In a previous article we introduced keratinocytes and looked at their biological functions and subtypes. Here, we explore some of the main reasons researchers study keratinocytes and the various approaches used. We focus on 2D assay formats, outlining their advantages and limitations, and provide a preview of how these compare with emerging 3D skin models. But first, let’s start with a brief recap on keratinocytes.
Why do keratinocytes matter so much in skin biology?
Because they make up the majority of the epidermis, keratinocytes are central to skin health and disease. They don’t just form a physical barrier; they actively communicate with other skin cells, respond to environmental stress, and play key roles in wound healing and immunity.
Comprising more than 90% of all epidermal cells, keratinocytes act as the body’s first line of defense. They protect against UV radiation by taking up melanosomes from melanocytes, help prevent invasion by pathogens, and minimize heat, solute, and water loss. Importantly, not all keratinocytes are equal. They are highly differentiated cells, and depending on the epidermal layer in which they reside, they express distinct keratin isoforms that contribute to epidermal structure, resilience, and overall skin toughness.
What kinds of studies use human keratinocytes?
Given their protective roles in skin health and barrier function, it’s not surprising that keratinocyte research often focuses on improving our understanding of skin diseases and aiding drug discovery efforts to develop new therapies. Some of the major research areas involving keratinocytes include:
What role do keratinocytes play in skin health and disease?
Skin barrier function and immunity. Keratinocytes are the predominant cell type in the epidermis, and together with melanocytes, Langerhans and Merkel cells, form the skin’s protective barrier. Beyond their structural role, keratinocytes are important in immune signaling, producing cytokines and chemokines that regulate inflammatory responses. By studying keratinocyte differentiation, metabolism and keratin production, researchers aim to dissect how defects in these processes contribute to inflammatory and autoimmune conditions such as atopic dermatitis, ichthyosis, psoriasis, and pemphigus (a group of rare blistering diseases), to better understand disease mechanisms and develop targeted therapies.
Wound healing. Keratinocytes are central to re-epithelialization, which is the final stage of wound healing in which the wound is resurfaced with new epithelium. Research into how keratinocytes migrate, proliferate, and interact with fibroblasts and immune cells may lead to new therapeutic applications to close and heal acute and chronic wounds, and to accelerate healing of burns, ulcers, and other skin traumas.
Aging research. Intrinsic aging processes and environmental stressors such as UV radiation alter keratinocyte biology by inducing oxidative stress, DNA damage, altered cell cycle regulation, and senescence. These changes reduce the cells’ regenerative capacity and weaken the skin’s barrier. Researchers in this area explore how aging impairs keratinocyte function and aim to develop ways to preserve skin resilience and boost repair.
Drug testing and toxicology. As the first cells exposed to topical treatments, e.g., steroid and anti-itch creams, cosmetics, and environmental chemicals, keratinocytes are widely used in in vitro assays to determine the safety (toxicity) and efficacy of investigational drugs.
Cancer research. Keratinocytes can undergo malignant transformation, giving rise to basal and squamous cell carcinomas. Their interactions with melanocytes also influence melanoma biology, making keratinocytes essential for studying tumor initiation, progression, and therapeutic response to cancer drugs.
Regenerative medicine. Keratinocytes form the basis of tissue-engineered grafts and skin substitutes for patients with burns, chronic wounds, and surgical defects. More recently, keratinocyte-derived exosomes have emerged as promising mediators of tissue repair and regeneration, expanding their potential in regenerative medicine.
What ethical, cost, and relevance-to-humans factors influence keratinocyte research?
What are the latest regulatory recommendations? One major driver in developing in vitro cell-based models is the global focus on reducing animal testing, in line with the 3Rs (replacement, reduction, refinement). Regulatory initiatives encourage the use of alternative models for medical research and drug safety testing. For example, in the European Union, this is reflected in OECD-validated assays and guidance from organizations such as EURL-ECVAM, while the U.S. Food and Drug Administration promotes the implementation of Alternative Methods, including New Approach Methodologies (NAMs).
