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HaCaT
HaCaT
HaCaT
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HaCaT is a spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin,[1][2] widely used in scientific research.[3] HaCaT cells are utilized for their high capacity to differentiate and proliferate in vitro.[4] Their use in research allows for the characterization of human keratinocyte using a model that is reproducible and addresses issues such as short culture lifespan and variations between cell lines that would otherwise be encountered. These cells have allowed the characterization of several processes, such as their utilization as a model system for vitamin D3 metabolism in the skin.[5]

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HaCaT

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HaCaT is a spontaneously immortalized, aneuploid cell line derived from the histologically normal adult of a 62-year-old Caucasian male, serving as a widely used model for epidermal biology due to its nontumorigenic nature and capacity for normal differentiation. Established through long-term cultivation without exogenous immortalization agents, it exhibits stable genetic markers, including unique chromosomes, and maintains a hypodiploid across extensive passaging, exceeding 140 passages without loss of proliferative potential. Unlike primary , which senesce after limited divisions, HaCaT cells provide a standardized, reproducible system for , expressing key epidermal markers such as keratins 1, 10, 14, involucrin, and . Developed by Petra Boukamp and colleagues in 1988, the line arose via spontaneous transformation during serial subculturing of keratinocytes from peritumoral skin, demonstrating that human adult epithelial cells can achieve immortality in vitro while retaining differentiation competence. When grafted onto nude mice, HaCaT cells form a well-stratified, orthokeratinized epidermis resembling normal human skin, complete with a functional basement membrane and absence of dyskeratosis. This differentiation is regulatable in culture by factors like extracellular calcium concentration (e.g., 0.07 mM for proliferation, 1.8 mM for stratification) and cell density, allowing precise modeling of keratinocyte responses. HaCaT cells have become a cornerstone in dermatological and toxicological studies, facilitating investigations into skin barrier function, , , and responses to irritants or pathogens without the variability of primary cells. They are particularly employed in assays for proinflammatory mediators (e.g., IL-8, VEGF, MMP-9), screening therapeutic compounds for conditions like and , and evaluating from chemicals such as sodium lauryl sulfate. Despite limitations, including reduced profiles compared to primary and inability to fully replicate skin heterogeneity, their ease of maintenance and ethical advantages over animal models underscore their enduring utility in advancing skin research.

Development and History

Origin and Derivation

The HaCaT cell line was derived from histologically normal isolated from full-thickness adult obtained from the distant periphery of a on the upper back of a 62-year-old donor. This source material ensured the cells originated from non-tumorigenic tissue, providing a basis for studying normal epidermal differentiation. The immortalization occurred spontaneously during long-term primary culture, without the use of viral , chemical , or other deliberate interventions. were maintained under low calcium (0.2 mM) conditions at an elevated temperature of 38.5°C to promote selective growth, leading to the emergence of a stable, non-senescent clone after extended passaging. This process resulted in a monoclonal population that has been propagated beyond 140 passages, demonstrating indefinite replicative potential. The cell line was established in the late 1980s at the German Cancer Research Center (DKFZ) in , . Transformation led to an aneuploid , initially hypodiploid, characterized by specific abnormalities including an of the long arm of (i(8q)) and other unique stable marker chromosomes, confirming its monoclonal origin. DNA fingerprinting verified the line's identity with the donor tissue, ruling out cross-contamination.

Initial Characterization and Publication

The HaCaT cell line was developed in the late 1980s as a spontaneously immortalized alternative to primary human , which have limited proliferative capacity and thus restrict long-term experimental studies on epidermal . This innovation addressed the challenges of obtaining stable, non-viral transformed models for investigating differentiation and skin physiology, contrasting with earlier attempts using or other oncogenes that often compromised normal cellular functions. The initial characterization of HaCaT cells was detailed in a seminal publication by Boukamp and colleagues in the Journal of . The study described the derivation of HaCaT from isolated from adult , which underwent spontaneous immortalization without exogenous genetic manipulation, achieving over 140 passages while retaining a hypodiploid aneuploid and monoclonal origin as confirmed by DNA fingerprinting. Notably, these cells exhibited keratinocyte-like differentiation, forming stratified epidermal structures with orderly keratinization when grafted onto nude mice, expressing key markers such as keratins 1 and 10, involucrin, and . Key initial findings highlighted HaCaT's transformed growth properties alongside preserved differentiation potential. The cells demonstrated anchorage-independent growth, forming colonies in soft and on plastic, indicative of a clonogenic, unlimited proliferative capacity. However, despite this transformed phenotype, HaCaT cells remained nontumorigenic upon subcutaneous injection into athymic nude mice, instead reforming normal, differentiated epidermal tissue without malignant invasion. Subsequent early validations in the early 1990s confirmed underlying genetic alterations contributing to immortality. A study by Lehman et al. identified missense mutations in both alleles of the (G to A transitions at codons 249 and 282), which likely impair wild-type function and facilitate the observed spontaneous immortalization without viral intervention. These findings positioned HaCaT as a valuable model for studying 's role in keratinocyte transformation, though activity was not addressed in these original reports.

