Human skin is organized into three principal layers—the epidermis, dermis, and hypodermis—connected by the dermo-epidermal junction, a specialized multilayered basement membrane that anchors the epidermis to the underlying dermis and regulates molecular exchange. The epidermis is an avascular, continuously renewing tissue composed of two main compartments: the interfollicular epidermis (IFE) and the pilosebaceous unit, which includes hair follicles and sebaceous glands. Epidermal stem cells represent a heterogeneous population of long-lived cells that sustain epidermal homeostasis and regeneration; within this broader group, KSCs specifically denote interfollicular epidermal stem cells committed to the keratinocyte lineage. The IFE is stratified into four sequential layers—basal, spinous, granular, and cornified—each corresponding to a defined stage of keratinocyte differentiation. KSCs reside in the basal layer immediately above the basement membrane, where they proliferate and initiate the upward migration that underpins continuous epidermal turnover. Derived from the embryonic ectoderm, keratinocytes constitute approximately 90% of epidermal cells and execute a tightly regulated program of proliferation, differentiation, and barrier formation (7).

KSCs of the skin reside in specialized microenvironments—niches—comprised of multiple cell types and key regulatory cues, including extracellular matrix (ECM) components, direct cell–cell interactions, and soluble growth factors. Homeostasis of the IFE depends on KSCs to continuously replace shed cells and to mount repair responses following injury. These SCs adhere directly to the basement membrane in the lower basal layer, where they divide by a process known as population asymmetry—typically yielding one SC and one transit-amplifying (TA) progenitor cell, although symmetric divisions (producing two SCs or two TA cells) can occur under specific demands (8). TA cells proliferate for a limited number of cycles before committing to terminal differentiation and migrating upward through the spinous, granular, and cornified layers to become corneocytes. This withdrawal from the cell cycle and progression through differentiation is collectively termed keratinization. Within the basal layer, a dynamic coexistence of slow-cycling SCs and more rapidly dividing progenitor cells maintains both long-term tissue renewal and everyday turnover (9). The Keratinocyte SCs and their differentiated progeny that are migrating upwards are organized into cone-like columns recognized as epidermal proliferation units (EPU)- reflecting the organized, hierarchical nature of epidermal maintenance and migration (10). Collectively, epidermal SCs exhibit the two hallmark properties of all stem cells: self-renewal—by division within the basal niche to preserve the stem cell pool—and differentiation—upon exit from the niche, initiating the terminal program that culminates in corneocyte formation.

Basal keratinocytes continuously choose between self-renewal (to sustain the proliferative basal compartment) and differentiation (to construct and repair the epidermal barrier). Extrinsic niche cues—integrins/ECM, EGFR ligands, Wnt, Notch, Ca²⁺/CaSR, TGF-β—bias this decision, while intrinsic programs— epigenetic memory, proteostasis/autophagy, DNA-damage checkpoints, Redox/KEAP1–NRF2 switch, asymmetric division—set thresholds and timing. Mechanistically, fate resolves through a small set of convergent “locks”: an identity switch (ΔNp63 vs. Notch/IRF6-KLF4/GRHL3/OVOL), a cell-cycle gate (E2F drive vs. p21/p27-RB arrest), and a mechanotransduction gate (nuclear YAP/TAZ under adhesion/tension vs. Hippo/LATS-mediated shutdown). In what follows, each pathway is discussed in terms of where it lands on these locks to push keratinocytes toward renewal or commitment (Figure 1).

Proper isolation epidermal KSCs that is fibroblast-free with minimal damage is a key step to ensure good supply of primary keratinocyte for cell culture. Obtaining skin biopsies from donors is the first step in acquiring and isolating primary keratinocytes. Various techniques and sources for skin biopsies are available and they are considered minimally invasive. Some of the most commonly used techniques are punch biopsy and shave biopsy (41). Alternatively, skin samples can be easily obtained during other medical interventions such as Cesarean patients, reconstructive abdominal plastic surgery, breast reduction and circumcision which is common to obtain neonatal cells. After harvesting the skin and soaking it in 70% ethanol for 1 min, samples should be immediately conserved in sterile pad soaked with saline solution or submerged is a saline solution. Commonly, Dulbecco's minimal essential medium (DMEM) or Minimum Essential Medium (MEM) supplemented with antibiotics and Fetal bovine serum (FBS) or fetal calf serum are used as a transport medium. Alternatively, HBSS or other media only supplemented with antibiotics can also be used. Tissue samples and transport media should be ideally kept at 4 C and samples can be stored for up to 24 h at 4 C in the transport medium before processing.

