] 5A-5B provide cross-sectional and perspective views, respectively, of an exemplary embodiment of compression unit 14. In some embodiments, compression unit 14 includes a microchannel 142 that interfaces with tissue so that a user can seal compression unit 14 to the tissue. In some embodiments, compression unit 14 includes an injection port 143 in fluid communication with microchannel 142. Using this embodiment, a user can clamp compression unit 14 against skin tissue sample 11 and then inject a liquid medical-grade adhesive into port 143, which enters microchannel 142 and seals the interface between tissue sample 11 and compression unit 14, allowing an air-liquid interface to be maintained.

[0078] V. High Content Model System As noted above, the ex vivo skin tissue support system can be in the form of alternative embodiments for a variety of different purposes. In some embodiments, for example, the insert 10 is conditioned to form a high-content model (HCM) system. As further described herein, HCM can include an ex vivo tissue model with a complexity and supportive environment that more closely mimics that experienced by in vivo human tissue in terms of functionality and immunocompetence.

[0079] HCM may include full-thickness skin biopsies with the incorporation of engineered vasculature and / or adipose tissue sourced from the same donor. HCM may also include modifications to the surrounding environment to increase longevity and maintain complexity, such as by providing nutrients that support vasculature growth and maintenance. As a result, upon application of one or more test compounds or biologics, HCM may be more accurate in mimicking tissue responses in living humans. Thus, HCM may also be used to detect numerous efficacy claims, complex biological responses, and mechanisms using multiple "high-content" readouts. The embodiments described herein may be further designed for compatibility with commercially available automation and analytical tools. Furthermore, HCM may serve as a long-lasting, authentic human skin-based model that can be used as a correlate for product consumer studies.

[0080] In some embodiments, the HCM described herein may include one or more of the following features: (1) full-thickness skin biopsies, (2) incorporation of engineered vasculature and adipose tissue from the same skin donor, (3) modification of the surrounding environment to increase longevity and maintain complexity, and (4) the ability to detect various efficacy claims, complex biological responses, and mechanisms using multiple high-content readouts. In some embodiments, the HCM system includes a recirculation system. Figures 6A-6D show various views of the recirculation system 6 according to some embodiments of the present disclosure. The HCM incorporates ex vivo tissue models into the recirculation system 6 for continuous perfusion by adapting the inlet and outlet channels to commercially available automation and analysis tools, including autosamplers. Furthermore, the recirculation system 6 enables continuous perfusion without the need for multiple reservoirs per sample or large reservoirs that may be required for a single flow pass through the sample. Figure 6A shows a cross-sectional view of the recirculation system 6. In some embodiments, the recirculation system 6 includes a recirculation cartridge 60, at least one insert 61, which can be any one of the inserts described herein for holding a tissue sample, e.g., tissue sample 11, an insert cap 62 for sealing the insert 61, a medium reservoir 63 for enabling medium recirculation into the insert 61, and multiple check valves 64 (e.g., two check valves), a reservoir cap 65, and a fluidic capacitor 66. The fluidic capacitor 66 can provide pressure for several hours with a short pressure charge, eliminating the need for a waste reservoir. The fluidic capacitor 66 can remain pressurized with ethanol or water for multiple days without leakage. Air bubbles rise to the top of the membrane 661 and are pushed out over time, making the membrane 661 also an effective bubble trap. Figure 6B shows a detailed cross-sectional view of the reservoir cap 64, which includes an integrated check valve 64, to enable fluid transfer between the medium reservoir 63 and the insert 61. Figure 6C shows a detailed cross-sectional view of the fluidic capacitor. The fluidic capacitor 66 allows for pressure-based perfusion of the system with short pulses from a pressure-generating device.One advantage is that multiple perfusion circuits can be controlled with a single air pump. When used in combination with two check valves 64, this can enable fluid recirculation using only a single reservoir. In some embodiments, a fluidic capacitor 66 includes at least one of a membrane 661, a fluid inlet 662, and a fluid outlet 663. The fluidic capacitor 66 is configured to provide pressure for several hours with a short pressure charge, eliminating the need for a waste reservoir. Air bubbles rise to the top of the membrane 661. Figure 6D provides a perspective view of the recirculation system 6, and Figure 6E provides a perspective view of a cartridge with multiple recirculation systems 6. In some embodiments, the recirculation systems 6 are configured to replicate the model system in HCM. In some embodiments, the HCM incorporates tissue samples containing vasculature and adipose tissue from the same donor. Benefits of high-content models include increased longevity, preservation of tissue sample complexity, and adaptation for use with authentic human skin samples containing varying skin cells, appendages, and structures. In some embodiments, the recirculation system 6 is adapted to be compatible with commercially available automation and analysis tools.

[0081] VI. High-Throughput Model Systems In various aspects, the present disclosure provides alternative embodiments to the HCM, such as high-throughput cartridge (HTC) systems. In contrast to the HCM, the HTC can include ex vivo tissue models, such as full- or partial-thickness tissue biopsies, while only being used to measure critical or selected toxicity and efficacy outcomes. The HTC embodiments described herein can be compatible with SBS microtiter plate formats and can provide at least 5x higher throughput than off-the-shelf biopsy models. Furthermore, low-humidity air can be applied to the HTC via the lid, so that a stronger humidity gradient can be created across the tissue for increased media transport.

[0082] 7A-7B, perspective and cross-sectional views, respectively, of a high-throughput cartridge (HTC) 7 are shown. The HTC 7 is generally intended for use in high-throughput screening of natural and / or synthetic component libraries. In some embodiments, the HTC 7 contains at least fibroblasts and keratinocytes. In some embodiments, each of a plurality of inserts 70 is inserted into one of a plurality of slots in the HTC 7. In some embodiments, the plurality of slots in the HTC 7 are arranged in a 96-well configuration. In some embodiments, the HTC 7 includes a lid 71. In some embodiments, the lid 71 has fluid channels for perfusing air over the tissue sample. In some embodiments, each insert in the HTC includes a basal medium well 72. The HTC 7 offers the benefits of high-throughput analysis and screening of tissue samples in complex systems.

[0083] HTC 7 can be configured in a "hanging drop" style, maintaining an air-liquid interface and transporting nutrients and waste products through a basal interface, as shown in Figures 7C-7G. In some embodiments, as shown in Figure 7C, there are reservoirs on either side of via 73, e.g., drip reservoir 74 and basal reservoir 75, to form a dual-reservoir system. By having reservoirs on both sides and adding fibroblast 3D culture, keratinocytes can be cultured in submerged culture for initial proliferation. The dual-reservoir system also allows HTC 7 to be rotated 90 degrees during epidermal maturation so that the fluid in the reservoirs does not exert significant hydrostatic pressure on the gel droplet in drip reservoir 74. For example, gel droplets containing fibroblasts can be generated and polymerized in the drip reservoir. Keratinocytes can be seeded outside the gel droplets, the system can be cultured in submerged conditions for initial growth, and medium can be removed from the dripping reservoir 74 and maintained on the basal reservoir 75, establishing an air-liquid interface. In some embodiments, the vias 73 have a larger diameter to increase the surface area between the dripping reservoir 74 and the basal reservoir 75. In some embodiments, a mixture of medium, gel, and cells is placed in the basal reservoir 75, and the mixture is transported by capillary action through the vias 73 to form gel droplets on the other side. The fixed geometry of the vias 73 and the basal reservoir 75 maintains the formed droplets in a stable and consistent geometry. The dripping reservoir 74 and the basal reservoir 75 are not symmetrical, so that the dripping reservoir 74 has specific fixed features for forming gel droplets. Once the gel in the gel droplet is crosslinked (via temperature, pH change, or UV crosslinking), a layer of keratinocytes can be seeded onto the surface of the gel droplet. In some embodiments, via 73 includes a porous layer 76, as shown in Figures 7D-7E. In some embodiments, the via has a smaller diameter to reduce the surface area between the drip reservoir 74 and the basal reservoir 75, as shown in Figure 7F. In some embodiments, via 73 has straight interior walls to improve imaging capabilities.

[0084] Figure 7G provides a close-up view of a single insert in an HTC system according to some embodiments of the present disclosure. Figure 7G is labeled with illustrative geometry throughout the insert, including the diameters of the drip reservoir, vias at the proximal end, center point, and distal end, as well as the basal reservoir. The geometry is designed to function regardless of orientation to allow for inversion of the system during cell seeding, differentiation, and maturation. This is provided by allowing the Eötvös number (Eo), also known as the Bond number (e.g., Bo), to be <1, as provided by the following formula:

[0085]

number

[0086] VII. Data Analysis Workflow (DAW) Data obtained from monitoring the ex vivo tissue support system described herein is incorporated for use in a data analysis workflow ("DAW"). In some embodiments, the DAW described herein includes a trained algorithm for predicting performance or assay results based on input characteristics, e.g., physiochemical, structural, or taxonomic properties, of a composition, compound, or biologic of interest, also referred to herein as a test agent. Test agents can be evaluated using a skin tissue sample or skin tissue array incorporated into the ex vivo tissue support system described herein, also referred to herein as an ex vivo tissue support system. In some aspects, the ex vivo tissue support system includes an insert including at least one opening and a wall; a sealing unit along the inner wall of the insert; a tissue sample, such as a skin tissue sample, positioned inside the insert via the sealing unit; and a porous layer positioned inside the insert, the porous layer configured to promote mass transport into the tissue sample and vascularization of the tissue sample. In some aspects, the porous layer has a first side facing the at least one opening of the insert and a second side facing the opposite side of the at least one opening. In some embodiments, the ex vivo dermal tissue support system is capable of supporting tissue viability for a longer period of time than previously reported such tissue support systems, for example, for more than one week or for more than two weeks. More details regarding the ex vivo dermal tissue support system are described herein.

[0087] In some embodiments, the present invention provides a method for skin tissue analysis, which may include a method for selecting a therapeutic agent.In some embodiments, the method includes: (a) contacting a skin tissue array with at least one test agent; (b) measuring a plurality of data sets after contacting the skin tissue array with the test agent; (c) mapping the measured plurality of data sets based on the scoring panel results; and (d) applying a machine learning algorithm to the mapped data sets to generate at least one threshold criterion for selecting a therapeutic agent from at least one test agent based on the mapped data.In some embodiments, the method forms an extraction-mapping-application workflow for simplifying the experiment of skin tissue samples or skin tissue sample arrays with test agents.