Biological Characteristics

Cellular Morphology and Growth Properties

HaCaT cells exhibit a characteristic epithelial morphology, forming a pavement-like of polygonal cells when cultured under standard conditions. These cells display a flattened, cuboidal in basal layers, transitioning to more flattened profiles in suprabasal compartments during differentiation, mimicking aspects of normal human . This morphology is maintained through contact inhibition, where proliferation ceases upon , restricting growth to the basal layer and preventing multilayered overgrowth. In terms of growth properties, HaCaT cells demonstrate robust proliferative capacity with a of approximately 24 hours in low-calcium media, enabling sustained expansion over hundreds of passages without . This high proliferation rate supports their utility as a stable model for long-term studies, though it is somewhat faster than primary , reflecting their immortalized nature. HaCaT cells possess inducible differentiation potential, forming stratified epithelial structures in response to elevated calcium concentrations (>0.1 mM) or air-liquid interface culture conditions. Under low calcium (<0.1 mM), they remain in a proliferative basal state, but switching to higher calcium triggers morphological changes, including cell flattening and multilayering, culminating in a stratified epithelium with granular and cornified layers. Upon these differentiation signals, HaCaT cells upregulate suprabasal keratins such as K1 and K10, which are expressed in organized patterns within the stratified layers, indicating functional epidermal maturation.

Genetic and Molecular Features

HaCaT cells harbor two heterozygous missense mutations in the TP53 gene: H179Y (c.535C>T) and R282W (c.843_844CC>TT), which disrupt the DNA-binding domain of the p53 protein and result in loss of its tumor suppressor function, contributing to the cells' immortalization without tumorigenic conversion. These UV-signature mutations, one on each allele, impair p53's ability to induce cell cycle arrest and apoptosis in response to DNA damage, while also conferring partial gain-of-function properties in certain contexts, such as altered transcriptional regulation. The mutations arose spontaneously during derivation from adult keratinocytes and are maintained across passages, underscoring their role in bypassing senescence. Cytogenetically, HaCaT cells exhibit stable with a modal number of approximately 45, reflecting a hypodiploid and monoclonal origin marked by unique structural abnormalities, including translocations leading to loss of arms 3p, 4p, and 9p, gain of 9q, and an 8q. Additional numerical changes include gains in 1, 7, 8, and 11, which support proliferative capacity without progression to , as evidenced by consistent karyotypic features over extended culture. This contrasts with diploid primary but remains non-random and non-progressive in standard conditions. At the molecular level, HaCaT cells retain key epithelial markers, constitutively expressing basal keratins K5 and K14, which maintain their proliferative, undifferentiated state, while suprabasal keratins K1 and K10 are inducibly upregulated upon differentiation cues like elevated calcium. They also express typical integrins, such as α3β1, α5β1, α6β4, and αvβ6, facilitating to extracellular matrix components like and fibronectin.43058-1/fulltext) Immortalization occurs independently of high (TERT) overexpression, though basal TERT expression confers detectable activity sufficient for telomere maintenance without . Epigenetically, HaCaT cells display hypermethylation in select promoter regions, such as those of KRT13 and , which silences differentiation-associated genes and alters stress response pathways, contributing to their altered regulatory landscape compared to primary cells.