The processing of the skin sample starts by several washing steps typically with antibiotic supplemented media or PBS. Under sterile conditions, the skin samples are scraped off hypodermis, flattened and cleaned off all subcutaneous elements such as adipose tissue. This is followed by few more washing before moving to the Keratinocyte isolation step. Majority of protocols use enzymatic methods that helps in separating epidermis from dermis, and this can be either a one step or two step procedure (Table 1). The initially introduced one step method relies on trypsin alone for digestion of finely minced skin and withdrawing single-cell suspension every 30 min (42). Commonly, a trypsinization flask is used to help in decanting the cell suspension while preventing undigested tissue from being poured out. Further improvements on this method involves the use vortexing or magnetic stirring, with the later showing improved cell isolation yield an colony forming efficiency in recent studies (43). Type II collagenase is also another enzyme used in single step isolations where cell suspension is collected and plated after overnight incubation of skin samples (44). Additionally, a recently optimized enzymatic combination employing hyaluronidase and collagenase I has demonstrated superior performance in terms of cell yield and viability (45). This protocol involves the digestion of finely minced epidermal tissue using hyaluronidase and collagenase I, facilitating efficient release of keratinocytes while preserving stem-like characteristics. The method has shown promise particularly in adult skin processing, which remains more resistant to enzymatic dissociation.

Improved two-step methods that are less fibroblast-contaminated with more cell yield have been later introduced (46). In the two-step method, dermo epidermal separation is achieved through a protease treatment before digestion with trypsin. While the two-step method separation can also be achieved with trypsin-HBS mixture, the usage of trypsin is associated with decrease in cell viability (47, 48). Hence, the use of neutral proteases such as dispase and thermolysin for dermo epidermal separation is favored (49). Actinidin-extracted from kiwi fruit- is another recently reported cysteine protease that was successfully implemented in the digestion and separation of dermis and epidermis (50, 51).

In addition to these established methods, several commercially available alternatives have been introduced for improved cell viability and xeno-free processing. TrypLE Select, a recombinant, animal-origin-free enzyme, serves as a gentler substitute for traditional trypsin. It is applied after dermo-epidermal separation and is reported to preserve cell surface epitopes better than conventional trypsin; however, it generally results in lower cell yields (52). Similarly, Accutase, another recombinant proteolytic enzyme mixture, has been employed for keratinocyte isolation due to its reduced proteolytic harshness and xeno-free composition (52). Although both TrypLE™ Select and Accutase® offer improved biocompatibility and reduced enzymatic damage relative to conventional trypsin, several studies have reported a modest reduction in keratinocyte yield when using these alternatives. Nonetheless, other investigations have demonstrated that TrypLE™ and Accutase® can achieve comparable cell yield and viability to those obtained with standard trypsin–EDTA digestion protocols (53, 54).

Liberase, a proprietary enzyme blend composed of highly purified collagenase isoforms and thermolysin, has also been used for primary keratinocyte isolation. It facilitates dermo-epidermal separation and partial tissue digestion with reduced variability and improved reproducibility compared to crude collagenase preparations. Depending on the specific formulation (e.g., Liberase DH or TM), it may be used alone or in combination with other proteolytic agents, and has demonstrated favorable outcomes in terms of keratinocyte viability, particularly in protocols involving adult human skin (55).