[0088] Referring to FIG. 8A, a diagram illustrating the data extraction workflow prior to executing the extraction-mapping-application workflow is provided for visualization purposes. In some aspects, the data extraction workflow includes applying multiple inputs to a skin tissue sample in an ex vivo tissue model obtained or cultured from the ex vivo tissue support system described herein, such as one or more of a high-content model (HCM) and a high-throughput cartridge (HTC) model, described in further detail below. In some aspects, the multiple inputs include one or more of the type of protocol used (cell-dependent and / or tissue-dependent), treatment (e.g., test agent), reagent used, and cells and / or tissue used. In some embodiments, non-limiting examples of substances required for cellular metabolism testing include transferrin, human serum albumin, putrescine, ethanolamine, carnitine, linoleic acid, and linolenic acid. In some embodiments, non-limiting examples of reducing agents include vitamin C, sodium selenate, sodium pyruvate, and glutathione. In some embodiments, non-limiting examples of antioxidants include at least one antioxidant selected from the group consisting of glutathione (reduced), dithiothreitol (DTT), vitamin E, vitamin K3, vitamin D2, or calciferol, niacin, niacinamide, and ascorbic acid. In some embodiments, non-limiting examples of reagents for lipid synthesis include one or more of cholesterol, linoleic acid, lipoic acid, and o-phosphorylethanolamine. In some embodiments, one or more reagents for hormone testing include one or more of progesterone, testosterone, hydrocortisone, and estrogen. In some embodiments, one or more reagents as growth factors include one or more of insulin and epidermal growth factor (EGF). In some embodiments, the reagents may also optionally contain antibiotics, such as penicillin and / or streptomycin.In some embodiments, the reagents used include Williams E medium, Glutamax, non-essential amino acid solution, insulin, transferrin, selenite, BSA, linoleic acid, hydrocortisone, vitamin C, serum, blood substitute, penicillin and / or streptomycin. In some embodiments, the reagents used include Williams E medium, Glutamax, EGF, transferrin, insulin, progesterone, testosterone, 17B-estradiol, o-phosphorylethanolamine, selenite, linoleic acid, BSA, triiodothyronine, hydrocortisone, cholera toxin, HEPES, and other components.

[0089] In some embodiments, data extraction involves contacting the ex vivo tissue model with a test composition, compound, or biologic. The contacting may be performed by one or more of topical application, subcutaneous application, systemic application, intradermal administration, infusion, perfusion, and injection. After contacting, data extraction may involve performing one or more in vitro assays to obtain corresponding clinical measurements, including, but not limited to, one or more of niacinamide, retinol, hyaluronic acid, glycolic acid, and ceramide. In some embodiments, the data includes measurements from one or more of skin surface, histological staining, immunostaining, biochemical assays, clinical sensors, and gene expression. As described herein, data generated from in vitro assays is used as input for machine learning to iteratively generate optimal algorithms for one or more of: verifying and generating product claims for skin concerns; conducting broad efficacy and / or toxicity screening of chemical libraries for quantitative comparison to product benchmarks; and performing virtual screening of therapeutic agents.

[0090] In some embodiments, the skin tissue array comprises a compartment for skin tissue growth, including an epidermal and / or dermal tissue layer, a porous layer, and an adipose tissue layer. In some embodiments, the porous layer comprises a structure with sufficient openings for vascularization from the adipose tissue layer into the epidermal and / or dermal tissue layer. In some embodiments, the skin tissue array is viable for a period of more than one week. In some embodiments, the skin tissue array is viable for a period of at least two weeks. In some embodiments, the scoring panel result is calculated according to the strength and effectiveness of each measurement value of the plurality of data sets measured as described above. In some embodiments, each of the at least one threshold criterion corresponds to at least one biological pathway.

[0091] In some embodiments, the extract-map-apply workflow can be used to identify structure-activity relationships and relationships, such as quantitative structure-activity relationships (QSAR), quantitative structure-property relationships (QSPR), structure-affinity relationships (SAFIR), etc., which can be used to significantly accelerate the therapeutic agent selection process. The therapeutic agent selection process disclosed herein implements an intelligent and data-enabled discovery process to optimize the selection of therapeutic agents based on the applied test agents. The therapeutic agent selection process can take advantage of recent advances in high-throughput therapeutic agent selection to rapidly search through a diverse parameter space in the extract-map-apply workflow cycle of experiments to provide agent-specific and robust therapeutic agent characteristics that provide the most stable and predictable behavior. Machine learning can be used to analyze the data to automatically search and identify structure-activity relationships and relationships.

[0092] 8B-8C provide diagrams illustrating example extraction-mapping-application workflows for data extraction, data mapping, and assertion quantification of test agents according to one or more embodiments of the present disclosure. In some embodiments of the present disclosure, data extraction may include data from one or more of the following sources: histological staining, immunostaining, biochemical assays, clinical sensors, skin surface analysis, and gene expression. In some embodiments of the present disclosure, data mapping may include one or more of epidermal differentiation, matrix density, epidermal thickness, barrier function, skin surface, dermal-dermal junction, dermal papillary thickness and projection, stem cell and regenerative activity, adipogenesis, melanogenesis, sagging pathway, wrinkling pathway, cellular senescence, cellular stress, inflammation, and apoptosis. In some embodiments of the present disclosure, assertion quantification may include analysis of one or more of anti-aging, hydration, toxicity, and irritation.

[0093] In some embodiments, the extraction-mapping-application workflow can be validated for one or more representative test agents, such as niacinamide, retinol, hyaluronic acid, glycolic acid, and ceramide. For each test agent, structure-activity relationship and relational machine learning models can be developed and validated with each new data set. These models can be utilized in subsequent de novo designs to create iterative workflows (e.g., the extraction-mapping-application workflows of Figures 8B-8C) once the accuracy of each test agent-specific model reaches or exceeds a threshold accuracy.

[0094] In some embodiments, the method for testing compositions includes selecting a therapeutic agent. In some embodiments, the method for testing compositions uses big data and diverse datasets, for example, generated from ex vivo tissue support systems and processed using the machine learning algorithms described herein, to substantiate and generate evidence-based skin care product claims for skin concerns, to conduct extensive efficacy and / or toxicity screening of chemical libraries for quantitative comparison to product benchmarks, and to train artificial intelligence (AI) models to perform virtual screening. In some embodiments, the structural prediction method described herein can use machine learning and computational intelligence techniques, such as deep neural networks, and a combination of supervised, semi-supervised, and unsupervised learning techniques. In some embodiments, the method for selecting therapeutic agents described herein uses supervised algorithms (non-limiting examples include linear region, random forest classification, decision tree learning, ensemble learning, bootstrap aggregation, etc.). In some embodiments, the method for selecting therapeutic agents described herein uses unsupervised algorithms (non-limiting examples include clustering or association). Iteration using machine learning using the methods described herein allows for robust simulations beyond the capabilities of singular data mode alone, which allows for much more predictive (and biologically relevant) algorithms to yield beneficial therapeutic agent selection.

[0095] The methods and systems described herein can utilize supervised machine learning models to uncover structure-activity relationships and relationships, for example, to develop therapeutic agent selection criteria. This can be achieved by iteratively applying an extract-map-apply workflow by implementing a robust, intelligent, high-throughput process, as described herein below. This workflow can utilize a diverse range of test agent features in conjunction with machine learning models to rank variable dependencies to reveal structure-function relationships that may otherwise be difficult to determine using hit- or miss-type rational design alone.

[0096] In some embodiments, the datasets used to train the machine learning algorithm include (a) a database filled with datasets generated from tests using one or more of the HTC and HCM systems, and (b) existing public databases, such as one or more of ChemSkin, SkinSensDB, PubChem, ClinicalTrial.gov, ChEMBL, OpenFDA, DrugBank, Binding DB, NCBI GEO, GO, Human Cell Atlas, and COCONUT. Depending on the test agent, the datasets may be sourced from in vivo or in vitro data, such as artificial skin systems, in vivo skin tissues, in vitro skin tissue models, laboratory-grown full-thickness skin systems, and / or other ex vivo skin models. In some aspects, model data from such databases may be continuously validated against the mapping of the test agent extraction-mapping-application workflow to extract structure-activity relationships and relationships.

[0097] In some embodiments, measurements generated from the skin surface include measurements from one or more of dermoscopy, megurometer, cutometer, and photography. In some embodiments, measurements generated from histological stains include measurements from one or more of hematoxylin and eosin (H&E), Masson's trichrome, and Movat pentachrome.

[0098] In some embodiments, measurements generated from gene expression include measurements from one or more of dermal differentiation, dermal-epidermal junction, dermal markers, matrix metalloproteinases (MMPs), MMP inhibitors, regeneration, wrinkles, sagging, inflammation, intrinsic apoptosis, extrinsic apoptosis, anti-apoptosis, immune cell markers, skin elasticity, skin rejuvenation, and antioxidant defense. In some embodiments, measurements generated from clinical sensors include measurements from one or more of moisture, oil, pH, dermoscopy, photography, melanin content, and ultrasound.

[0099] The clinical measurements obtained during data extraction can then be pooled together in a database for one or more of algorithm training and / or mapping. In some embodiments, the database can be constructed in such a way that data can be fed into the training set for machine learning algorithms with minimal user interaction. In some embodiments, mapping includes capturing one or more features, such as epidermal differentiation, epidermal thickness, epidermal-dermal junction, barrier function, cellular stress, cellular senescence, dermal markers, MMPs, MMP inhibitors, regeneration, wrinkles, sagging, inflammation, intrinsic apoptosis, extrinsic apoptosis, anti-apoptosis, matrix density, dermal papilla thickness and projection, stem cell and regenerative activity, adipogenesis, melanogenesis, immune cell markers, skin elasticity, skin rejuvenation, and antioxidant defense.

[0100] The DAW may use one or more of the following as input data obtained from one or more of HCM and HTC, which is a set for improved predictive algorithms compared to others using specific data modes: skin surface imaging, histological staining, immunostaining, biochemical assays, transcriptomics, proteomics, biochemical data, and clinical sensor data.

