Abstract

Recent innovations in skin microphysiological systems (MPSs) have gained momentum following regulatory advances such as the FDA Modernization Act 2.0 and the global shift toward alternatives to animal testing. This review highlights the development of three major technologies—3D bioprinting, skin organoids, and skin-on-a-chip—and their roles in replicating human skin physiology for research and preclinical applications. We examine how these platforms model complex skin functions, including epidermal barrier formation, vascular and immune interactions, and disease phenotypes such as psoriasis, atopic dermatitis, melanoma, and viral infections. In addition to summarizing their utility in toxicological screening and therapeutic evaluation, we explore how current OECD test guidelines may guide future validation efforts. Finally, we discuss emerging strategies for integrating automation and machine learning-based image analysis to enable scalable, high-content screening of skin MPS models across diverse applications.

Introduction

Conventional methods for evaluating skin irritancy and corrosiveness, such as animal testing and Transwell-based human skin equivalents (HSEs), remain widely used but have intrinsic limitations. These models often fail to capture the structural and functional complexity of human skin, including its dynamic responses to long-term stimuli, vascular interactions, and immune involvement. In contrast, microphysiological systems (MPSs) are in vitro models that replicate the three-dimensional (3D) architecture, cellular diversity, and functional responses of human tissues under physiologically relevant conditions. These platforms often incorporate elements such as fluid perfusion, mechanical stimulation, or multicellular co-culture to reproduce tissue-level behaviors. Skin MPS are a subset of MPS that specifically model human skin, including its stratified structure, barrier function, and interactions with vascular, lymphatic, and immune components1,2. Skin MPS encompasses three major approaches: skin-on-a-chip devices, bioprinted skin constructs, and skin organoids. By capturing these integrated features, skin MPS provides more physiologically relevant, dynamic, and reproducible platforms than conventional 2D cultures or static Transwell-based HSEs3,4.

The enactment of the FDA Modernization Act 2.0 and the Modernization of Cosmetics Regulation Act (MoCRA) represents a pivotal shift in preclinical testing, formally supporting non-animal approaches, including MPS and organotypic models5,6. These reforms align with global initiatives such as the EU Cosmetics Regulation and REACH, reflecting an accelerating movement toward ethical and human-relevant testing7,8,9. However, widespread adoption of skin MPS still faces significant real-world barriers. High validation costs, limited inter-laboratory reproducibility, and slow regulatory acceptance hinder their routine use in regulatory submissions. Achieving equivalency or superiority to legacy animal models requires harmonized protocols, standardized readouts, and robust regulatory-grade datasets, which are resource-intensive to generate and maintain. These challenges underscore that while legislative changes support MPS, practical implementation remains an ongoing developmental process.

Amid these challenges, skin MPS are emerging as versatile platforms for mechanistic skin biology studies, long-term toxicology, and disease modeling, with clear potential for regulatory-compliant and industrial applications. This review highlights recent advances in skin-on-a-chip systems, 3D bioprinted skin, and skin organoids, emphasizing their advantages over conventional models and their role in enabling the transition away from animal testing. It also incorporates regulatory and industrial perspectives to provide a realistic framework for translating these advanced in vitro platforms into real-world preclinical and cosmetic testing.

Current OECD test methods

The OECD guidelines provide a standardized framework for evaluating the preclinical safety of cosmetic and pharmaceutical substances for skin and eye irritation. Key methods include OECD Test Guideline (TG) 430, which measures skin corrosion through transepithelial electrical resistance (TER), and TG 431 and TG 439, which assess skin corrosion and irritation using reconstructed human epidermis (RHE) models. For ocular safety, TG 437 and TG 438 rely on bovine and chicken eye tissues to assess irritation and corrosion, while TG 492 employs reconstructed human cornea-like epithelium (RhCE) models. These tests have successfully established global standards for hazard identification and risk assessment, but their ability to fully capture human tissue complexity is limited.

A major limitation of current OECD test methods is the absence of vascularization and immune system components. Human skin and eyes are highly vascularized and immunologically active tissues. Vascular networks provide nutrient delivery, waste clearance, and systemic signaling, which are essential for tissue repair and recovery after chemical exposure. Immune cells such as macrophages and T cells mediate inflammation, regulate barrier function, and contribute to both acute and chronic responses. Standard RHE or RhCE models in TG 431, TG 439, and TG 492 lack these features, which prevent accurate modeling of inflammation, delayed hypersensitivity, or chronic toxicity. Consequently, compounds that may trigger vascular leakage, prolonged immune activation, or systemic effects can go undetected in these static tests10,11,12.