While the two-step methods are well-established and proved to work well for neonatal tissue, further improvement is still required for adult tissue isolations as it is more challenging. Furthermore, long and non-optimal digestion conditions in addition to enzyme batch differences can lead to low keratinocyte yield and viability. One proposed solution is the use of one step digestion isolation followed by a selective media that only enriches for keratinocytes, thus omitting the dermo-epidermal separation step and quickly processing the skin samples (56). Other techniques utilized for further enrichment of high-colony forming keratinocytes after isolation include density gradient centrifugation, gravity-assisted cell sorting, and cell sorting with specific marker antibodies (46). However, methods of isolation that don't rely on enzymatic digestion at all also been proposed and successfully implemented. In explant isolation method, keratinocytes migrate out of the plated skin explants and grow on the culture vessel which later on can be passaged out (57). The simple explant method takes advantage of the time lag between the migration of keratinocyte vs. fibroblast to isolate rapidly growing primary keratinocyte cells that are fibroblast-contamination free.

Currently, two general approaches are used for the 2D culture of keratinocytes in vitro: the first involves the use of a feeder layer of either murine 3T3 fibroblasts or human dermal fibroblast (HDF). The second, truly feeder-free strategy, combines serum-free, chemically defined media with a wide range of basement membrane–mimetic substrates and coatings, or even biomimetic hydrogels—to foster keratinocyte attachment, proliferation, and differentiation without the use of animal-derived feeder layers (Table 2) (58). Since 1975, the method described by Rheinwald and Green has been mainly employed to culture keratinocytes. This method is based on the co-culture of human keratinocytes that is in contact with irradiated, non-proliferating murine 3T3 fibroblast that acts as a feeder cell layer. The feeder cells support the growth of keratinocyte through a complex but still ill-defined mechanism. Even though various types of murine and human fibroblast have been reported, thee in vitro–stabilized murine 3T3-J2 cell line has the most established track record and is considered the gold standard in supporting keratinocyte culture. The feeder layer is prepared by exposing the 3T3 cells to high dose of gamma rays to render them non-proliferating. The keratinocytes then are seeded onto the monolayer of the irradiated 3T3 cells in a basal medium supplemented with fetal bovine serum (FBS) and other components. Additionally, a high dose of adenine is typically added to inhibit the proliferation of the contaminating fibroblast in the initial cultures (59). For further propagation, cells are passaged into new culture vessels prepared with 3T3 feeder monolayers. Rheinwald and Green's culture technique is well-established and has yielded positive results in clinic settings. However, the use of the feeder co-culture system brings some disadvantages and risks as it relies on animal derived cells and components such as FBS. This could expose the patients to toxins, zoonotic pathogens and immunogenic agents, hence limiting their use in treatments and other clinical applications. Therefore, efforts have been established to develop serum-free medium and to replace murine feeder cells with HDF for safe and effective culturing of keratinocytes.

As opposed to the murine 3T3 feeder system, an alternative HDF based feeder method has been developed to mitigate the use of animal derived cells. Similar to the 3T3 cells, post mitotic or irradiated HDF feeder layer is established before culturing the keratinocytes. Further efforts also reported the use of co-culturing system that utilizes non-irradiated autologous HDFs for keratinocyte expansion (60). This system is much safer and omits the need to test for infectious diseases, however, HDF feeder co-culture remains an undefined system due to the use of FBS which contains serum. Consequently, a research extension of this culture system further improved the conditions with the use of non-irradiated HDF layer and serum free media for keratinocyte expansion. Stark et al. demonstrated that primary human keratinocytes, when co-cultured atop collagen-embedded dermal fibroblasts in a serum-free, chemically defined medium, could form well-stratified, orthokeratinized epithelia expressing differentiation markers (keratins 1/10, involucrin, filaggrin) at par with traditional serum-containing systems (61). Building on this, Higham et al. (2003) enhanced keratinocyte attachment and expansion using plasma-polymer-coated surfaces combined with irradiated dermal fibroblasts, providing a chemically defined feeder layer alternative (62). Later, Mujaj et al. (2010) introduced a recombinant-protein-based, serum-free co-culture medium capable of supporting the parallel expansion of both keratinocytes and fibroblasts, further advancing defined culture systems without serum or animal-derived components (63). Most recently, Ghio et al. (2023) developed Surge SFM, a chemically defined, serum-free medium supplemented with fibroblast feeders, which maintains a proliferative K19⁺ epithelial stem cell population across passages, supports full epidermal stratification, and enables long-term graft persistence in vivo (64). Collectively, these advances reflect a progressive refinement of co-culture strategies toward clinically applicable, xeno-free skin regeneration platforms.