[0101] In some embodiments, the monitoring comprises: (a) contacting the ex vivo tissue model with a test composition, compound, or biological agent; (b) generating a gene or protein expression / pathway map for the ex vivo tissue model, wherein the gene or protein expression / pathway map comprises data related to the transcription of one or more genes; (c) comparing the gene or protein expression / pathway map for the ex vivo tissue model with a control gene or protein expression / pathway map; and (d) identifying the test composition, compound, or biological agent as effective for providing a feature, benefit, or function to the ex vivo tissue model if the gene or protein expression / pathway map for the ex vivo tissue model and the control gene or protein expression / pathway map are consistent. Each of the gene or protein expression / pathway maps can be obtained via one or more of bulk RNA-seq and microarrays.Each gene or protein expression / pathway map includes TJP1, OCLN, KRT(1,2,5,9,10,14), EGFR, TGM(1,3,5), DSP, LORICRIN, FLG, CASP14, CLDN1, IVL, ITGB(1,4,5), LAMA(4,5), LAMC1, COL4A1, COL5A1, COL1A1, COL3A1, COL17A1, COL7A1, COL9A1 , VCAN, VIM, COL12A1, FBN1, ACTA2, THY1, fN1, FAP, HAS2, TGFB1, HNF1A-AS1, SMIM6, HPSE, EMILIN1, MMP(1, 2, 3, 7, 9-17, 19-21, 23B, 24-26, 28), TIMP(1-4), FGF(2, 7, 9), LMN81, MK167, KRT(15, 19), TP63, SOK2, MNAT1, P SMA1, PSMD2, PSMC2, XPC, PSMB(3, 5), DOB1, PMM2, POLE4, GMDS, CYBRK1, VEGFA, CMAS, RAD23B, RPTOR, TSC1, DD B1, CAB39, RPS6KA2, PIK3R1, PIK3CA, GFPT2, ERCCB, TNF, IL35G, IL12A, IL1B, S100A7, IL33, CXCLB, IL6, ATM, The pathway maps may include expression datasets from one or more genes or proteins selected from DFFB, MDM2, TPS3, BAD, BAX, DBC3, AJFM1, CHEK2, PMASP1, BID, CASP(3, 7-9), BCL2, BMF, TNFSF10, TRADD, TNFRSF1A, FADD, BCL2, BCL2A1, BCL2L1, MCL1, BCL2L10, BCL2L2. Each of the gene or protein pathway maps may include pathway datasets from one or more of epidermal development, peptide cross-linking, retinoic acid metabolism, keratinocyte differentiation, flavonoid glucuronidation, xenobiotic glucuronidation, and epidermal cell differentiation.

[0102] In some embodiments, one or more selection criteria are positively or negatively correlated with the condition.The selection criteria include at least one threshold for quantifying data.In some embodiments, the condition includes one or more of anti-aging, skin moisturizing, toxicity, brightening, discoloration, pigmentation, UV protection, cleansing, permeability, inflammation, anti-inflammatory, antibacterial, wound healing, skin barrier, epidermal thickness, dermal matrix, cell stress, cell aging, sagging, and wrinkles.

[0103] In some embodiments, the input data for skin surface imaging includes a scoring result when comparing an image of an untreated skin surface with a treated skin surface, where the treated skin surface is treated with a test composition described herein. In some embodiments, the transcriptomics data includes gene expression data for one or more of the characteristics described herein. In some embodiments, the input data includes pathology scoring for one or more of the following conditions: parakeratosis, hyperkeratosis, acanthosis, dyskeratosis, keratinocytes, spongiosis, ballooning, edema, and follicles. In some embodiments, the input data may be from cells associated with the microbiome, which may be useful for correlating skin data with test agents used for one or more of maintaining, restoring, and activating the microbiota.

[0104] In some embodiments, measurements of test agents from any of the databases can be mapped as shown in Figures 8B-8C. These measurements can be compared to the mappings for each feature from all machine learning models to establish structure-activity relationships and associations. The generated mappings can be highly quantitative and systematic, capturing different strengths and potencies using the scoring system described herein, thereby providing potential insight into multiple functionalities. Finally, the best-performing test agents can be further characterized by one or more of the above conditions.

[0105] VIII. Data Mapping and Analysis In various embodiments, the present disclosure provides a method for mapping data from ex vivo tissue models, using various standard techniques and assay panels, and analyzing the obtained readings for reliable comparison and / or ranking of test compounds and biological agents, for example, through scoring, to extract specific features and / or characteristics when contacted with at least one test compound or biological agent.The methods described herein can be used to provide validation of ex vivo tissue models with known active ingredients (for example, cosmetics, natural ingredients, hair care, plant extracts / oils, sun / light protection, fruit acids, vitamins / proteins, etc.) used in skin care testing, thus setting the basis for selecting the most sensitive measurement readings, normalizing measurement scales, evaluating toxicity, and selecting safe therapeutic ranges for active ingredients, and benchmarking data for comparing new ingredients.

[0106] In some embodiments, the methods described herein include monitoring at least one of cellular and tissue characteristics, including at least one of efficacy, toxicity, pharmacodynamics, and longevity of the tissue sample, to contribute to the DAW. The cellular and tissue characteristics provide the benefit of generating large amounts of data for downstream analysis, rather than relying solely on existing databases. In some embodiments, the monitoring step is performed in response to a test composition, compound, or biologic applied to the ex vivo tissue model within the ex vivo tissue support system. In some embodiments, the ex vivo tissue support system includes an HCM or HTC system, as described in more detail below. The ex vivo tissue model can be at any stage of development or growth within the ex vivo tissue support system.

[0107] In some embodiments, monitoring at least one of the cellular and tissue characteristics includes monitoring at least one of skin structure, anti-aging, skin moisturization, brightening, discoloration, pigmentation, UV protection, cleansing, permeability, inflammation, anti-inflammatory, antibacterial, wound healing, skin barrier, epidermal thickness, dermal matrix, cellular stress, cellular senescence, sagging, wrinkles, sensitization, irritation, erosion, and phototoxicity. Table 1 provides a summary of exemplary characteristics. In some embodiments, at least one of the cellular and tissue characteristics is measured when stimulated with an active ingredient. Table 2 provides exemplary active ingredients used to benchmark results from the ex vivo tissue support system described herein. In some embodiments, monitoring includes using at least one of staining and imaging, gene expression, sensing, dermoscopy, and ultrasound. In some embodiments, the monitoring step includes ex vivo assays, which include at least one of hematoxylin and eosin (H&E), other histology and immunostaining (e.g., immunofluorescence (IF)), biochemistry and immunoassays, and gene expression analysis.In some embodiments, the monitoring step comprises measuring at least one of dermal markers, such as fibrillin, vimentin, collagen I, collagen III, and elastin; epidermal-dermal junction markers, such as integrin B4, laminin 5, and collagen VII; epidermal differentiation markers, such as filaggrin, trichrome, involucrin, transglutaminase, and cytokeratin; lipid markers, such as sebaceous lipids, epidermal lipids, such as ceramides, cerebrosides, and phospholipids; epidermal extracellular matrix components, such as glycosaminoglycans (GAGs) and hyaluronic acid, proteoglycans, ECM receptors, and proteases; markers of epidermal adhesion and intercellular cell junctions, such as tight junctions and adhesion proteins, such as claudins, occludin, and desmoglein; epidermal-dermal junctions, such as integrin V, collagen IV, and collagen VII; gap junctions, such as connexins; and molecular channels, such as aquaporins. In some embodiments, the monitoring step comprises measuring at least one of Pro Col1a, matrix metalloproteinases (MMPs), serine proteases, reactive oxygen species (H2DCFDA), LC3, AGEs; DNA damage, miR-23a-3p (for hyaluronan synthase 2), miR126 (endothelial); free radical production, and advanced glycation end products (AGEs). In some embodiments, the monitoring step includes measuring melanocyte location, Ki 67, gp-100, Fontana-Masson, loricrin, keratin-1, keratin-14, melanogenesis markers (such as TYR, TRP-1, Pmel17, MITF, etc.); tyrosinase activity and expression; cyclobutane pyrimidine dimer (CPD), active caspase 3; fluorescence and LCMS for permeability; cytokines (IL-17, 2, 4, 6, 12, 8, 10, TNF-α, IFN-γ, TGF-β); staining for keratin 14, vimentin, involucrin, and filaggrin. Table 1 provides information on example features measured, as well as the endpoints and assays used to measure the features.Table 2 provides illustrative active ingredients used for testing with the ex vivo model. Table 1 [Table 1-1] [Table 1-2] [Table 2]

[0108] In some embodiments, the input features are scored or classified. In some embodiments, the scoring or classification is based on at least one of (1) the percent change in each feature from normal and (2) the method used to obtain the features. In some embodiments, the percent change score ranges from 0 to 4, with a score of 0 indicating no change, 1 being given if the change is <25%, 2 being given if the change is 26-50%, 3 being given if the change is 51-75%, and 4 being given if the change is 76-100%. In some embodiments, a combination of method classifications is applied to the features. In some embodiments, the tool and endpoint inputs are incorporated into the trained algorithm. Such tools and endpoint inputs include one or more of ultrasound, H&E, dermoscopy, photography, melanin content, Corneometer, Cutometer, Megzameter, TEWL, pH meter, thermometer, oil sensor, Periodic Acid Schiff (PAS), Trichrome, elastin, Collagen IV, Collagen VII, Procollagen I, Laminin 5, Hyaluronan Synthase (HAS), Vimentin, Filaggrin, Keratin 10, Keratin 14 (basal), Lipid (adipocyte), Ki67, Keratin 15, CD34, TUNEL, p53, CPD (UV damage mediated), ROS, MMP, L-2, IL-4, IL-6, IL-8, IL-10, IL-17, IL-18, IL-22, IL-27, TNF-α, IFN-γ, TGF-β, MTT, RNA seq and LC-MS. In some embodiments, tool and endpoint inputs are multiplied by a factor.

[0109] In some embodiments, the ranking may be performed by one or more of a grade and a number. Table 3 provides an example ranking system that uses grades to quantify effectiveness in addressing a particular endpoint. [Table 3]

[0110] Skin care compositions can generally be prepared by conventional methods, such as those known in the art of making compositions and topical compositions. Such methods typically involve mixing the ingredients in one or more steps to a relatively homogeneous state, with or without heating, cooling, applying a vacuum, etc. The compositions are preferably prepared to optimize the stability (physical stability, chemical stability, photostability, etc.) and / or delivery of the active ingredients.

[0111] The compositions may be in a variety of product forms, including, but not limited to, solutions, suspensions, lotions, creams, gels, toners, sticks, pencils, sprays, aerosols, ointments, cleansing liquid washes and solid bars, shampoos and hair conditioners, pastes, foams, powders, mousses, shaving creams, wipes, strips, patches, electrically powdered patches, wound dressings and adhesive bandages, hydrogels, film-forming products, facial and skin masks (with and without insoluble sheets), makeup, e.g., foundations, eyeliners and eye shadows, viral and non-viral gene delivery agents, microneedle patches, and the like.

[0112] IX. Systems and Computer Implementation In various aspects, the present disclosure provides a method for skin tissue analysis, which relies on the use of a database associated with the ex vivo tissue support system described herein to compile and organize any evaluation or test data obtained.Due to the automation and thus the scalability of the ex vivo tissue support system for high-throughput data, it is highly desirable to have a simplified workflow for the data obtained in the laboratory, which is added and organized into a screening library for user access and understanding, whether for the purpose of training intelligent data analytics or for clinical trials.