Research Applications

Modeling Skin Biology and Diseases

HaCaT cells have been extensively utilized to model normal skin biology through three-dimensional (3D) organotypic cultures, which replicate key aspects of epidermal stratification and . In these setups, HaCaT are cultured at an air-liquid interface on a matrix, often with dermal fibroblasts, leading to the formation of a multilayered that expresses differentiation markers such as keratins, , and loricrin. This stratification mimics the epidermal architecture, enabling studies of cornified envelope formation and intercellular junctions. Although HaCaT-derived models exhibit decreased compared to primary , often lacking a complete , they provide a stable platform for long-term investigations into epidermal and permeability, as demonstrated in scaffold-based cultures that enhance tissue-like organization. In disease modeling, HaCaT cells serve as a versatile tool for simulating pathological skin conditions. For psoriasis, cytokine cocktails such as M5 (comprising TNF-α, IL-17A, IL-22, IL-1α, and oncostatin M) or imiquimod (IMQ) are applied to induce hyperproliferation, altered differentiation, and inflammatory responses, recapitulating lesional features like increased keratinocyte turnover and chemokine secretion. Wound healing processes are investigated via scratch or Transwell migration assays, where HaCaT cells demonstrate collective migration and re-epithelialization, influenced by factors like growth factors or oxygen gradients, providing insights into keratinocyte motility during tissue repair. UV-induced damage is modeled by exposing HaCaT monolayers to UVB radiation (typically 20-100 mJ/cm²), which triggers apoptosis, reactive oxygen species (ROS) production, and DNA damage, allowing evaluation of protective mechanisms against photoaging and photocarcinogenesis. HaCaT cells contribute significantly to , particularly in studying (SCC) pathogenesis. While non-tumorigenic in their basal state, HaCaT cells transfected with oncogenes like H-Ras exhibit enhanced invasiveness, as assessed in invasion assays that measure matrix degradation and motility, mirroring aggressive SCC behavior. In xenograft models, subcutaneously injected HaCaT variants form tumors in immunodeficient mice, enabling analysis of tumor growth, vascularization, and response to therapies, with amphiregulin-overexpressing lines showing rapid proliferation and high Ki-67 indices. These models highlight HaCaT's utility in dissecting epithelial-mesenchymal transition and metastatic potential in cutaneous SCC. Specific applications include HPV infection models, where HaCaT cells transfected with HPV genomes (e.g., HPV16 or HPV11) replicate viral early and immune evasion, facilitating studies on oncoprotein effects like E6/E7-mediated deregulation. In atopic dermatitis research, HaCaT cells stimulated with TNF-α and IFN-γ exhibit upregulated proinflammatory cytokines (e.g., IL-6, IL-8) and impaired barrier integrity, simulating chronic inflammation and Th2-skewed responses observed in lesional skin.

Use in Toxicology and Pharmacology

HaCaT cells are widely employed in toxicology to evaluate the cytotoxic effects of skin irritants through standard viability and damage assays. The MTT assay, which measures mitochondrial dehydrogenase activity as an indicator of cell viability, has been used to assess the impact of surfactants such as sodium dodecyl sulfate (SDS) on HaCaT keratinocytes, revealing dose-dependent reductions in metabolic activity following 48-hour exposures. Similarly, lactate dehydrogenase (LDH) release assays quantify membrane integrity loss, demonstrating elevated LDH leakage in HaCaT cells exposed to zinc oxide nanoparticles at concentrations above 50 μg/mL, indicating cytotoxicity via plasma membrane disruption. Reactive oxygen species (ROS) detection, often via DCFDA fluorescence, further characterizes oxidative stress; for instance, amorphous nanosilica particles induce significant ROS production in HaCaT cells at 10-70 nm sizes, leading to DNA damage and apoptosis. In , HaCaT cells facilitate screening for topical drug efficacy and safety, particularly in and contexts. Permeability testing involves evaluating how enhancers like affect HaCaT monolayers, where non-cytotoxic concentrations (e.g., 1-5%) promote drug flux without compromising cell viability, as assessed by CCK-8 assays. For evaluation, HaCaT models simulate cytokine-induced inflammation; extracts from Acer truncatum leaves, applied at 50-200 μg/mL, reduce TNF-α and IL-6 secretion in sodium lauryl sulfate (SLS)-stimulated HaCaT cells by modulating pathways, supporting their potential in topical formulations. HaCaT-derived models contribute to regulatory frameworks for hazard assessment, aligning with guidelines on . The KeratinoSens , utilizing a HaCaT-derived line stably transfected with an ARE-driven reporter, is outlined in OECD Test Guideline 442D for detecting sensitizers by measuring Nrf2 pathway , offering a validated alternative to with an accuracy of 77% for human sensitization potential. Representative studies highlight HaCaT's role in specific applications. In , acylglutamate-based exhibit low (IC50 > 1000 μM) in HaCaT MTT assays, informing safer ingredient selection. For UV filters, morin-Schiff base derivatives protect HaCaT cells from UVB-induced ROS and at 10-50 μM, enhancing photostability without toxicity. In , agents like induce HaCaT via ROS-mediated pathways, with IC50 values around 1-5 μM, aiding in predicting skin side effects such as .

Cultivation Protocols

Standard Culture Conditions

HaCaT cells are routinely maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (FBS), 4 mM L-glutamine, 100 units per ml penicillin, and 100 μg per ml to support their proliferation and viability. This nutrient-rich medium provides essential , vitamins, and growth factors necessary for the cells' metabolic needs, while the antibiotics help prevent microbial contamination during routine handling. Variations may include the use of high-glucose DMEM (4.5 g/L) to accommodate the cells' rapid growth rate, but the core composition remains consistent across standard protocols. Incubation occurs at 37°C in a humidified atmosphere with 5% CO₂ to mimic physiological conditions and maintain pH stability in the bicarbonate-buffered medium. The humidified environment prevents evaporation of the culture medium, ensuring consistent osmolarity and nutrient availability over the culture period. These parameters promote , with HaCaT cells typically reaching high densities without significant phenotypic drift under controlled conditions. For initial plating, a seeding of 1–2 × 10⁴ cells per cm² is recommended to achieve even formation and avoid overcrowding. This allows for attachment and initial expansion within 24–48 hours, optimizing experimental reproducibility. Passages are performed every 3–5 days when cultures reach 70–80% , corresponding to the cells' of approximately 24–48 hours. Monitoring ensures cells remain in an active proliferative state, preventing overgrowth that could induce unwanted differentiation.