The development of clinically translatable keratinocyte culture systems began in earnest in 1983 with Boyce and Ham's landmark serum-free, feeder-free medium (MCDB 153), which replaced Rheinwald and Green's murine feeder- and serum-dependent method by leveraging low calcium (0.1–0.3 mM) to maintain proliferative undifferentiated states while suppressing fibroblasts (65). This formulation introduced defined supplements like epidermal growth factor (EGF), insulin, hydrocortisone, phosphoethanolamine, and monoethanolamine. However, it retained a critical limitation: optimal clonal growth required bovine pituitary extract (BPE), an undefined component prone to batch variability and safety risks 16. In subsequent decades, commercial media such as Lonza's KGM (based on MCDB-153), Gibco's DK-SFM (Defined Keratinocyte-SFM) and Gibco's EpiLife® EDGS reduced BPE dependency but did not eliminate it entirely; animal based components were used or BPE was retained in lower amounts alongside hormones and lipids in a “partially defined” environment (66, 67).

Significant progress occurred with media such as CELLnTEC's CnT-07, which were completely BPE-free, serum-free, and xeno-free, relying solely on chemically defined growth factors such as transferrin and trace elements. This supported strong keratinocyte expansion while preserving stem/progenitor phenotypes and yielding stratified epidermal constructs (67, 68). A further leap came with Gibco's EpiLife® base medium, combined with the animal-origin-free Supplement S7, creating a fully defined, xeno-free system that extends primary human keratinocyte lifespan and supports efficient expansion as confirmed in recent studies (67, 69).

Ongoing improvements focus on optimizing growth factor cocktails, fine-tuning calcium concentrations, and developing novel growth surfaces to enhance keratinocyte proliferation, clonogenicity, and downstream stratification. A recent study used fibroblast-produced extracellular matrix (ECM) for xenogeneic-free keratinocyte expansion (70). Proteomicly, The ECM closely resembled the core matrix composition of natural dermis. Indeed, the keratinocytes rapidly proliferated on these matrices, retaining their small sizes and expressing the early-stage markers. Further characterization revealed high colony forming efficiency compared to collagen I grown cells. Additionally, keratinocyte sheets grown on the novel matrix displayed more stable cell-cell junctions and demonstrated more robustness. Another effort has successfully developed a chemically defined, xeno-free, feeder free culture system using biologically relevant recombinant human laminins (LNs) as culturing surface (71). LN proteins are naturally occurring in the basement membrane; they contribute to the physical structure and also serve as ligands for cellular receptors and signaling. Several types of LNs have been identified in the basement membrane. In particular, LN-421 and LN-511 has shown to support the growth of keratinocytes in vivo as a replacement for a feeder lay-er. The laminin system showed comparable gene expression profile, colony-forming efficiency and differentiation capacity to the 3T3-coculture system. Because it is a serum and xeno free system, there are no batch-to-batch variances occurring when culturing the cells, which typically is the case in culture media containing FBS and bovine purity extract.

Keratinocyte culture on biomimetic substrates has emerged as a compelling alternative to traditional plastic surfaces. In a seminal 2021 study, researchers demonstrated that primary keratinocytes grown on soft hydrogels tuned to ∼50 kPa stiffness—mimicking the mechanical properties of skin—exhibited notably altered cell architecture, increased density, and enhanced nuclear biomechanics, alongside upregulated expression of mechanotransduction proteins like components of the LINC complex (72). When these mechanically conditioned cells were used to generate 3D epidermal models, the resulting tissue displayed improved stratification and organization compared to those derived from keratinocytes expanded on rigid plastic. This finding highlights how substrate stiffness alone—independent of biochemical cues—can profoundly influence keratinocyte behavior, suggesting that mechanically biomimetic culture surfaces may significantly enhance the physiological relevance and performance of engineered skin constructs.