[0113] In some embodiments, the method for skin tissue analysis comprises: receiving a plurality of data sets, wherein the data sets are measured after contacting skin tissue array with a plurality of test agents; after contacting skin tissue array with test agents, measuring a plurality of data sets; based on scoring panel results, mapping the measured plurality of data sets, wherein scoring panel results are calculated according to the strength and effectiveness of each measurement value of the measured plurality of data sets; and applying machine learning algorithm to the mapped data sets to generate at least one threshold criterion for selecting therapeutic agents from at least one test agent based on mapped data, wherein each at least one threshold criterion corresponds to at least one biological pathway.Example thresholds will be further discussed in the following Example 7.

[0114] FIG. 9 shows a block diagram of a system 100 that some embodiments may operate to receive multiple datasets. The system 100 may analyze the datasets for multiple test agents for therapeutic agent selection. The system 100 may include a user computing device 110, which may be a desktop or laptop personal computer, a mobile smartphone, a server, or other suitable device. The user computing device 110 may include a user interface 111 through which a user 102 may interact with the user computing device 110. For example, the user 102 may use the user interface 111 to interface with a test agent database 130 or a test agent analysis facility 121 of the server computing device 120. For example, the user 102 may operate the user interface 111 to initiate analysis of the datasets from the test agent database 130 and to display the analysis results, e.g., to calculate scoring panel results based on the strength and effectiveness of each measurement in the dataset, or to generate at least one threshold criterion for therapeutic agent selection in the interface 111. The user 102 may additionally or alternatively operate the user interface 111 to input a data set obtained from the study drug database 130, e.g., output to the user 102 in another interface. These values ​​may be provided to the study drug analysis facility 121. As a further example, the user 102 may operate the user interface 111 to initiate an analysis of a study drug by the study drug database 130 and the provision of analysis results (e.g., scoring panel results or threshold criteria) from the study drug database 130 to the study drug analysis facility 121. Results of the analysis of the results (received from the study drug database 130 or from the interface 111) by the study drug analysis facility 121 may be output to the user interface 111, e.g., by being received at the user interface 111 and displayed on the device 110.In some embodiments, as described above, user interface 111 may include a web interface, e.g., one or more web pages, to which values ​​may be output and which may display the results of analysis by test drug analysis equipment 121, although embodiments are not so limited. User interface 111 may accept input in a variety of different forms, for example, via voice recognition, text entry, or other means, although embodiments are not limited in this respect.

[0115] System 100 may include a server computing device 120 that may include a test drug analysis facility 121 configured to analyze factors (e.g., derived from a dataset, e.g., by test drug database 130) for user 102 to rank or select test drugs. In some embodiments, test drug analysis facility 121 may receive information about the factors from test drug database 130 and / or from user interface 111. In some embodiments, test drug analysis facility 121 may output selected test drugs that meet predetermined criteria as therapeutic agents.

[0116] System 100 may include a network 140 for facilitating communication between test medication database 130, user computing device 110, and server computing device 120. Network 140 may be or include any one or more wired and / or wireless, local area and / or wide area networks, including one or more enterprise networks and / or the Internet.

[0117] 9 includes a user interface on device 110 separate from sample analyzer 112, it should be understood that embodiments are not so limited. In other embodiments, user interface 111 may be an interface to test drug database 130 and may be operated by user 102. Additionally or alternatively, test drug analysis equipment 121 is shown on a different computing device than user computing device 110 and test drug database 130, but embodiments are not so limited. In other embodiments, analysis equipment may be implemented on a client computing device or test drug database 130. In some embodiments, user interface 111 may not be separate from test drug analysis equipment 121, but instead may be implemented as a single program or software application. In some embodiments, test drug database 130 may include user interface 111 and test drug analysis equipment 121, and interface 111 and equipment 116 may be implemented within the same program or application that runs against test drug database 130.

[0118] Computer-executable instructions for carrying out the techniques described herein (whether implemented in one or more functional units or in any other manner) may, in some embodiments, be encoded on one or more computer-readable media to provide the media with functionality. Computer-readable media include magnetic media, such as hard disk drives, optical media, such as compact discs (CDs) or digital versatile discs (DVDs), persistent or non-persistent solid-state memory (e.g., flash memory, magnetic RAM, etc.), or any other suitable storage medium. Such computer-readable media may be implemented in any suitable manner, including as computer-readable storage medium 1103 of FIG. 10 (i.e., as part of computing device 1100), described below, or as a standalone, separate storage medium. As used herein, "computer-readable medium" (also referred to as "computer-readable storage medium") refers to a tangible storage medium. A tangible storage medium is non-transitory and has at least one physical structural component. "Computer-readable medium," as used herein, refers to at least one physical structural component having at least one physical characteristic that can be altered in some way during the process of creating the medium with embedded information, recording information thereon, or any other process that encodes information in the medium. For example, the magnetization state of a portion of the physical structure of the computer-readable medium can be altered during the recording process.

[0119] In some, but not all, implementations in which the techniques may be embodied as computer-executable instructions, these instructions may be executed on one or more suitable computing devices operating in any suitable computer system, including the example computer system of Figure 9, or one or more computing devices (or one or more processors of one or more computing devices) may be programmed to execute the computer-executable instructions. A computing device or processor may be programmed to execute the instructions if the instructions are stored in a manner accessible to the computing device or processor, for example, in a data store (e.g., an internal cache or instruction register, a computer-readable storage medium accessible via a bus, a computer-readable storage medium accessible via one or more networks and accessible by the device / processor, etc.). The functional equipment containing these computer-executable instructions may be integral with and may direct the operation of a single general-purpose programmable digital computing device, a cooperative system of two or more general-purpose computing devices that share processing power and jointly perform the techniques described herein, a single computing device or a cooperative system of dedicated computing devices (co-located or geographically distributed) that execute the techniques described herein, one or more field programmable gate arrays (FPGAs) for implementing the techniques described herein, or any other suitable system.

[0120] 10 shows one exemplary implementation of a computing device in the form of a computing device 1100 that may be used in a system that performs the techniques described herein, although others are possible. It should be understood that FIG. 10 is not intended to be a depiction of the components necessary for a computing device to perform an analytical facility in accordance with the principles described herein, nor is it intended to be a comprehensive depiction.

[0121] The computing device 1100 may include at least one processor 1101, a network adapter 1102, and a computer-readable storage medium 1103. The computing device 1100 may be, for example, a desktop or laptop personal computer, a personal digital assistant (PDA), a portable smartphone, a server, a wireless access point or other networking element, or any other suitable computing device. The network adapter 1102 may be any suitable hardware and / or software that enables the computing device 1100 to communicate wired and / or wirelessly with any other suitable computing devices over any suitable computing network. A computing network may include wireless access points, switches, routers, gateways, and / or other network equipment and any suitable wired and / or wireless communication medium(s) for exchanging data between two or more computers, including the Internet. The computer-readable medium 1103 may be adapted to store data processed and / or instructions executed by the processor 1101. The processor 1101 enables the processing of data and the execution of instructions. Data and instructions may be stored on a computer-readable storage medium 1103 .

[0122] The data and instructions stored on the computer-readable storage medium 1103 may include computer-executable instructions that perform techniques operating according to the principles described herein. In the example of Figure 10, the computer-readable storage medium 1103 stores computer-executable instructions that perform various facilities and store various information as described above. The computer-readable storage medium 1103 may store analytical facilities 1104.

[0123] Although not shown in FIG. 10 , a computing device may further have one or more components and peripherals, including input and output devices. These devices may be used, among other things, to present a user interface. Examples of output devices that may be used to provide a user interface include a printer or display screen for visual presentation of output and a speaker or other sound-generating device for audible presentation of output. Examples of input devices that may be used for a user interface include a keyboard and pointing devices, such as a mouse, touchpad, and digitizing tablet. As another example, a computing device may receive input information via voice recognition or in other audible formats. [Example]

[0124] Example 1 Preparation of ex vivo tissue models The purpose of this example is to incorporate adipose stem cells (ASCs) into the insert.

[0125] Step 1. Prepare Fibricol and 10x Minimum Essential Medium (MEM) on ice. Chill serological pipettes and pipette tips. Place NaOH solution in a biological safety cabinet (BSC).

[0126] Step 2. Prepare ASC cells: thaw and resuspend in adipose nutrient medium and spin down at 200 g for 5 min.

[0127] Step 3. Prepare gel solution (ratio = Fibricol (8): 10xMEM (1): ASC suspension (1)).

[0128] Step 3a. On ice, mix fibricol (4 mL) and 10x MEM (500 uL) using a cold pipette / tip in a cold 15 mL conical tube.

[0129] Step 3b. Add 2 uL of 5N NaOH, mix thoroughly, and monitor the color of the medium. Repeat this step until a color change to a pH of approximately 7.2-7.4 is observed (a total of 5 uL of NaOH has been added).

[0130] Step 4. After centrifugation, aspirate the supernatant and resuspend the cell pellet (500,000 cells total) in 1 mL of adipocyte nutrient medium.

[0131] Step 5. Add 500uL of cell suspension to the gel solution on ice and mix thoroughly.

[0132] Step 6. Invert the insert (first side down) in the 6-well plate (mesh / porous portion on top) and gently add 500uL of gel + cell solution to the center. Monitor the solution spreading and use the pipette tip to spread the solution over the surface of the insert.

[0133] Step 7. Incubate the well plate in a CO2 incubator for 40 minutes.

[0134] Step 8. Invert the insert back in the 6-well plate (first side up) and add 3 mL of mixture medium.

[0135] Step 9. After 3 hours, place the tissue into the insert.

[0136] Example 2 Histological analysis of tissues The purpose of this example is to demonstrate histological analysis of tissues achieved using HCM and HTC in accordance with embodiments of the present disclosure. Skin tissues were grown and analyzed for various molecular markers, demonstrating the complexity of the resulting tissues and the effects of various test agents, compounds, or biologics of interest on the tissues from a histological overview. These results provide an illustrative benchmark for skin product testing, thereby evaluating the potential efficacy or toxicology of skin products. Figures 11A-11F provide a series of photomicrographs containing histological samples from ex vivo tissue model samples subjected to test conditions in which different stains were applied (SC = skin cream; N = ). Figures 11A-11B provide photomicrographs using H&E staining, in which the samples were treated with base cream, 1% retinol, 0.5% retinol, 0.3% retinol, retinol serum, vitamin C serum, niacinamide serum, or hyaluronic acid serum. In some embodiments, the retinol serum included bukachiol. In some embodiments, the retinol serum included a retinol substitute. Figures 11C-11D provide photomicrographs using Masson's trichrome staining, in which samples were treated with base cream, or 1% retinol, or 0.5% retinol, or 0.3% retinol, or retinol serum, or vitamin C serum, or niacinamide serum, or hyaluronic acid serum. Figures 11E-11F provide photomicrographs using Movat pentachrome staining, in which samples were treated with base cream, or 1% retinol, or 0.5% retinol, or 0.3% retinol, or retinol serum, or vitamin C serum, or niacinamide serum, or hyaluronic acid (HA) serum.