How can we sustain the cost? Which aspects can be modeled in vitro using cell based models? Although assay development can be very costly upfront, once established, the assays can be standardized, adapted for high-throughput, and potentially reused across multiple projects. High-quality in vitro assays can be used to study skin functions such as barrier integrity, elasticity, blood flow, mitochondrial activity, and regenerative capacity, as well as cellular responses to drugs and environmental factors, offering long-term savings, scalability, and human-relevant data compared with repeated in vivo studies.
What methods do researchers use to model the skin?
2D monolayers – the traditional approach
Keratinocyte or fibroblast 2D monolayers grown on cell culture plates, with or without the addition of extracellular matrix components to provide cues of the native 3D skin environment, remain the most widely used in vitro models to study the skin. These are straightforward and cost-effective to establish, support high-throughput applications, and are widely used in early-stage drug toxicity screening and in vitro studies of molecular mechanisms involved in aging, homeostasis and other processes. However, their predictive power is limited because they lack the cellular complexity and 3D structural organization of native skin, do not recapitulate the complex cell-cell or cell-matrix communication that occurs in skin, and lack a proper skin barrier.
Why culture keratinocytes in 2D/monolayer?
A wide range of biochemical, histochemical, and immunoassay assays have been developed to study keratinocyte biology in 2D monolayers, each designed to capture specific aspects of skin function or stress response. Widely used examples include:
Inflammatory signaling. Keratinocytes produce and secrete pro-inflammatory cytokines including certain interleukins, TNF, chemokines, interferons and Thymic Stromal Lymphopoietin (TSLP). These mediators recruit specialized leukocytes to the site of injury or infection for a coordinated response to the specific threat. ELISAs and multiplex bead-based assays are commonly used to measure cytokines secreted under various conditions, to provide insights into inflammatory skin diseases such as psoriasis and atopic dermatitis, as well as screening of candidate inflammation-modulating compounds.
Mitochondrial health. Cellular metabolism and stress pathways can be probed using mitochondrial assays. For example, oxidative stress and membrane potential are often assessed using fluorescent dyes such as TMRM or JC-1, which accumulate in mitochondria in proportion to the membrane potential. A drop in fluorescence (TMRM) or a shift from red to green emission (JC-1) indicates depolarization, providing a readout of oxidative stress or mitochondrial dysfunction. These assays shed light on how keratinocytes respond to stress, aging, and regenerative signals.
UV and aging. Exposure to UV light is widely used as a model to study extrinsic aging of the skin, since keratinocytes are the first cells to absorb UVB radiation. Typical readouts include DNA damage (via γH2AX foci staining or comet assays to detect DNA fragmentation), oxidative stress, and markers of cellular senescence such as SA-β-galactosidase (measured histochemically) and p16 (measured via immunoassay). These assays can also be combined with exposure to antioxidants or compounds that alter DNA repair processes to investigate keratinocytes’ protective strategies. Intrinsic skin processes are assayed using similar readouts that reflect keratinocyte proliferation, differentiation, and barrier integrity.
Barrier function. Keratinocyte-driven barrier properties can be evaluated using transepithelial electrical resistance (TEER), which quantifies ionic permeability, or dye penetration assays, which measure diffusion of tracer molecules across the keratinocyte layer. These methods are often applied to cytokine-treated keratinocyte cultures or co-culture models to mimic in vivo barrier disruption observed in inflammatory skin diseases such as atopic dermatitis or psoriasis. Barrier recovery assays following mechanical or chemical disruption are also used to investigate how drugs, biomaterials, or environmental exposures influence barrier repair.
Wound healing. Keratinocyte-based scratch or migration assays are widely used to model re-epithelialization (wound closure) by tracking the migration of cells into a defined gap, while transwell migration assays allow analysis of directed cell movement. These in vitro systems are applied to study impaired healing in chronic wounds, ulcers, and burns, and to evaluate candidate therapies that aim to accelerate closure and restore barrier function.
What are 3D in vitro skin models, and why are they important?
In this article we focused primarily on keratinocyte monolayers and their use in 2D assay formats. However, 3D skin models are becoming increasingly important for replicating the structural and functional complexity of human skin. The table below offers a quick side-by-side comparison of 2D and 3D approaches.