Hassanzadeh et al. (2018) developed a novel feeder-free approach for culturing human keratinocytes using adipose-derived mesenchymal stem cell (Ad-MSC) conditioned medium, eliminating the need for animal-derived components or feeder layers (73). This method supported robust keratinocyte proliferation and the formation of multilayered epidermal sheets, with preserved expression of stem/progenitor markers (P63, K14, K19, and α6 integrin) alongside differentiation markers (K10, involucrin, and filaggrin). Marcelo et al. described the epithelial “pop-Up” keratinocyte (ePUK) method, which uses repeated high-volume feeding with serum- and fatty acid–free Epilife medium to harvest proliferative keratino-cytes in suspension from confluent cultures (74). The ePUK approach enriches for highly clonogenic, migratory cells in a truly feeder-free context, improving both expansion speed and graft take in experi-mental skin models.

Despite significant advances in chemically defined keratinocyte culture systems, these platforms still face critical limitations that hinder their clinical translation. Current serum-free media often fail to consistently support proper epidermal stratification, producing thin or dysfunctional skin equivalents lacking the robust barrier function of native tissue (64, 75). Additionally, these systems remain prohibitively expensive due to their reliance on recombinant growth factors and specialized supplements. Different commercial media formulations (e.g., KGM-CD, EpiLife, DK-SFM) generate keratinocytes with distinct morphological and functional characteristics, including variations in proliferation rates, differentiation potential, and stem cell retention (76). The media-specific differences mean that cells cultured in one system frequently cannot be used interchangeably with those grown in another, complicating standardization across research and clinical applications.

Rapid expansion phases — commonly required to generate clinically relevant cell numbers — impose replication stress that increases the probability of both structural and sequence-level genomic lesions. Aneuploidy, the gain or loss of whole chromosomes, is a hallmark of genomic instability and is frequently observed in human pluripotent stem cells (hPSCs) during long-term culture (77). Although less documented in primary keratinocytes, the underlying mechanisms are highly relevant to any rapidly dividing epithelial population in vitro. Trisomy 12 is a particularly common whole-chromosome abnormality, often arising not from a single rare event but simultaneously in a high percentage of cells (∼2%) during critical transition passages. The lack of serum-derived protective factors and the reliance on simplified growth factor cocktails may exacerbate these mitotic errors (78). TP53 (p53) alterations merit particular attention in the context of in vitro keratinocyte expansion because TP53 mutant clones are present as frequent, small clones in normal human epidermis and because p53 dysfunction confers both replicative advantages and altered responses to DNA damage. Several lines of evidence indicate that (1) heterozygous or functionally inactivating TP53 mutations can permit clonal expansion of epithelial cells in vivo, (2) p53 loss or dysfunction in keratinocytes increases replication stress and can permit accumulation of structural genomic abnormalities, and (3) mutant p53 alleles may be positively selected under strong proliferative culture conditions (i.e., during rapid expansion), thereby creating a population-level enrichment of mutant clones that would be missed by crude phenotypic assays (79). These findings imply that culture processes that favour extreme or prolonged proliferative selection can inadvertently select for TP53-compromised cells or other mutations that confer growth advantage.

Methods of SC isolation are now well-stablished while the in-vitro culture systems of epidermal KSC are improving with new media formulations that are serum and Xeno free. Consequently, different assay has been developed to study the KSC and evaluate their capacity to self-renew, proliferate and differentiate. These assays are important in characterizing the SCs, and also assess their viability for grafting and other clinical applications.