[0137] The results in Figures 11A-11F show the complexity of the tissue in both structure and viability. For example, staining revealed features such as elastic fibers, nuclei, collagen, reticular fibers, mucin, fibrin, muscle, cytoplasm, and keratin. For example, retinol 1% (SC) produced higher magnitude side effects of intraepidermal clefting and necrotic keratinocytes; retinol 0.5% (SC) produced intermediate magnitude side effects of intraepidermal clefting, necrotic keratinocytes, and melanocyte activation; retinol 0.3% (SC) produced lower magnitude side effects of intraepidermal clefting, necrotic keratinocytes, and melanocyte activation; retinol serum (N) produced higher levels of collagen production and necrotic keratinocytes; vitamin C serum (N) produced higher levels of collagen production and thicker epidermis; niacinamide serum (N) produced intermediate side effects of necrotic keratinocytes and subepidermal clefting, and HA serum produced lower levels of collagen production, uniform collagen distribution, and necrotic keratinocytes.

[0138] Example 3 Gene or protein expression maps The purpose of this example is to provide a genomics analysis of tissue achieved using HCM and HTC according to embodiments of the present disclosure. Skin tissue was grown and analyzed for various genetic markers to display the resulting genetic profile of the tissue sample and the effects of various test agents, compounds, or biologics of interest on the tissue based on a gene mapping configuration. In particular, each set of gene expression levels displayed in the mapping indicates the expression changes obtained upon exposure to the test agent. These results provide further illustrative benchmarks for skin product testing, thereby evaluating the potential efficacy or toxicology of skin products. Figures 12A-12F are gene expression heat maps for several different study conditions according to one or more embodiments of the present disclosure. Figure 12A provides gene expression maps for epidermal differentiation and the epidermal-dermal junction, and the left branch of each heat map shows the clustering of samples based on gene expression similarity. Figure 12B provides gene expression maps for dermal markers and MMPs. Figure 12C provides gene expression maps for MMP inhibitors and regeneration. Figure 12D provides gene expression maps for wrinkle-related genes and sagging-related genes. Figure 12E provides gene expression maps for inflammation and apoptosis (intrinsic). Figure 12F provides gene expression maps for apoptosis (extrinsic) and anti-apoptotic genes. Each gene expression map is quantified for each of the following treatments: base cream, retinol 1%, retinol 0.5%, retinol 0.3%, retinol serum, vitamin C serum, niacinamide serum, or HA serum.

[0139] Example 4 Experimental Design for Path Analysis The purpose of this example is to provide an experimental design for gene pathway analysis. The cell and model source for this experimental design is human skin biopsy. The applications for testing are anti-aging, vehicle testing, UV, and "improvement."

[0140] The anti-aging treatment regimen involves application of retinol cream, multiple retinoic acid (pure) formulations (e.g., three retinoic acid formulations labeled RA, F1; RA, F2; and RA, F3), and vehicle to human skin biopsies.

[0141] The treatment regimen for UV involves application of UV to human skin biopsies. The component conditions tested include a sunscreen labeled UV protection (prior to treatment with UV), a no-treatment option labeled UV damage, and multiple post-treatment options (e.g., two post-treatment options) labeled UV damage recovery.

[0142] The treatment regimen for UV includes no treatment with application of the vehicle component and the retinol component.

[0143] Endpoints measured during each treatment regimen include one or more of the following: ultrasound, dermoscopy, photography, corneometer, cutometer, TEWL, megurometer, pH, IHC staining: H&E, PAS, trichrome, elastin, filaggrin, keratin 10, keratin 14, Ki67, transglutaminase 1, collagen IV, collagen VII, laminin 5, fibrillin, procollagen I, HAS, vimentin, lipids; for the UV group: active caspase 3, TUNEL, cyclobutane pyrimidine dimer; efflux assay: MMP, serine protease, ROS; for the UV group: IL-2, 4, 6, 8, 10, 17, 18, 22, 27, TNF-α, IFN-gamma, TGH-beta; MTT bulk for pathway analysis; RNA-seq; and LCMS for absorption and metabolism.

[0144] Example 5 Tissue lifespan analysis The purpose of this example is to provide an analysis of skin tissue culture longevity according to aspects of the present disclosure.

[0145] Figures 13A-13B provide a comparison of histology results for fresh human skin tissue in inserts using media containing the components in Table 4 between day 0 and approximately 3 weeks in culture, respectively, demonstrating proof-of-concept of tissue viability within the inserts during that time interval. Figure 14 further provides an analysis of selected genes involved in skin development and biological function, performed via comparison of z-scores in expression heat maps. As shown, the skin tissue cultures of this example had comparable gene expression levels to fresh tissue (day 0) after 17 days in culture. The retinol complex serum and retinol cream applied to the skin tissues exhibited gene expression patterns distinct from the baseline group.

[0146] [Table 4]

[0147] Table 5 provides several media mixtures that were applied to skin samples to evaluate the effect of various test conditions on skin tissue. Histologically stained tissue sections were analyzed via microscope to determine the best media conditions, as provided in Table 6, using experimental criteria for epidermal and dermal structure as well as testing consistency between skin tissue sample groups using two samples per group.

[0148] [Table 5-1] [Table 5-2] [Table 5-3]

[0149] [Table 6-1] [Table 6-2]

[0150] Figures 15A-15G show histology results using each of the media mixes in Table 6. The best media condition, shown in Figure 15E, was determined based on epidermal and dermal structure and consistency between the two replicates. Additional test conditions included initially treating the skin samples with 0.1 mM calcium chloride on the surface and then applying higher concentrations of calcium chloride (0.5 mM and 1 mM) in the media. However, none of the test conditions were observed to have any beneficial effect.

[0151] 16A-16B show histology results using Medium Mix 12 provided in Table 7 between day 0 and 4 weeks in culture, respectively. Based on epidermal and dermal structure, Medium Mix 12 was shown to result in the longest culture duration among all commercially available models.

[0152] Tests using the above conditions produced healthy, viable skin morphology in culture for up to four weeks, a four-fold increase over existing commercially available ex vivo models. By enabling long-term treatment studies beyond conventional approaches, complex biological interactions and clinically relevant phenomena can be better understood. Further studies with longer tissue culture lifespans are likely to emerge from further experimentation.

[0153] Skin tissue cultures were further observed to survive for up to 6 weeks after seeding. Figures 17A-17C show photographic images of culture samples that survived at 6 weeks, including samples subjected to Medium Mix 9, Medium Mix 9 with 5% human platelet lysate, and Medium Mix 9 with 5% human platelet lysate and 10 ng / mL EGF and 10 ng / mL bFGF, respectively.

[0154] Example 6 Biopsy occlusion and transepidermal water loss (TEWL) analysis The goal of this example is to incorporate a sealed sensor into the insert. As shown in the photograph in Figure 18A and the histological stain image in Figure 18B, sealing the biopsy edges using Loctite super glue not only allowed for measurement of changes in barrier function with high sensitivity, but also prevented leakage of topically applied substances into the dermis through the biopsy edges, allowing for accurate measurement of changes in skin barrier function using the clinical sensor. Table 7 provides the adhesives tested and the corresponding results of their use.

[0155] [Table 7]

[0156] Figures 19A-19B show transepidermal water loss (TEWL) measurements on model skin that was unsealed or sealed using Loctite Super Glue, respectively. TEWL measurements were obtained using a Courage+Khazaka Multi Probe Adapter MPA 10. Accurate measurement of TEWL is facilitated by sealing the biopsy edges to the insert; this sealing was observed to enhance the sensitivity of detecting changes in skin barrier function. For example, Figures 19A-19B show that the expected reduction in barrier function caused by SDS treatment was observable via TEWL detection only after sealing the biopsy edges. Figure 20 shows a comparison of the effects of a barrier enhancer (niacinamide) and a barrier disrupter (SDS) on skin sealed with Loctite Super Glue over a 6-day period on TEWL measurements.

[0157] Example 7 Exemplary Data Mapping Analysis The purpose of this example is to provide a visualization of a data map using data from an exemplary test agent. Figure 21A shows exemplary data types contributing to the data map, while Figures 21B-21C show a comparison between data maps generated from retinol and retinol substitutes, respectively. The data map in Figure 21A was derived from multimodal data using specific thresholds for each data type. The data included, for example, gene set enrichment analysis (GSEA) based on a Net Enrichment Score (NES) threshold of 1.2 or greater, differential gene expression data (DGE) based on a logarithmic fold change (logFC) threshold of 5 or greater, pathology scores based on a 20% improvement from vehicle threshold, and lactate dehydrogenase (LDH) release data based on a percent release threshold compared to a dissolved control. Table 9 provides a list of exemplary histopathological parameters used to score the compiled experimental data from the test agent for its respective evaluation, and the scores are used as pathology score inputs into the data map. Table 9 can also be used to assess tissue and / or cell viability. In some embodiments, a skin tissue sample can be considered viable if each of epidermal damage, eczematous dermatitis, hyperpigmentation, epidermal thickness, solar elastosis, and collagen deposition is each scored as 1 compared to a control skin tissue sample on day 0. In some embodiments, a skin tissue sample can be considered viable if, compared to a control skin tissue sample on day 0, the epidermal damage score does not exceed 2, the eczematous dermatitis score does not exceed 1, and the scores for hyperpigmentation, solar elastosis, and collagen deposition do not exceed 1, respectively.

[0158] The resulting data maps show a four-fold increase in skin barrier function and a two-fold reduction in irritation when using the retinol substitute compared to retinol.

[0159] [Table 8]

[0160] Example 8 Exemplary Endothelial Cell Growth Assay in Porous Layers The purpose of this example is to evaluate the use of 3D-printed gyroid support structures in an experimental design for endothelial cell incorporation. HDMECs were obtained from PromoCell and seeded onto Transwell inserts with 0.4 μm pore size at a density of 5.383e+4 cells / insert or 7.69e+5 cells / mL. The inserts were coated with different ECM compositions shown in Table 10 and then imaged using CD31 staining after 14 days of culture.