Stay tuned, because we will dig deeper into 3D in vitro human skin models, their applications, and specialized research approaches, such as patient-derived models, gene editing, and tumor spheroids, in a future article!
| Feature | 2D Models | 3D Models |
| Structural complexity | Simple, flat monolayer, usually comprising one or two cell types. | Stratified, multi-layer structure that aims to mimic the epidermis and dermis. |
| Cellular interaction | Limited cell-cell and cell-matrix communication. | More accurately replicates interactions between different skin cell types and the extracellular matrix, but lacks neural compartment precluding the study of sensations such as pain or itch. |
| Assays of biological functions | Facilitates the study of basic phenotypes such as cytotoxicity, proliferation, migration, and simple stress readouts, e.g., oxidative stress, mitochondrial activity. | Allows investigation of broader functional phenotypes including barrier integrity (e.g., transendothelial electrical resistance or TEER), differentiation and stratification, inflammatory signaling, and response to topical drugs or other agents. |
| Frequently used applications | High-throughput compound screening, cytotoxicity testing, and mechanistic assays, including:
scratch assays to analyze keratinocyte or fibroblast migration on 2D surfaces in the context of wound healing. ECIS (Electric Cell-Substrate Impedance Sensing) assays for measuring cell viability, attachment, and motility. Assays to study signaling cascades, e.g., c-Src activation as a readout for skin irritation. Melanocyte-keratinocyte co-cultures for pigmentation studies to gain insights into melanogenesis and the mechanisms of melanin transfer. Cytokine profiling to study cytokine-induced gene expression profiles, e.g., exposing 2D skin cell cultures to specific cytokine cocktails to study the cellular and molecular aspects of diseases such as atopic dermatitis. |
Regulatory toxicology, skin irritation tests, and efficacy studies for drugs and cosmetics, including:
OECD-validated assays for skin corrosion (TG 431) and irritation (TG 439) using Human Skin Equivalents and Reconstructed Human Epidermis. Permeation studies using Skin-on-a-Chip platforms to assess percutaneous absorption and transdermal transport. Disease modeling through cytokine-treated 3D constructs to study inflammatory conditions such as psoriasis and atopic dermatitis. Wound healing assays analyzing re-epithelialization processes using excisional and burn wound models. Cancer research using 3D tumor spheroids to study melanoma invasion and test therapeutic compounds targeting cellular and molecular mechanisms of skin malignancies. |
Get in touch with Tempo!
If you are working with or considering working with keratinocytes, and would like to learn more about the considerations mentioned above, or have any other question about working with this cell type, please do not hesitate to get in touch with us here. We are here to support your projects!
References:
- Quílez C, Bebiano LB, Jones E, et al. Targeting the Complexity of In Vitro Skin Models: A Review of Cutting-Edge Developments. J Invest Dermatol. 2024 Dec;144(12):2650-2670.
- Moniz T, Costa Lima SA, Reis S. Human skin models: From healthy to disease-mimetic systems; characteristics and applications. Br J Pharmacol. 2020 Oct;177(19):4314-4329.
- Hofmann E, Fink J, Pignet AL, et al. Human In Vitro Skin Models for Wound Healing and Wound Healing Disorders. Biomedicines. 2023 Mar 30;11(4):1056.
- Simmons J, Gallo RL. The Central Roles of Keratinocytes in Coordinating Skin Immunity. J Invest Dermatol. 2024 Nov;144(11):2377-2398.
- Gallegos-Alcalá P, Jiménez M, Cervantes-García D, Salinas E. The Keratinocyte as a Crucial Cell in the Predisposition, Onset, Progression, Therapy and Study of the Atopic Dermatitis. Int J Mol Sci. 2021 Oct 1;22(19):10661.
Karen O’Hanlon Cohrt is an independent Science Writer with a PhD in biotechnology from Maynooth University, Ireland (2011). After her PhD, Karen relocated to Denmark where she held postdoctoral positions in mycology and later in human cell cycle regulation, before moving to the world of drug discovery. Karen has been a full-time science writer since 2017, and has since then held numerous contract roles in science communication and editing spanning diverse topics including diagnostics, molecular biology, and gene therapy. Her broad research background provides the technical know-how to support scientists in diverse areas, and this in combination with her passion for learning helps her to keep abreast of exciting research developments as they unfold. Karen is currently based in Ireland, and you can follow her on Linkedin here.