The self-renewal and differentiation of epidermal KSCs are assayed using three different basic techniques. The first method examines the clonal growth ability of individual cells in culture through colony forming assays (CFA) or clonogenic assays (96). Cells are evaluated by culturing them at clonal density and then subcloned. Specifically, unlike undifferentiated cells that expresses SC markers differentiated cells can't form colonies (97). CFA of keratinocyte SC has led to identification of three types of clones, each demonstrating different proliferative potential. This offers another tool to monitor the comparative efficacy of different culture conditions in maintaining growth potential of SC by analyzing the clonal composition (98). The second method uses lineage tracing to genetically label a stem cell and trace it's progeny, which gives us information on how the cells behave in different conditions, the number of all progenies of the founder cell, their differentiation status and location. Using fluorescence labelling and other reporters, in situ monitoring and fate mapping of epidermal SC was possible in intact undamaged tissue (99, 100). Nowadays, large number of clones in complex tissues can be traced by using induced heritable DNA barcodes or naturally occurring somatic mutations that can be read using next generation sequencing (101).

The third method uses in vitro skin forming assay to assess the functionality of keratinocytes and their capacity to keratinize and form the epidermis. Simple epidermis reconstruction assays are achievable using cell culture inserts with porous membranes. After culturing the cell monolayer on the membrane, the inserts are lifted for air-liquid interface and the culture media is supplemented with extra calcium, thus facilitating the stratification of the cells and the formation of the epidermis layers.

Additional methods of assessment and characterization has also been described in literature. In wound-healing, migration of keratinocytes is essential for the complete reepithelization of the damaged site. Hence, a simple in vitro scratch-wound assay is utilized to assesses the migratory capacity of keratinocytes (102). Simply, cells are grown on a monolayer until 80% confluency. This is followed by removal of media growth factors and the mechanical disruption of cells by scratching the monolayer with a tool. By observing the cells under a microscope and periodically measuring the scratch gap, the migratory capacity of the cells can be evaluated.

In vivo methods for assessing Epithelialization, skin reconstitution and formation of hair follicles by keratinocytes has also been described (103–105). The tracheal grafting method involves inoculating rat tracheas with keratinocytes and fibroblasts from newborn mice then implanting back into the mice. The tracheas are then monitored for 2–4 weeks to study epithelialization and hair follicle reconstitution. The other method involves the injection of SCs into the dermo-epidermal junction of newborn mouse skin graft. The grafts are then placed and stitched on the back of athymic mice and monitored for epithelialization and hair growth. Diett et al. reported the use of a silicon chamber with a full thickness wound on the upper back of immunocompromised mice (106). The chamber is injected with dermal and epidermal SCs and the reconstituted tissue is assessed after two weeks. These methods allow researchers to investigate the dynamics of epithelialization and the formation of hair follicles in vivo, providing valuable insights into skin biology and potential applications in regenerative medicine.

Since the introduction of cultured epithelial autografts (CEAs) in the early 1980s, keratinocyte-based therapies have become fundamental in modern burn care and wound management. Today, these products fall into four main categories—each designed to address specific clinical needs. In the following sections, we will briefly review representative products and the key advances within each category (Figure 3).

Solid Epidermal and Multilayered Keratinocyte-Based Sheets

Cultured keratinocyte sheets have long constituted the cornerstone of cell-based wound therapy. Autologous confluent sheets—such as Epicel®—are expanded from patient biopsies over 2–3 weeks, providing durable reconstitution of full-thickness epidermis in extensive burns (FDA-approved for ≥30% TBSA) with take rates around 75% (107). Off-the-shelf allogeneic composite sheets (Apligraf®, OrCel®, StrataGraft®) incorporate keratinocyte and fibroblast layers on biodegradable scaffolds, delivering metabolic and extracellular matrix cues that expedite re-epithelialization without permanent engraftment. More recently, fully biomimetic tri-layered skin substitutes—combining epidermal, dermal, and hypodermal analogs—have emerged. These constructs integrate keratinocyte-seeded hydrogels with fibroblast and adipocyte layers or basement membrane–mimetic films, aiming to replicate full skin architecture and improve outcomes in deep wounds (108). While still largely preclinical, these stratified platforms demonstrate promising re-epithelialization, vascularization, and appendage regeneration, heralding the next generation of durable, anatomically faithful skin replacements. 2.