[0161] Figures 22A-22B provide images of cells coated onto 3D-printed support structures. Figures 23A-23H provide images of inserts coated with conditions 1-8, respectively, from Table 10, after 14 days of culture in CELLnTEC-Promocell media mix (media mix 12 described in Table 7) according to the manufacturer's instructions. Figures 24A-24H provide images of inserts coated with conditions 1-8, respectively, from Table 10, after 14 days of total culture, when cells were pretreated with endothelial growth medium for 8 days and then switched to CELLnTEC-Promocell media mix for 6 days. No significant changes were observed between the two media groups.

[0162] [Table 9-1] [Table 9-2]

[0163] Figure 25 provides an image of an F-actin stained insert containing adipose-derived stem cell incorporation in a 3D printed gyroid support structure, where 10 human adipose-derived stem cells (HASCs) from Obatala were added per 100 μL. 4 Cells were seeded at a density of 1000 x 1000 and cultured for 18 days. Cells were maintained in CELLnTEC and Promocell media mix (media mix 12), and ObaGel was used as the culture matrix. ObaGel was found to increase endothelial vascular network formation compared to other ECM conditions tested.

[0164] Example 11 Exemplary Skin Inflammation Assay The purpose of this example is to test and replicate the dynamic changes in skin across various types of clinically relevant stimuli, including skin irritation, intradermal injection, cosmetic product treatment, and antioxidant defense. Skin irritation approximations evaluated in this manner include topical chemical treatment 261, burn treatment 262, and UV treatment 263, shown in Figures 26A-26C, respectively. Chemical irritation was approximated via application of 2% SDS for 24 hours, burn treatment was approximated via contact with a hot metal rod at 100°C for 45 seconds, and UV irritation was approximated via application of UVA and UV light for 30 minutes. Figure 27 provides an exemplary protocol showing that each stimuli was applied between days 5 and 6 of an 8-day culture using normal skin tissue in inserts. Skin tissue was evaluated and scored using the parameters in Table 9 provided above. Included are protocols, histological parameters, histological staining and scoring results, immune and inflammatory responses, antioxidant defense evaluation, and more.

[0165] Figures 28A-28D provide H&E-stained tissues for a first selection of untreated, chemical-treated, burn-treated, and UV-treated skin tissue samples, respectively. Pathological analysis of the extent of damage was performed to assess epidermal damage, eczematous epidermis, and epidermal thickness, respectively, after application of the damaging stimulus, and is shown in Figures 29A-29C. Application of 2% SDS resulted in significant epidermal thickening, as evidenced by extensive dyskeratinocytes, moderate eczematous (atopic) epidermis, and pathological epidermal thickening (acanthosis), compared to the control group. Both the burn group and the UV-irradiated group showed significant epidermal damage. UV irradiation caused epidermal thinning compared to the control group.

[0166] A second selection of samples was cultured and subjected to irritating stimuli, whereby the chemically stimulated group was subjected to 2% SDS for 24 hours, the burned group was subjected to 100°C exposure for 90 seconds, and the UV-stimulated group was subjected to UV-B for 30 minutes. Figures 30A-30D show insert images of untreated, chemically treated, burned, and UV-treated skin tissues, respectively, after staining with Masson's trichrome. Figure 31A provides an analysis of collagen deposition using histopathological scoring to determine the impact of the injury stimuli on each group. As a result of this analysis, both the burned and UV-irradiated groups were found to have reduced collagen deposition compared to the untreated group. No significant changes in collagen deposition were observed in the SDS-treated group. Figure 31B provides an analysis of lactate dehydrogenase (LDH) release, which is widely used as a marker for assessing cell death and toxicity. The analysis showed a significant increase in LDH release from all treatment conditions compared to untreated samples. The highest LDH release occurred from the SDS-treated group.

[0167] Inflammatory responses were performed and recorded for each of the first and second selections of samples for the different treatment groups. Figures 32A-32B provide gene expression heat maps for each of the first and second selections of samples for the different treatment groups. The heat map in Figure 32A includes genes representing Langerhans cells, dendritic cells, plasma cells, M1 and M2 macrophages, neutrophils, mast cells, T cells (CD8+, CD4+, regulatory), natural killer (NK) cells, B cells, and monocytes. The heat map in Figure 32B further includes genes representing memory T cells, gamma delta T cells (GdT), and eosinophils. Figures 33A-33C further provide immune cell profiles of IL-1β, IL-6, and IL-8, respectively, for each treatment group at different time points. This study found that increased pro-inflammatory cytokines were released after the samples were subjected to burn injury compared to the other treatment groups.

[0168] Immune cell profiling was further performed on skin tissue samples treated using the protocol of FIG. 27 using intradermal injection 340 of lipopolysaccharide (LPS) treatment, as shown in FIG. 34A. FIG. 34B provides exemplary H&E staining of intradermal injections of 1 μg / mL LPS and 100 ng / mL TNF-α. FIG. 34C provides immune cell profiles of IL-1β resulting from 1 μg / mL LPS, 10 μg / mL LPS, and 1 μg / mL LPS + TNF-α. IL-1β is a pro-inflammatory cytokine important for the host response to infection. Various concentrations of LPS and TNF-α were tested to optimize acute and localized inflammatory conditions. The observed 14.7-fold increase in IL-1β levels in the LPS + TNF-α treatment group compared to the saline control treatment group indicated the onset of an inflammatory response.

[0169] Immune cell profiling was further performed on skin tissue samples treated with various cosmetic products using the protocol in Figure 27. Exemplary heat maps of profiling are shown in Figures 35A-35B for two different donors: a 53-year-old Caucasian male and a 54-year-old Caucasian female, respectively. Examples of such cosmetic products include retinol cream (0.3% Skinceutical), hyaluronic acid serum (Natrium), and vitamin C serum (Natrium), compared to untreated and day 0 controls. The expression patterns of various immune cell markers were evaluated after treatment of skin samples from the donors with four different cosmetic products. Both donors showed significant increases in markers for T cells, monocytes, M2 macrophages, B cells, NK cells, and neutrophils compared to other treatments and controls. This is an example of expression pattern analysis based on various treatments and specific donors.

[0170] Immune cell profiling was further performed on skin tissue samples treated using the protocol in Figure 27 with various skin care ingredients or UV treatments to evaluate antioxidant defenses in the skin. Figure 36A provides a heat map of the evaluated skin care ingredients, including 5% ascorbic acid, 0.5% retinyl acetate, and 1% hyaluronic acid. Figure 36B provides profiles of superoxide dismutase (SOD) activity from each of the various UV treatments, including 73 seconds of UVB, 5 minutes of UVB, and 20 minutes of UVA, as well as chronic treatment conditions, such as 4 x 73-second intervals of UVB, 4 x 5-minute intervals of UVB, and 4 x 20-minute intervals of UVA. The profiling in Figure 36B shows a significant effect of chronic UVB exposure over longer periods on SOD activity, suggesting a need for additional catalysis of the dismutation of superoxide radicals to molecular oxygen and hydrogen peroxide.

[0171] Example 12 Exemplary Protocol for Perfusion Controller The purpose of this example is to provide an exemplary configuration and protocol for operating the perfusion controller of Figure 1G in conjunction with the ex vivo skin tissue system of Figures 1C-1D. Figure 37 shows a diagram of the configuration of the perfusion controller 30 for the ex vivo skin tissue system 1, which further includes an air pump 31 with a closed-loop pressure feedback system, a three-way valve 32, and connections therebetween (e.g., electrical wires 33, pneumatic tube 34). The pneumatic tube 34 can be for applying either positive pressure or vacuum.

[0172] Based on the UI input, the perfusion controller 30 sends a pressure set point and duration to the air pump 31. The air pump 31 applies vacuum or positive pressure to the conical tubing, drawing fluid through the insert 10. An exemplary protocol for operation is: vacuum pressure 30 seconds on, 30 seconds off, repeated for 6 hours, positive pressure for 2 minutes (to pump collected effluent back into the input tubing), repeating the entire sequence.

[0173] definition As used herein, "about" and its grammatical equivalents, when used herein in connection with a reference numerical value and its grammatical equivalents, can include a range of values ​​from that value that is plus or minus 10%. For example, the amount "about 10" encompasses the amounts 9 to 11. The term "about," when used in connection with a reference numerical value, can also include a range of values ​​from that value that is plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0174] As used herein, "cell" refers to a biological cell. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protist cells, cells derived from plants, algae cells, fungal cells, fungal protoplast cells, animal cells, etc. Sometimes the cell does not originate from a natural organism; for example, the cell may be synthetically produced and sometimes referred to as an artificial cell.

[0175] While various features of the present disclosure may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may for clarity be described herein in terms of separate embodiments, various aspects and embodiments may also be implemented in a single embodiment.

[0176] While exemplary embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments described herein, or combinations of one or more of the embodiments or aspects described herein, may be used in implementing the present disclosure. It is intended that the following claims define the scope of the disclosure, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. an insert including at least one opening and a wall; a skin tissue sample sealed inside the insert; and a porous layer positioned inside the insert, the porous layer having a first side facing the at least one opening of the insert and a second side facing an opposite side of the at least one opening, the porous layer comprising a structure and arrangement relative to the skin tissue sample to promote bulk transport of reagents and / or fluids into the skin tissue sample to support viability of the skin tissue sample for at least one week. An ex vivo skin tissue system comprising:

2. The ex vivo skin tissue system of claim 1 , further comprising an adipose layer positioned inside the insert.

3. The ex vivo skin tissue system of claim 2 , wherein the porous layer is in contact with at least one of the fat layer and the tissue sample.

4. The ex vivo skin tissue system of claim 3 , further comprising at least one of an inlet channel and an outlet channel positioned laterally relative to the porous layer.

5. 3. The ex vivo skin tissue system of claim 2, further comprising a gel layer in contact with the second side, the gel layer configured to receive one or more of adipose-derived cells, dermal cells, and endothelial cells.

6. The ex vivo skin tissue system of claim 5 , wherein the gel layer comprises a gel material.

7. The ex vivo skin tissue system of claim 5 , wherein the gel layer comprises an extracellular matrix.

8. The ex vivo skin tissue system of claim 2 , further comprising an adhesive film configured for at least one of contacting and sealing the fat layer.

9. The ex vivo skin tissue system of claim 1 , further comprising a cell composition seeded within the porous layer between the first side and the second side.

10. The ex vivo skin tissue system of claim 9 , further comprising a gel layer in contact with the second side.

11. 10. The ex vivo skin tissue system of claim 9, wherein the seeded cells comprise one or more of endothelial cells, primary cells, iPSCs, keratinocytes, fibroblasts, melanocytes, resident immune cells, circulating immune cells, stem cells, adipocytes, and microorganisms.

12. The ex vivo skin tissue system of claim 1 , wherein the insert includes a first thread along an inner surface of the wall perpendicular to the first side of the porous layer.

