How HaCaT Cells Are Utilized In Skin Biology Studies?

In medical research and dermatological biology, investigation mostly depends on in vitro models and how they reproduce human skin activity correctly.

Owing to the advancements through multiple studies, HaCaT cells have evolved into the gold standard among the several cell lines now in use. From wound healing to carcinogenesis, these immortalized human keratinocytes are crucial since they provide the ideal mix between physiological relevance and experimental convenience.

That being said, models of epidermal cells, too, are essential for researching the biology of the skin. The scientific community has traditionally relied on primary cell cultures from leftover tissue, usually from the foreskin of neonates. These cells exhibit several traits of primary cells, such as pathogenicity, differentiation kinetics, barrier construction, and/or protein expression.

However, the question still persists: why have HaCat cells influenced skin biology investigations? Let’s hover around the subject and draw a few study-based influences.

HaCaT Cells as a Reliable In Vitro Differentiation Model

The skin is a constantly renewing organ that actively contributes to the host’s defenses and dynamically regulates the human body’s outside-inside-outside interactions.

Ninety-five percent of the cells of the epidermis are keratinocytes (KCs), for they primarily serve the epidermis’ structural and barrier functions. However, it is also widely acknowledged that HaCat cells play a part in wound healing and the start and continuation of immunological and inflammatory reactions in the skin.

KCs develop into highly differentiated, nucleus-free corneocytes under homeostatic conditions after differentiating and maturing from proliferating nucleated basal cells. The expression of structural proteins, including lipids and keratins (K), is indicative of each step of differentiation. While K1 and K10 are found on more developed suprabasal cells, and involucrin, loricrin, and keratolinin are found in cells of the top spinous layer, K5 and K14, for instance, are only expressed in the basal layer.

Many cytokines and growth factors stimulate terminal differentiation, usually linked to developing peripheral envelopes high in lipids and proteins. Here, one of the most significant initiators of KC differentiation is the calcium (Ca2+) gradient in the epidermis, which rises from the basal to the granular layer.

On the other hand, vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) ligands are produced by resting KCs; however, the expression of cytokines and chemokines is altered when bacterial products or direct injury from chemicals or UV light activate them. The cytokine/chemokine network is considerably more intricate in skin conditions, including psoriasis and atopic dermatitis, and autocrine/paracrine loops have been reported.

Moving on, let’s discuss how HaCat cells are employed and utilized in skin biology studies.

1. Use in Cosmetic and Toxicological Testing

Particularly in evaluating novel skincare products, sunscreens, and chemical compounds, the HaCaT approach greatly helps the cosmetic business. Safety evaluations, including skin irritation, phototoxicity, and permeability studies, make great use of these cells since they simulate typical keratinocyte behavior and skin barrier qualities.

HaCaT cells are also utilized under criteria created by regulatory agencies such as the OECD in genotoxicity testing and skin sensitizing research. Without turning to animal research, their consistent and repeatable reactions make them a dependable model for preclinical testing.

2. Isolation of Human Keratinocytes from Skin Biopsies

A researcher can choose to isolate the primary KCs using nonlesional skin biopsies taken from adult psoriatic patients who had not been receiving topical or systemic treatments for at least six months. Further into the investigation, they utilize dispase (Gibco BRL, Gaithersburg, MD, USA) to treat the 0.4 mm punch biopsy in order to separate the epidermal layer from the basement membrane.

Human keratinocyte growth factors (Gibco BRL) can also be added to the primary cells after they have been plated on 6-well tissue culture plates (Costar), coated with a coating matrix (type I collagen, Gibco BRL), and cultured in a particular keratinocyte-serum-free medium at low Ca2+ concentrations (<0.07 mM).

Scholars use trypsinization to separate the cells after the monolayer has achieved 60% to 70% confluence. Moreover, they also employ keratinocyte cultures from the third and fourth passages in every experiment.

3. Role in Wound Healing and Regenerative Medicine

Another key area where HaCaT cells are quite helpful is wound healing. Perfect for mimicking the early phases of re-epithelialization, these cells can move, multiply, and react to growth factors like EGF and TGF-β. HaCaT cells are widely employed in tissue engineering and regenerative medicine to create synthetic skin equivalents for graft research and biomaterial testing.

Evaluation of new wound dressings, scaffolds, and topical treatments benefits especially from HaCaT-based three-dimensional skin models. More physiologically relevant than conventional monolayer cultures, these models replicate epidermal stratification and barrier development.

4. Analysis of DNA Content

Following the manufacturer’s instructions, the commercial fluorimetric “FluoReporter Blue Fluorometric DNA Quantitation Kit” (Molecular Probes, Life Technologies, Carlsbad, CA, USA) is employed to analyze the amount of DNA in 96-well plates. After thawing, the studies necessitate 200 μL of distilled water in each well of the plates, containing treated HaCaT cells.

Following three freezing-thawing cycles, 100 μl can later be moved into a 96-well plate and utilized for the test. Simultaneously, the process allows the development of standard calibration curves using calf thymus DNA diluted in TE buffer at escalating concentrations (0–1000 ng/100 μl).

Final Thoughts

From investigations on inflammation and wound healing to cosmetic testing and carcinogenesis research, HaCat cells provide a flexible and reliable model that closes the basic science gap with therapeutic uses. Although not without restrictions, their matchless contribution to forward dermatological science is unparalleled.

HaCaT cells are not only a handy substitute for any lab looking at epidermal biology; they are a necessary instrument that helps us to make discoveries that still define human skin health and illness.