Ocular models like TG 437 (BCOP) and TG 438 (ICE) rely on non-living animal tissues that cannot regenerate or mount immune responses, and therefore cannot predict cumulative or repeated exposure effects13. Similarly, phototoxicity tests like TG 498 capture only short-term damage, overlooking long-term outcomes such as DNA repair, skin aging, or carcinogenesis14. Without vascular and immune features, current OECD methods provide only surface-level hazard information, which is insufficient for predicting real-world human responses, especially under repeated or chronic exposure.

These limitations highlight the urgent need to incorporate dynamic, vascularized, and immunocompetent models into regulatory testing15. Next-generation skin MPS—including skin-on-a-chip, bioprinted constructs, and skin organoids—offer multi-cellular interactions, perfusion, and immune integration that can overcome the critical gaps of current OECD methods. Table 1 summarizes the key OECD guidelines for skin and ocular safety assessments and the specific physiological gaps that next-generation platforms are designed to address.

Transition to skin MPS in addressing OECD method limitations

The current OECD guidelines provide a structured framework for evaluating skin and ocular irritation, corrosion, and phototoxicity, but they are primarily based on simplified static models and non-human tissues. This results in a limited ability to capture the complex biological interactions that occur in human skin. Key limitations include the inability to model immune cell recruitment, vascular perfusion, tissue repair, and chronic exposure responses. Conventional methods also lack integration with real-time sensing or controlled fluidic flow, which reduces precision and reproducibility.

To meet regulatory requirements within these constraints, preclinical testing has traditionally relied on animal models and HSEs16,17. Animal models provide systemic responses but frequently fail to translate to human outcomes, contributing to drug attrition rates of more than 80% in clinical trials for dermatological compounds18,19. HSEs, while widely adopted, typically consist of an epidermal layer of keratinocytes over a fibroblast-embedded dermal matrix and can reproduce differentiation markers such as filaggrin and keratic 10. However, they lack vasculature and immune components, making them unable to model inflammation, systemic absorption, or long-term regeneration20,21. Furthermore, static Transwell cultures require labor-intensive handling and show batch-to-batch variability, with reported inter-laboratory reproducibility below 70% for irritancy tests22,23.

Advanced MPS overcomes these limitations by recapitulating key structural and dynamic features of native human skin (Fig 1a, b). Skin-on-a-chip platforms incorporate microfluidic perfusion, which supports continuous nutrient delivery and waste removal, and have demonstrated up to a 90% reduction in variability for barrier integrity measurements compared with static HSEs24,25. Bioprinted skin allows precise spatial placement of keratinocytes, fibroblasts, melanocytes, and even endothelial cells, achieving layered architecture and enabling high-throughput drug screening with over 85% reproducibility across runs26. Skin organoids self-organize from stem or progenitor cells and can capture disease-relevant microanatomy such as basal cell clustering and hair follicle-like structures, allowing chronic toxicity testing that is not possible with current OECD models (Fig. 1c).

Fig. 1: Schematic representation of skin MPS as advanced in vitro test platform.

These platforms recapitulate the multilayered architecture of human skin using primary or cell line-derived keratinocytes, fibroblasts, and endothelial cells, and support integrated assay systems for dynamic response tracking. a Human skin structure composed of epidermis, dermis, and vasculature, with representative cell types. b Skin MPS design mimicking multilayered skin architecture under perfusable conditions. c Skin MPS analysis incorporating high-content imaging, real-time viability tracking, and automated readouts. Created with BioRender.com

By providing human-relevant architecture, dynamic physiological cues, and higher reproducibility, skin MPS enhances predictive accuracy and reduces reliance on animal testing. Figure 2 summarizes how these platforms occupy a unique space with both high physiological relevance and increasing scalability, representing a clear step beyond conventional OECD-guided methods. The following sections detail their core principles, representative applications, and recent innovations.

Fig. 2: Comparative overview of preclinical models as new approach methods.