Keratinocyte Suspensions and Spray Systems

Clinical translation of keratinocyte-derived exosomal therapies is limited by the difficulty of achieving GMP-compliant, scalable manufacturing and by the intrinsic heterogeneity of extracellular vesicle preparations, which varies with cell source, culture conditions, and isolation methods. This variability complicates the establishment of robust identity, purity, stability, and batch-to-batch comparability criteria, while the absence of standardized, quantitative potency assays further challenges regulatory approval (116). Consequently, widespread adoption will require harmonized MISEV-guided characterization frameworks and validated GMP manufacturing pipelines tailored to EV-based therapeutics (116).

Since keratinocytes have historically been synonymous with grafts for burns and chronic wounds, it's easy to overlook their wider therapeutic potential. In reality, over the past decade researchers have begun harnessing both live keratinocyte transplants and their secreted products (exosomes, antimicrobial peptides, immunoregulatory vesicles, etc.) for applications as diverse as pigment restoration in vitiligo, modulation of autoimmune skin diseases, vaccine adjuvancy, peripheral nerve regeneration, ocular surface repair, and even targeted antimicrobial therapies. What follows is a concise overview of these cutting-edge, non-wound-care uses—each representing a novel frontier in keratinocyte biology and translational medicine (Figure 4).

Dermatology

Stable vitiligo, characterized by well-demarcated depigmented patches, has found effective treatment through autologous melanocyte–keratinocyte transplantation—a procedure that suspends patient-derived epidermal cells (both keratinocytes and melanocytes) and applies them to depigmented sites after skin preparation. Keratinocytes in this mix serve a crucial role, providing a nurturing microenvironment that supports melanocyte engraftment and melanin production—far beyond the simple replacement of lost cells (117). Over multiple clinical studies, approximately 36% of treated lesions achieve excellent repigmentation (≥ 75%–80%), with long-term data showing sustained results in over 50% of cases at 24 to 72 months post-treatment (118). A large retrospective analysis of 2,283 patients revealed excellent repigmentation in 58.8%–66% of cases, with higher success in segmental vitiligo, disease stability ≥ 6–12 months, and absence of Koebner (119). Overall, autologous melanocyte–keratinocyte transplantation has emerged as a robust, long-lasting, and well-tolerated alternative for stable vitiligo patients unresponsive to medical therapies. It achieves impressive aesthetic results through the natural synergy between keratinocytes and melanocytes and has been validated by both randomized controlled trials and extensive real-world outcomes extending beyond six years.

Researchers have begun repurposing keratinocyte-derived exosomes as a novel cell-free immunotherapy for inflammatory skin diseases like psoriasis. In a breakthrough preclinical study, ∼120 nm exosomes sourced from A-431 keratinocytes and loaded with the JAK inhibitor tofacitinib (“Exo-TFC”) displayed lower cytotoxicity and more potent suppression of psoriatic cytokine genes—TNF-α, IL-6, IL-23, and IL-15—compared to equivalent doses of free drug in vitro (120). When topically applied in an imiquimod-induced psoriasis mouse model, Exo-TFC induced superior lesion regression relative to free tofacitinib (120). This approach leverages inherently biocompatible keratinocyte vesicles, enabling efficient drug delivery, controlled release, and improved skin targeting through exosomal surface markers such as CD9. Additionally, keratinocyte exosomes have been shown to modulate neutrophil-mediated inflammation, although their effects can be pro- or anti-inflammatory depending on cellular context and stimuli (121). Although still in the preclinical stage, Exo-TFC exemplifies a paradigm shift, offering a promising, cell-free strategy for targeted immunomodulation in skin diseases—a novel frontier demonstrating how keratinocyte products can be engineered into next-generation dermatological treatments. 2.