13. 10. The ex vivo skin tissue system of claim 1, wherein the tissue sample is sealed using an adhesive, the adhesive being selected from super glue and UV curable glue.

14. The ex vivo skin tissue system of claim 1 , wherein the tissue sample comprises at least one of an epidermis layer, a dermis layer, and a subcutaneous tissue layer.

15. The ex vivo skin tissue system of claim 1 , wherein the porous layer comprises a porosity gradient.

16. The ex vivo skin tissue system of claim 1 , wherein the porous layer comprises a plurality of pores, each of the plurality of pores being gyroid.

17. The ex vivo skin tissue system of claim 1 , further comprising at least one standoff along the second side of the porous layer.

18. The ex vivo skin tissue system of claim 1 , wherein an inner surface of the wall perpendicular to the first side of the porous layer comprises a plurality of pores.

19. 20. The ex vivo skin tissue system of claim 18, wherein each of the plurality of pores is a gyroid.

20. 20. The ex vivo skin tissue system of claim 18, wherein the plurality of pores penetrate the wall to a desired thickness.

21. 10. The ex vivo skin tissue system of claim 1, further comprising a growth medium in contact with the tissue sample.

22. 22. The ex vivo skin tissue system of claim 21, wherein the growth medium is maintained at between 32 and 37 degrees Celsius.

23. 22. The ex vivo skin tissue system of claim 21, wherein the growth medium comprises at least one test agent.

24. 10. The ex vivo skin tissue system of claim 1, further comprising at least one test agent contacted with the tissue sample, wherein the contacting is by one or more of topical application, subcutaneous application, systemic application, intradermal administration, infusion, perfusion, and injection.

25. an insert including at least one opening and a wall; a sealing unit along the inner wall of the insert; a tissue sample positioned inside the insert via the sealing unit; and a porous layer positioned inside the insert, the porous layer configured to facilitate bulk transport of reagents and / or fluids into the tissue sample, the porous layer having a first side facing the at least one opening in the insert and a second side facing opposite the at least one opening. An ex vivo skin tissue system comprising: An ex vivo skin tissue system capable of supporting tissue viability for periods of more than one week.

26. 26. The ex vivo skin tissue system of claim 25, further comprising an adipose layer positioned inside the insert.

27. 26. The ex vivo skin tissue system of claim 25, further comprising a cell composition seeded within the porous layer between the first side and the second side.

28. 28. The ex vivo skin tissue system of claim 27, further comprising a gel layer in contact with the second side.

29. 28. The ex vivo skin tissue system of claim 27, wherein the seeded cells comprise one or more of endothelial cells, primary cells, iPSCs, keratinocytes, fibroblasts, melanocytes, resident immune cells, circulating immune cells, stem cells, adipocytes, and microorganisms.

30. 26. The ex vivo skin tissue system of claim 25, wherein the insert includes a first thread along an inner surface of the wall perpendicular to the first side of the porous layer.

31. 31. The ex vivo skin tissue system of claim 30, wherein the sealing unit includes a compression unit, the compression unit including a second thread along its periphery configured to mate with the first thread.

32. 32. The ex vivo skin tissue system of claim 31, wherein the compression unit provides either (1) a complete occlusion or (2) a partial occlusion of the tissue sample.

33. 32. The ex vivo skin tissue system of claim 31, wherein the compression unit comprises an injection port in fluid communication with the microchannel.

34. 26. The ex vivo skin tissue system of claim 25, wherein the sealing unit is an adhesive, and the adhesive is selected from super glue and UV curable glue.

35. 27. The ex vivo skin tissue system of claim 26, wherein the porous layer is in contact with at least one of the fat layer and the tissue sample.

36. 36. The ex vivo skin tissue system of claim 35, further comprising at least one of an inlet channel and an outlet channel positioned laterally relative to the porous layer.

37. 26. The ex vivo skin tissue system of claim 25, wherein the tissue sample comprises at least one of an epidermis layer, a dermis layer, and a subcutaneous tissue layer.

38. 26. The ex vivo skin tissue system of claim 25, wherein the porous layer comprises a porosity gradient.

39. 26. The ex vivo skin tissue system of claim 25, wherein the porous layer comprises a plurality of pores, each of the plurality of pores being gyroid.

40. 26. The ex vivo skin tissue system of claim 25, further comprising at least one standoff along the second side of the porous layer.

41. 26. The ex vivo skin tissue system of claim 25, wherein an inner surface of the wall perpendicular to the first side of the porous layer comprises a plurality of pores.

42. 42. The ex vivo skin tissue system of claim 41, wherein each of the plurality of pores is a gyroid.

43. 42. The ex vivo skin tissue system of claim 41, wherein the plurality of pores penetrate the wall to a desired thickness.

44. 27. The ex vivo skin tissue system of claim 26, further comprising a gel layer in contact with the second side, the gel layer configured to receive one or more of adipose-derived cells, dermal cells, and endothelial cells.

45. 45. The ex vivo skin tissue system of claim 44, wherein the gel layer comprises a gel material.

46. 45. The ex vivo skin tissue system of claim 44, wherein the gel layer comprises an extracellular matrix.

47. 27. The ex vivo skin tissue system of claim 26, further comprising an adhesive film configured for at least one of contacting and sealing the fatty layer.

48. 26. The ex vivo skin tissue system of claim 25, further comprising a growth medium in contact with the tissue sample.

49. 49. The ex vivo skin tissue system of claim 48, wherein the growth medium is maintained at between 32 and 37 degrees Celsius.

50. 49. The ex vivo skin tissue system of claim 48, wherein the growth medium comprises at least one test agent.

51. 26. The ex vivo skin tissue system of claim 25, further comprising at least one test agent contacted with the tissue sample, wherein the contacting is by one or more of topical application, subcutaneous application, systemic application, intradermal administration, infusion, perfusion, and injection.

52. providing an insert including a porous layer, the porous layer including a structure for facilitating fluid mass transport; positioning and sealing the dermal tissue sample against a first side of the porous layer along an interior wall of the insert to promote mass transport into the dermal tissue sample; Culturing the skin tissue sample, thereby creating an ex vivo skin model; and perfusing a reagent through the ex vivo skin model to support viability of the skin tissue sample for a period of at least one week. A method comprising:

53. 53. The method of claim 52, further comprising depositing a fat layer on one or more of the first side, interior region, and second side of the porous layer.

54. 53. The method of claim 52, wherein the porous layer is bioprinted.

55. 53. The method of claim 52, wherein the porous layer is configured to promote vascularization of the tissue sample.

56. 54. The method of claim 53, further comprising seeding a cell composition within the porous layer between the first side and the second side.

57. 57. The method of claim 56, wherein the seeding step is performed using one or more of bioprinting, manual or automated liquid handling, and perfusion.

58. 57. The method of claim 56, wherein the cell composition comprises at least one of endothelial cells, primary cells, iPSCs, keratinocytes, fibroblasts, melanocytes, resident immune cells, circulating immune cells, stem cells, preadipocytes, and adipocytes.

59. 59. The method of claim 58, wherein the resident immune cells comprise at least one of Langerhans cells, dendritic cells, macrophages, mast cells, monocytes, and lymphocytes.

60. 59. The method of claim 58, wherein the circulating immune cells comprise at least one of neutrophils, T lymphocytes, B lymphocytes, natural killer cells, monocytes, and dendritic cells.

61. 53. The method of claim 52, wherein the reagents comprise at least one of culture medium, non-essential amino acid solution, insulin, transferrin, selenite, bovine serum albumin, linoleic acid, hydrocortisone, vitamin C, serum, blood substitute, penicillin, and streptomycin.

62. 54. The method of claim 53, further comprising contacting the second side with a gel layer, the gel layer configured to receive at least one of adipose-derived cells, dermal cells, and endothelial cells.

63. 63. The method of claim 62, wherein the gel layer comprises a gel material.

64. 63. The method of claim 62, wherein the gel layer comprises an extracellular matrix.

65. 63. The method of claim 62, wherein the gel layer is bioprinted.

66. 54. The method of claim 53, wherein the porous layer is in contact with at least one of the fat layer and the tissue sample.

67. 67. The method of claim 66, further comprising at least one of an inlet channel and an outlet channel positioned laterally relative to the porous layer.

68. 53. The method of claim 52, wherein the tissue sample comprises at least one of the epidermis layer, the dermis layer, and the subcutaneous tissue layer.

69. 53. The method of claim 52, wherein the porous layer comprises a porosity gradient.

70. 53. The method of claim 52, wherein the porous layer comprises a plurality of pores, each of the plurality of pores being in the shape of one or more of gyroid, Schwarz, x-cell, and cross.

71. 54. The method of claim 53, further comprising at least one standoff along the second side of the porous layer.

72. 53. The method of claim 52, wherein an inner surface of a wall perpendicular to the first side of the porous layer comprises a plurality of pores.

73. 73. The method of claim 72, wherein each of the plurality of pores is a gyroid.

74. 73. The method of claim 72, wherein the plurality of pores penetrate the interior wall to a desired thickness.

75. 54. The method of claim 53, further comprising a gel layer in contact with the second side, the gel layer configured to receive at least one of the fat layer and the tissue sample.

76. 76. The method of claim 75, wherein the gel layer comprises a gel material.

77. 76. The method of claim 75, wherein the gel layer comprises an extracellular matrix.

78. 53. The method of claim 52, wherein the sealing step includes a compression unit, the compression unit including a microchannel.

79. 79. The method of claim 78, wherein the compression unit comprises an injection port in fluid communication with the microchannel.

80. 54. The method of claim 53, further comprising sealing the fat layer with a gas permeable film.

81. 53. The method of claim 52, wherein an oscillatory pressure is applied to the fluid to facilitate filling and emptying of the fluid.

82. perfusing the composition through the insert; culturing the tissue sample; performing one or more of applying an environmental condition and monitoring an environmental condition; providing at least one sensor for obtaining at least one functional readout; monitoring at least one of a cellular characteristic and a tissue characteristic using the at least one sensor; and Scoring the at least one of a cellular feature and a tissue feature.

53. The method of claim 52, further comprising:

83. 83. The method of claim 82, wherein one or more of culturing the tissue sample and monitoring the at least one cellular characteristic and tissue using the at least one sensor are performed for at least two weeks.

84. 83. The method of claim 82, wherein the composition is adapted to maintain and / or promote a disease state.

85. 83. The method of claim 82, wherein the composition is adapted to promote cell differentiation.

86. 83. The method of claim 82, wherein the composition is adapted for one or more of promoting tissue complexity, maintaining multiple cell types, and allowing cell-cell and / or cell-tissue interactions to occur.

87. 83. The method of claim 82, wherein the environmental conditions include at least one of temperature, humidity, gas composition, contamination, and light exposure.