This illustration compares various preclinical test models in terms of scalability, reproducibility, and physiological relevance to the human body. The models range from traditional approaches such as 2D cell culture and animal testing to more advanced technologies, including 3D cell culture (e.g., human skin equivalent, HSE), 3D bioprinting, organ-on-a-chip systems, and organoids. The diagram highlights a paradigm shift toward MPS that more accurately recapitulates human tissue architecture and function. Created with BioRender.com

3D Bioprinting technologies for skin research

Fundamentals of 3D bioprinting

3D bioprinting is an advanced biofabrication technique that constructs 3D biological tissues by the precise layer-by-layer deposition of bioinks containing living cells and biomaterials27,28,29. This approach enables the spatial organization of multiple cell types and biomaterials to recreate the structural and functional complexity of native tissues30,31. In the context of skin research, 3D bioprinting provides a platform to generate constructs that recapitulate the stratified architecture of the epidermis and dermis and, in some cases, incorporate hypodermal components32. This capability supports applications in regenerative medicine, pharmaceutical testing, and the development of physiologically relevant in vitro skin models.

Bioinks requirements and functional roles in skin bioprinting

Bioinks are the core materials of 3D bioprinting, comprising living cells embedded in biomaterial matrices that provide both structural support and biochemical cues essential for tissue development33. For constructing in vitro skin models, bioinks must meet several requirements: (i) biocompatibility to maintain high cell viability and promote proliferation and differentiation, (ii) printability and rheological stability to achieve precise structural fidelity, and (iii) mechanical and degradative properties that support long-term tissue integrity without compromising nutrient diffusion34.

Each biomaterial contributes uniquely to meeting these requirements. Natural biomaterials such as collagen and alginate dominate skin bioprinting due to their excellent biocompatibility and ability to create hydrogels suitable for keratinocyte and fibroblast encapsulation35,36,37. Collagen promotes cell adhesion and extracellular matrix (ECM) deposition, while alginate allows rapid ionic gelation under mild conditions. Hyaluronic acid (HA) and gelatin are widely employed to enhance hydration, provide bioactive motifs, and support dermal matrix formation38,39. Fibrin contributes to wound-healing–related studies by supporting angiogenesis and tissue remodeling40, whereas polyethylene glycol (PEG) and its derivatives are used to fine-tune mechanical properties and degradation rates41. In clinical settings, acellular dermal matrices such as AlloDerm and Integra serve as gold standards for skin reconstruction and wound healing, and their biomechanical and biochemical characteristics continue to inform the design of next-generation bioinks for regenerative and in vitro applications. A survey of bioink usage in recent skin bioprinting studies is shown in Fig. 3, highlighting collagen (26%) and alginate (24%) as the most prevalent components, followed by hyaluronic acid (11%), gelatin (11%), fibrin (5%), and PEG (3%). The remaining 21% includes other materials such as decellularized ECM and composite hydrogels. These trends underscore the importance of balancing biological fidelity, printability, and structural stability when designing bioinks for engineered skin models.

Fig. 3: Distribution of commonly used bioink components for skin bioprinting.

The pie chart illustrates the proportion use of various biomaterials in skin bioprinting. Collagen and alginate represent the most frequently used components, followed by hyaluronic acid, gelatin, fibrin, polyethylene glycol (PEG), and other materials. These data highlight prevailing trends in bioink formulation for engineered skin tissue models, reflecting preferences based on biocompatibility, printability, and structural support

Engineered bioinks for enhanced fidelity

Engineered bioinks are advanced formulations developed to overcome the limitations of single-component bioinks by integrating biological and mechanical functionality42,43. These bioinks often combine natural polymers (collagen, gelatin) with synthetic scaffolds (PEG or PEG derivatives) to simultaneously achieve tunable stiffness, controlled degradation, and bioactive signaling44,45. Functional enhancements include the incorporation of growth factors, ECM fragments, and peptides, which guide epidermal differentiation, dermal remodeling, and basement membrane formation46,47,48. Rheological optimization, using viscosity modifiers or thixotropic agents, improves extrusion fidelity and prevents cell sedimentation, while tailored crosslinking strategies ensure structural stability.