Maintenance and Subculturing Techniques

HaCaT cells are typically subcultured when they reach 70-80% to prevent overgrowth and maintain proliferative capacity. The procedure begins by aspirating the spent medium and rinsing the monolayer with () to remove residual serum. Cells are then detached using 0.05% trypsin-EDTA solution, incubated for 5 minutes at 37°C until the cells round up and detach from the surface. The reaction is neutralized by adding complete containing (), followed by at 300-500 × g for 5 minutes to pellet the cells. The supernatant is discarded, and the cell pellet is resuspended in fresh medium, with viable cells counted using exclusion before seeding at a density of 1:3 to 1:10 ratio depending on the vessel size, typically achieving in 3-5 days. For cryopreservation, HaCaT cells are harvested similarly via trypsin-EDTA detachment and resuspended at 1-5 × 10^6 cells/mL in freezing medium composed of 90% FBS and 10% (DMSO). Aliquots of 1 mL are transferred to cryovials, which are initially cooled at -80°C in a controlled-rate freezing container for 24 hours to minimize formation, then stored in vapor phase for long-term preservation. Upon thawing, vials are rapidly warmed in a 37°C water bath, diluted in pre-warmed complete medium, centrifuged to remove DMSO, and seeded immediately to recover >80% viability. Quality control during maintenance includes regular screening for contamination using PCR-based detection kits, as infections can alter growth and without overt signs. Additionally, the identity is confirmed periodically through staining for differentiation markers such as 14 (basal) or 10 (suprabasal), ensuring retention of epidermal characteristics. HaCaT cells exhibit remarkable long-term stability, retaining proliferation, differentiation potential, and genetic features without up to passage 200 or beyond, as demonstrated by consistent marker expression and chromosomal stability over extended cultivation.

Advantages and Limitations

Key Advantages Over Primary Cells

HaCaT cells, being a spontaneously immortalized human line, offer the key advantage of unlimited proliferative capacity, enabling over 140 passages without or loss of differentiation potential, in contrast to primary which are limited to approximately 20-40 population doublings before entering replicative . This immortality allows for long-term experiments and consistent cell availability, avoiding the often seen in extended cultures of primary cells. The genetic stability of the HaCaT line, despite its , minimizes variability across experiments and passages, providing a reproducible model that circumvents the donor-to-donor heterogeneity inherent in primary derived from biopsies. This standardization enhances the reliability of results in studies requiring uniform cellular responses, such as those investigating epidermal . Furthermore, HaCaT cells are more cost-effective and accessible than primary , as they can be maintained in standard culture media without the need for growth factors, feeder layers, or repeated procurement of fresh tissue, thereby reducing both financial and logistical burdens. Ethically, their use eliminates the ongoing requirement for invasive biopsies, promoting animal- and human-free alternatives in research.

Potential Limitations and Considerations

While HaCaT cells offer a stable model for research, their genetic alterations, particularly mutations in the TP53 gene (H179Y and R282Q), can significantly impact cellular responses compared to normal . These mutations lead to a loss of wild-type tumor suppressor function, impairing DNA damage-induced and potentially conferring resistance to genotoxic stresses such as UV radiation or chemotherapeutic agents. For instance, due to these mutations, HaCaT cells show reduced apoptotic sensitivity to and other DNA-damaging agents compared to primary , which may not accurately reflect primary cell behavior . Additionally, as a homogeneous immortalized cell line, HaCaT lacks the full cellular heterogeneity of native , including interactions with stromal fibroblasts, components, and immune cells that are crucial for physiological processes like and . This limitation restricts the model's ability to replicate complex microenvironments, particularly under inflammatory or disease conditions where and immune modulation play key roles. HaCaT cells also possess a low but notable tumorigenic potential; when transplanted into immunodeficient nude mice, they typically form benign epidermal cysts or papillomas rather than invasive carcinomas, which can confound interpretations in cancer progression studies by mimicking early, non-malignant stages. To address these limitations and enhance clinical relevance, researchers are advised to validate HaCaT-derived findings using primary or models, which better capture tissue-specific heterogeneity and dynamic interactions.

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