Immunomodulation

Researchers have recently explored the innovative use of engineered keratinocytes as a skin-based vaccination platform. A compelling preclinical example involves keratinocytes genetically modified to overexpress the endoplasmic reticulum stress response factor XBP1, enabling them to create a locally pro-inflammatory skin environment that effectively enhances vaccination efficacy (123). In murine models, transient overexpression of XBP1 in epidermal keratinocytes triggered elevated production of cytokines and chemokines, recruited key immune cells—such as CD103⁺ dendritic cells, plasmacytoid dendritic cells, γδ T-cells, and innate lymphoid cells—and significantly amplified both antigen-specific cellular and humoral immune responses compared to antigen delivery alone. This translated into robust protection against Zika virus and induced therapeutic immunity in a melanoma model (124). The team further demonstrated that XBP1-modified keratinocytes generated a similarly immunogenic milieu in human skin explants, offering a strong foundation for translational potential.

Keratinocytes offer promising avenues for tumor immunomodulation by expressing key immune checkpoint molecules—particularly PD-L1 and the non-classical HLA-G1. Upon exposure to inflammatory signals like IFN-γ and TNF-α, these cells significantly upregulate PD-L1 and, to a lesser extent, HLA-G1, effectively inhibiting CD4⁺ T-cell proliferation through PD-1 engagement; PD-L1 blockade reverses this effect, underscoring its functional importance (125). Moreover, keratinocyte stem/progenitor populations (CD49f^high) exhibit heightened PD-L1 and HLA-G expression alongside secretion of TGF-β and IL-10—attributes resembling immunoprivileged cells—which suggests a role in creating a tolerogenic microenvironment (126). These findings suggest two translational strategies: (1) modifying keratinocyte expression of PD-L1 or HLA-G to locally modulate anti-tumor immunity—potentially improving immune surveillance in squamous cell carcinoma, and (2) exploiting keratinocyte-associated antigens, such as keratins 6, 14, and 17, which act as tumor-associated antigens and have been linked to better outcomes in non–small-cell lung cancer treated with checkpoint inhibitors. Together, these discoveries reveal keratinocytes as emerging tools in skin-targeted cancer immunotherapy, capable of both dampening and redirecting immune responses in the tumor milieu. 3.

Neurological applications

keratinocytes can also exacerbate nerve sensitization. In a rat model of nerve injury, implants of human keratinocytes at the injury site caused dramatic hyperexcitability of nearby neurons: the transplanted keratinocytes secreted excess NGF, leading to spontaneous firing and pain behaviors (129). This indicates a dual role: while certain keratinocyte factors encourage growth, they must be carefully controlled to avoid pathological pain. Understanding this keratinocyte–neuron crosstalk has opened potential for topical pain therapies (e.g., targeting keratinocyte NGF signaling) and warns that keratinocyte-based nerve grafts must be designed to prevent chronic pain (129). 4.

Regenerative medicine (Non-Cutaneous Epithelia)

keratinocyte-based tissue engineering has also been successfully adapted for repair of oral and other non-cutaneous mucosal epithelia. Unlike skin-derived grafts, these approaches utilize keratinocytes directly harvested from mucosal tissues, such as buccal or tongue epithelium, preserving their innate compatibility with the target site. In one rodent model, autologous oral mucosal keratinocytes cultured as three-dimensional cell sheets integrated seamlessly into deep tongue wounds, promoting rapid re-epithelialization with reduced fibrosis and a mature p63⁺ basal layer reminiscent of normal oral mucosa (131). Similarly, application of these mucosal sheets to human intraoral surgical defects demonstrated faster healing, thicker epithelial coverage, and minimal scarring compared to conventional treatments (132). Notably, when transplanted to post-endoscopic submucosal dissection sites in the esophagus, these grafts virtually eliminated stricture formation and accelerated closure—highlighting their potential to repair mucosa in anatomically distinct regions. This “site-matched” strategy extends to the urinary tract, where oral keratinocytes seeded onto bladder-derived scaffolds reconstructed functional urethral mucosa in rabbits with durable epithelial architecture and reduced stricture risk (133). These findings underscore a paradigm shift: by sourcing keratinocytes from mucosal, rather than cutaneous, origins, researchers are expanding the regenerative reach of epithelial grafting into diverse non-skin tissues—leveraging cell-autonomous characteristics to rebuild mucosal barriers with fidelity and function. 5.

Infectious and Antimicrobial Applications