Physical crosslinking (ionic or thermal) provides rapid yet reversible gel formation42,49, chemical crosslinking enhances mechanical robustness, and enzymatic crosslinking using agents such as transglutaminase preserves high cell viability50,51. In particular, photo-crosslinkable bioinks such as gelatin methacryloyl (GelMA) and collagen methacrylate (ColMA) have been widely adopted due to their tunable mechanical properties, high printability, and compatibility with UV-based curing52,53,54,55 By adjusting the degree of methacrylate or UV exposure time, researchers can finely control matrix stiffness to modulate cell-specific behavior. For example, stiffer GelMA substrates promote keratinocyte differentiation and stratification, while softer hydrogels may be preferred for fibroblast migration and ECM remodeling56,57. However, crosslinked biomaterials that favor one lineage (e.g., keratinocyte differentiation) may impair fibroblast migration or immune cell infiltration, highlighting the need for balanced tuning of biophysical parameters58.

These engineered bioinks directly support functional outcomes that traditional bioinks cannot reliably achieve, including sustained barrier formation, robust dermal–epidermal junction (DEJ) development, and the integration of vascular or immune components. By aligning material properties with biological performance, engineered bioinks are accelerating the fidelity and translational potential of 3D bioprinted skin for applications in drug testing, regenerative medicine, and next-generation in vitro models59,60.

3D Bioprinting for HSE

3D bioprinting has significantly advanced the fabrication of human skin equivalents (HSEs) by enabling precise, layer-by-layer deposition of bioinks containing keratinocytes, fibroblasts, and other skin-relevant cell types61,62. Unlike standard OECD-defined reconstructed skin models, which typically reproduce the epidermis and dermis in static culture inserts, bioprinted HSEs offer enhanced structural control and the potential for functional complexity. Specifically, bioprinting allows the deliberate recreation of the epidermis, dermis, and in some models the hypodermis, while establishing a well-defined DEJ with basement membrane proteins and interlayer signaling essential for barrier formation63,64,65.

To achieve functional skin construction, bioprinted models meet several key requirements: (i) accurate stratification of keratinocytes and fibroblasts to recapitulate the native epidermal and dermal compartments66, (ii) integration of ECM components that support cell adhesion and maturation, (iii) development of a functional DEJ for interlayer communication and basement membrane formation, and (iv) incorporation of elements such as vascular and neural components to maintain nutrient delivery, waste removal, and long-term tissue viability67,68,69,70. However, the limited ability of current 3D bioprinting techniques to fully replicate the spatial microarchitecture of native skin may restrict cell-cell and cell-matrix interactions, posing a bottleneck in recapitulating multi-cellular communication within skin MPS platforms.

Advanced 3D bioprinting techniques are defined as methods that allow high-resolution, multi-material printing with precise spatial control to reproduce layered skin structures while also enabling the addition of complex features such as vascular channels71, neural elements, and sebaceous appendages72. These innovations enhance the physiological relevance and translational potential of bioprinted HSEs for applications in wound healing73, grafting63, cosmetic testing74, aging75, and preclinical drug evaluation76,77,78. Recent studies have further integrated patient-derived cells, enabling personalized skin models for studying genetic disorders or individualized therapeutic responses. Complementary imaging and characterization tools, including multiphoton microscopy and Raman spectroscopy, validate the morphological and biochemical fidelity of these constructs, ensuring that bioprinted HSEs meet the functional benchmarks required for advanced in vitro skin models67,79,80,81,82 (Fig. 4).

Fig. 4: 3D Bioprinted HSEs and key advantages.

a Representative constructs containing only human epidermal keratinocytes (HEKa), only human dermal fibroblasts (HDF), or a co-culture (CC) of both cell types, demonstrating the adaptability of the printing approach for different cellular compositions. b Hematoxylin and eosin (H&E) staining showing stratified epidermal layers and organized dermal compartments. c Schematic of the construct design illustrating sequential deposition of dermal and epidermal layers. They enable reproducible fabrication of full-thickness skin with physiological architecture, supports high cell viability and balanced proliferation in co-culture, and facilitates applications in drug efficacy/safety testing, cosmetic evaluation, and replacement of animal models through simplified and cost-effective production

Skin organoids

Skin organoids represent a significant advance in in vitro skin research, offering 3D, multicellular systems that closely mimic the structure and function of human skin. Often derived from stem cells or reaggregated primary cells, these organoids are cultivated through precise differentiation protocols that simulate embryonic development83, using specific growth factors and tailored culture conditions. This approach enables the formation of stratified squamous epithelium, marking a pivotal advance in skin biology and facilitating the development of sophisticated 3D skin models84. Today, skin organoids serve as invaluable tools for disease modeling, compound screening, and regenerative medicine.