88. 83. The method of claim 82, wherein the step of monitoring at least one of the cellular and tissue characteristics comprises monitoring at least one of irritation, corrosion, and phototoxicity.

89. 83. The method of claim 82, wherein monitoring at least one of the cellular and tissue characteristics comprises monitoring at least one of anti-aging, skin moisturization, brightening, discoloration, pigmentation, UV protection, cleansing, permeability, inflammation, anti-inflammatory, antibacterial, wound healing, skin barrier, epidermal thickness, dermal matrix, cellular stress, cellular senescence, sagging, and wrinkles.

90. 83. The method of claim 82, wherein each of the at least one sensor comprises at least one clinical sensor.

91. 83. The method of claim 82, wherein each of the at least one sensor comprises a viscoelastic sensor, a wettability sensor, a pH sensor, an oil content sensor, a barrier function sensor, a pigmentation sensor, and a skin surface sensor.

92. 83. The method of claim 82, wherein said scoring step comprises calculating the percent change in each characteristic from normal or vehicle conditions.

93. 83. The method of claim 82, wherein said scoring step comprises characterizing the method used to obtain said features.

94. 83. The method of claim 82, wherein an oscillatory pressure is applied to the perfused composition to facilitate filling and draining of the perfused composition.

95. 1. A method for skin tissue analysis, comprising: receiving a plurality of data sets, the plurality of data sets comprising measurements after contacting a skin tissue array with at least one test agent, the skin tissue array comprising a compartment for skin tissue growth comprising the ex vivo skin tissue system of any one of claims 1 to 51; mapping the plurality of datasets based on scoring panel results, wherein the scoring panel results are calculated according to the strength and validity of each measurement of the mapped plurality of datasets; and generating at least one threshold criterion for identifying a drug from the at least one test agent based on the mapped plurality of datasets, wherein each of the at least one threshold criterion corresponds to at least one biological pathway. A method comprising:

96. 96. The method of claim 95, wherein the skin tissue array comprises one or more of an epidermal tissue layer and a dermal tissue layer.

97. 96. The method of claim 95, wherein the skin tissue array comprises one or more tissue layers from a biopsy, donor, or graft.

98. 96. The method of claim 95, wherein the skin tissue array comprises an adipose layer, the adipose layer underlying the porous layer.

99. 96. The method of claim 95, wherein the skin tissue array comprises an adipose layer, the adipose layer being within the porous layer.

100. 96. The method of claim 95, wherein the plurality of datasets comprises measurements from one or more of skin surfaces, histological staining, immunostaining, biochemical assays, clinical sensors, gene expression, and protein expression.

101. 101. The method of claim 100, wherein the measurements generated from the skin surface include measurements from one or more of dermoscopy, megusometer, cutometer, and photography.

102. 101. The method of claim 100, wherein the measurements generated from the histological stains include measurements from one or more of hematoxylin and eosin (H&E), Masson's trichrome, and Movat pentachrome.

103. 101. The method of claim 100, wherein the measurements generated from one or more of the gene expression and protein expression comprise one or more of dermal differentiation, dermal-epidermal junction, dermal markers, matrix metalloproteinases (MMPs), MMP inhibitors, regeneration, wrinkles, sagging, inflammation, intrinsic apoptosis, extrinsic apoptosis, anti-apoptosis, immune cell markers, skin elasticity, skin rejuvenation, and antioxidant defense.

104. 101. The method of claim 100, wherein the measurements generated from the clinical sensors include measurements from one or more of moisture, barrier function, sebum, pH, dermoscopy, photography, melanin content, and ultrasound.

105. 96. The method of claim 95, wherein the at least one threshold criterion is either positively or negatively correlated with a condition.

106. 106. The method of claim 105, wherein the condition comprises one or more of anti-aging, skin moisturizing, toxicity, brightening, discoloration, pigmentation, UV protection, cleansing, permeability, inflammation, anti-inflammatory, antibacterial, wound healing, skin barrier, epidermal thickness, dermal matrix, cellular stress, cellular senescence, sagging, and wrinkles.

107. 96. The method of claim 95, wherein the skin tissue array is viable for a period of more than one week.

108. 96. The method of claim 95, wherein the at least one biological pathway comprises one or more of skin surface, barrier function, epidermal thickness, matrix density, epidermal differentiation, epidermal-dermal junction, papillary dermal thickness and projection, stem cell and regenerative activity, adipogenesis, melanogenesis, sagging pathway, wrinkle pathway, cellular senescence, cellular stress, inflammation, and apoptosis.

109. 96. The method of claim 95, wherein the at least one threshold criterion is generated by applying a machine learning algorithm to the mapped plurality of data sets.

110. 110. The method of claim 109, wherein the machine learning algorithm is further configured to perform one or more of: verifying and generating product claims for skin concerns; conducting broad efficacy and / or toxicity screening of chemical libraries for quantitative comparison against product benchmarks; and performing virtual screening of the at least one test agent.

111. 111. The method of claim 110, wherein the machine learning algorithm is either an unsupervised algorithm or a supervised algorithm.

112. 96. The method of claim 95, wherein the porous layer is configured to promote mass transport into each tissue sample in the skin tissue array.

113. 96. The method of claim 95, wherein the porous layer comprises a porosity gradient.

114. 96. The method of claim 95, wherein the porous layer comprises a plurality of pores, each of the plurality of pores being in the shape of one or more of a gyroid, a Schwarz, an x-cell, and a cross.

115. 96. The method of claim 95, further comprising contacting the skin tissue array with the at least one test agent, wherein the contacting is one or more of topical application, subcutaneous application, systemic application, intradermal administration, infusion, perfusion, and injection.

116. 1. A method for ex vivo skin tissue analysis, comprising: Providing an ex vivo skin tissue system according to any one of claims 1 to 51; perfusing the composition through the ex vivo skin tissue system; culturing the ex vivo skin tissue system; performing one or more of applying an environmental condition and monitoring an environmental condition; providing at least one sensor; monitoring at least one of a cellular characteristic and a tissue characteristic using the at least one sensor; and Scoring the at least one of a cellular feature and a tissue feature. A method comprising:

117. 117. The method of claim 116, wherein one or more of said culturing and said monitoring steps are carried out for at least two weeks.

118. 117. The method of claim 116, wherein the composition is adapted to maintain and / or promote a disease state.

119. 117. The method of claim 116, wherein the composition is adapted to promote cell differentiation.

120. 117. The method of claim 116, wherein the composition is adapted for one or more of promoting tissue complexity, maintaining multiple cell types, and allowing cell-cell and / or cell-tissue interactions to occur.

121. 117. The method of claim 116, wherein the environmental conditions include at least one of temperature, humidity, gas composition, contamination, and light exposure.

122. 117. The method of claim 116, wherein the step of monitoring at least one of the cellular and tissue characteristics comprises monitoring at least one of irritation, corrosion, phototoxicity, viability, and metabolic activity.

123. 117. The method of claim 116, wherein monitoring at least one of the cellular and tissue characteristics comprises monitoring at least one of anti-aging, skin moisturization, brightening, discoloration, pigmentation, UV protection, cleansing, permeability, inflammation, anti-inflammatory, antibacterial, wound healing, skin barrier, epidermal thickness, dermal matrix, cellular stress, cellular senescence, sagging, and wrinkles.

124. 117. The method of claim 116, wherein each of the at least one sensor comprises a viscoelastic sensor, a moisturization sensor, a pH sensor, an oil content sensor, a barrier function sensor, a pigmentation sensor, and a skin surface sensor.

125. 117. The method of claim 116, wherein said scoring step comprises calculating the percent change in each characteristic from normal or vehicle conditions.

126. 117. The method of claim 116, wherein the step of scoring comprises characterizing the method used to obtain the features.

127. 117. The method of claim 116, wherein an oscillatory pressure is applied to the perfused composition to promote filling and draining of the perfused composition.

128. 1. An ex vivo skin tissue system comprising one or more inserts, each of said one or more inserts comprising: a drip reservoir at the proximal end; a basal reservoir at the distal end; a via sandwiched between the drip reservoir and the base reservoir; and a tissue sample positioned inside at least one of the drip reservoir and the basal reservoir; Including; The ex vivo skin tissue system, wherein the vias are configured to promote one or more of mass transport and cell growth from seeded cells into the tissue sample, the vias having a first side facing the opening of the drip reservoir and a second side facing the opening of the basal reservoir, and the ex vivo skin tissue system is capable of supporting tissue viability for a period of more than one week.

129. 129. The ex vivo skin tissue system of claim 128, further comprising an adipose layer positioned inside at least one of the drip reservoir and the basal reservoir.

130. 130. The ex vivo skin tissue system of claim 129, wherein the via is in contact with at least one of the fat layer and the tissue sample.

131. 129. The ex vivo skin tissue system of claim 128, wherein the tissue sample comprises at least one of the epidermis layer, the dermis layer, and the subcutaneous tissue layer.

132. 129. The ex vivo skin tissue system of claim 128, wherein the vias comprise a porosity gradient.

133. 129. The ex vivo skin tissue system of claim 128, wherein the via comprises a porous layer, the porous layer comprising a plurality of pores.

134. 134. The ex vivo skin tissue system of claim 133, wherein each of the plurality of pores is in the shape of one or more of a gyroid, a Schwarz, an x-cell, and an intersection.

135. 129. The ex vivo skin tissue system of claim 128, further comprising a gel layer in contact with the second side, the gel layer configured to receive at least one of adipose-derived cells, dermal cells, and endothelial cells.

136. 136. The ex vivo skin tissue system of claim 135, wherein the gel layer comprises a synthetic gel material.

137. 136. The ex vivo skin tissue system of claim 135, wherein the gel layer comprises an extracellular matrix.

138. 129. The ex vivo skin tissue system of claim 128, wherein the seeded cells comprise one or more of primary cells, iPSCs, keratinocytes, fibroblasts, melanocytes, endothelial cells, resident immune cells, circulating immune cells, stem cells, adipocytes, and microorganisms.

139. 129. The ex vivo skin tissue system of claim 128, wherein the drip reservoir, base reservoir and vias are sized to have a Bond number <1.

140. 129. The ex vivo skin tissue system of claim 128, wherein the via comprises an axially straight interior wall.

141. 129. The ex vivo skin tissue system of claim 128, further comprising a growth medium in contact with the tissue sample.

142. 142. The ex vivo skin tissue system of claim 141, wherein the growth medium is maintained at between 32 and 37 degrees Celsius.

143. 142. The ex vivo skin tissue system of claim 141, wherein the growth medium comprises at least one test agent.

144. 129. The ex vivo skin tissue system of claim 128, further comprising at least one test agent in contact with the drip reservoir.

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