Potential applications of skin organoids in personalized modeling

Skin organoids enable the development of personalized models by leveraging patient-derived or genetically modified cells, offering genetically relevant platforms for studying skin biology and therapeutic responses. These systems provide enhanced fidelity in disease modeling through three core features: (i) the ability to recapitulate the 3D architecture of skin, including appendages such as hair follicles and sebaceous glands; (ii) the use of patient-derived or genetically modified cells for personalized and genetically relevant disease modeling; and (iii) the capacity for long-term culture, supporting studies on chronic disease progression, regenerative processes, and sustained drug responses85. These models capture individual-specific genetic backgrounds, making them powerful tools for investigating inherited disorders, patient-specific immunopathology, and tailored treatment strategies86,87. Recent advances have further improved their biological relevance by incorporating immune cells such as Langerhans cells and T-cells88, enabling the modeling of complex inflammatory skin diseases, including psoriasis, eczema, and allergic contact dermatitis. Notably, human induced pluripotent stem cells (iPSC)-derived organoids have demonstrated the formation of a functional DEJ, where basal keratinocytes adhere to a laminin-332 and collagen IV-rich basement membrane via type I hemidesmosomes and integrin β1-based adhesion complexes, closely mimicking native skin histology.

The inclusion of skin appendages allows organoids to model previously inaccessible conditions, such as alopecia89 and acne, which depend on follicular and sebaceous gland biology90,91. Furthermore, using patient-derived cells introduces a layer of personalization, making organoids powerful tools for studying inherited skin disorders and personalized therapeutic responses92. This is especially relevant when immune components are included, enabling the investigation of patient-specific immunopathology and responses to immunomodulatory treatments93. Additionally, the extended culture longevity of skin organoids facilitates long-term studies, making them valuable for modeling chronic skin disease, wound healing, and regeneration. Their complex 3D structure supports drug penetration and metabolism studies across multiple layers, improving the translational relevance of preclinical testing.

Technological innovations and methodological enhancements

Recent advances in skin organoid research have given rise to three distinct categories of organoids: (i) basic skin layer organoids, (ii) appendage-enhanced organoids, and (iii) disease-specific organoids. Each type contributes uniquely to skin biology, regenerative medicine, and practical applications such as safety assessment.

Basic skin layer organoids, typically derived from iPSCs, replicate essential skin compartments, including stratified epidermal and dermal structures. These organoids allow researchers to study key biological processes such as epidermal differentiation, barrier formation, and wound healing in a physiologically relevant 3D setting94,95,96 (Fig. 5a). Long-term culture systems support adult epidermal stem cell maintenance and basal-apical polarity, making these organoids particularly useful for studying tissue regeneration and homeostasis97.

Fig. 5: Advances in skin organoid models: from basic epidermal layers to appendage formation and disease-specific applications.

a Basic Skin Layer Organoids: Timeline and process of epidermal organoid differentiation from induced pluripotent stem cells (iPSCs). b Appendage-Enhanced Organoids: Histological and immunohistochemical analyses of skin organoids at Day 2 and Day 6, showing the expression of markers such as H&E, TCHH, and HPSE1. c Disease-Specific Organoids: Confocal microscopy images of melanoma patient-derived organoids cultured in Matrigel and Collagen I, displaying marker expression, including HMB-45, α-SMA, Vimentin, and ICAM-1. Images reprinted from references96,108,179 with permission

Appendage-enhanced organoids represent a major step forward in physiological relevance. They contain structures such as hair follicles98,99,100,101 and sebaceous glands90,102, which are formed through self-organizing epithelial–mesenchymal interactions that recapitulate embryonic skin development. In iPSC–derived models, signaling pathways including Wnt, BMP, and FGF are modulated to guide follicle development. Dermal condensate formation supports follicle-like structures, while sebaceous glands arise through regionally guided differentiation[102](https://www.nature.com/articles/s41378-025-01149-1#ref-CR102 “Lee, J. et al. Hair follicle development in mouse pluripotent stem cell-derived skin organoids. Cell Rep 22