Human keratinocytes (HaCaT) are an immortalized epidermal keratinocyte cell line widely used in the study of mechanisms in skin biology, oxidative stress, cell proliferation, and apoptosis.
detailed information about HaCaT cells:
The origin and characteristics of HaCaT cells
Source and Background: The Hacat cell line was established by German scientist N.E. Fusenig in 1989. The cells were derived from the normal skin surrounding the lesion of a 62 year old male melanoma patient. These cells exhibit typical characteristics of keratinocytes, including the expression of keratin and differentiation markers such as involucrin, and are able to maintain a certain level of proliferation and migration ability in vitro culture [9,21].
Morphology and characteristics: These cells grow adherent and have an epithelial cell like morphology, capable of expressing keratin, keratinized cell cross-linked outer membrane protein, and intermediate filament related protein. They are important tools for studying epidermal keratinocyte formation.
Biological characteristics: Hacat cells have partial to complete differentiation phenotypes and can proliferate indefinitely in vitro without natural apoptosis, solving problems such as short cell lifespan and potential variations in cell line culture.
Culture conditions
Culture medium: It is recommended to use DMEM+10% FBS+1% P/S as the basic culture medium, with a culture environment of 95% air and 5% carbon dioxide, and a temperature of 37 ℃.
Passage ratio: The passage ratio is usually 1:2 to 1:4, and the liquid exchange cycle is 2-3 days. The specific operation needs to be adjusted according to the cell density and status.
Freezing conditions: The freezing substrate is 50% basic culture medium+60% FBS+10% DMSO, stored in liquid nitrogen.
Application Fields
Skin biology research: HaCaT cells are used to study the proliferation, differentiation, apoptosis, and epidermal barrier function of keratinocytes.
Phototoxicity and UV research: HaCaT cells are sensitive to UV radiation and can be used to study the effects of UV on skin cells and develop sunscreen products.
Drug screening and toxicity testing: HaCaT cells are used to evaluate the effects of drugs on keratinocytes, such as antioxidants, anti-inflammatory drugs, etc.
Tumor transformation research: HaCaT cells are used as a model for studying tumor transformation.
4. Functions and Features
Proliferation and differentiation: HaCaT cells have stable proliferation ability, but under certain conditions (such as low calcium environment), they may exhibit morphological and functional changes.
Migration ability: Studies have shown that gene knockout or drug treatment can enhance the migration ability of HaCaT cells.
Antioxidant and anti-inflammatory effects: Certain compounds, such as Nrf2 activators, can significantly enhance the antioxidant capacity of HaCaT cells.
5. Precautions
During the cultivation process, attention should be paid to controlling the passage density and digestion time to avoid excessive cell adhesion or floating death.
The storage conditions are liquid nitrogen vapor phase, and the environment needs to maintain air (95%) and carbon dioxide (5%).
Before use, check the cell status to ensure there are no abnormalities.
6. Potential Issues and Disputes
Although HaCaT cells are widely used in research, their immortalized properties may bring some controversy. For example, studies have shown that low concentrations of sodium arsenite treatment may lead to malignant transformation of HaCaT cells. Therefore, strict control of experimental conditions is required during use.
Oxidative stress and protective effects
HaCaT cells are highly sensitive to oxidative stress. Research has shown that ursolic acid (arbutin) can significantly reduce hydrogen peroxide induced oxidative damage in HaCaT cells by increasing the activity of antioxidant enzymes such as SOD and CAT to reduce malondialdehyde (MDA) content [1]. In addition, selenium has also shown a protective effect against oxidative stress in HaCaT cells, reducing reactive oxygen species levels and enhancing antioxidant capacity [16].
UV damage and repair
UVB radiation induces oxidative stress and apoptosis in HaCaT cells, leading to a decrease in SOD and GSH Px activity and an increase in MDA content [6]. However, natural ingredients such as water extract of Polygonatum sibiricum can protect HaCaT cells from UVB damage by increasing antioxidant enzyme activity and reducing MDA content [6].
The impact of drugs and compounds
Artemisinin (ART):
Low concentrations of ART (0.01-0.5mg/mL) can inhibit the proliferation of HaCaT cells and promote the secretion of TGF - β 1 [2].
Compound glycyrrhizin (CG):
CG can inhibit the proliferation of HaCaT cells and significantly reduce CXCR2 expression within a certain concentration range (50-200 μ mol/L).
Tacrolimus:
Appropriate concentrations of tacrolimus can promote the proliferation of HaCaT cells and the secretion of stem cell factor (SCF).
Photodynamic therapy (PDT) combined with low-dose photodynamic therapy (ALA-PDT) can promote the proliferation of HaCaT cells, while high-dose PDT can inhibit their proliferation [4,7].
Migration and Migration Capability
HaCaT cells have a certain migration ability, and serum levels have a significant impact on their migration characteristics. Low serum medium (2% FBS) can significantly enhance the migration ability of HaCaT cells [8].
Gene expression and signaling pathways
HaCaT cells exhibit active gene expression in multiple signaling pathways. For example, Toll like receptors (TLRs) are constitutively expressed in HaCaT cells, particularly TLR4 and TLR2 [17]. In addition, FAK phosphorylation status also plays an important role in HaCaT cell migration and keratinocyte formation [9].
Other research directions
HaCaT cells are also used in fields such as skin inflammation, immune response, and skin tissue engineering. For example, Candida albicans infection can upregulate the expression of β - defentin-2 in HaCaT cells, thereby exerting anti infective effects [12].
HaCaT cells, as an important in vitro model, provide valuable tools for studying skin biology. By conducting in-depth research on its reactions in oxidative stress, UV damage, drug action, and signaling pathways, theoretical foundations and technical support can be provided for the treatment of skin diseases and the development of skin tissue engineering.
References
1. Lei Shuyuan, Xu Jiao, Gu Jianmei et al. Protective effect of arbutin, an active component of Cornus officinalis, on skin keratinocyte damage [J]. Traditional Chinese Medicine Information, 2016.
4. Qiu Haixia, Gu Ying, Liu Fangguang et al. Effects of porphyrin monomethyl ether photodynamic therapy on proliferation of human keratinocyte in vitro [J]. Chinese Journal of Laser Medicine, 2006.
6. Wang Yeqiu, Chen Qiaoyun, Zhang Ning et al. Protective effect of Solomon's seal on UVB-induced HaCaT cell damage [J]. Chinese Journal of Cosmetic Medicine, 2012.
7. Yang Hang, Tan Yang, Tang Hui et al. Effects of low-dose ALA-PDT on keratinocyte growth and cytokine secretion in humans [J]. Chinese Journal of Cosmetic Medicine, 2017.
9. Transmembrane Collagen XVII Modulates Integrin Dependent Keratinocyte Migration via PI3K/Rac1 Signaling. [PMID: 24505282]
10. Generation and Culturing of Primary Human Keratinocytes from Adult Skin.[PMID: 29286419]
11.Human epithelial cells induce human melanocyte growth in vitro but only skin keratinocytes regulate its proper differentiation in the absence of dermis. [PMID: 2460471]
12. Duan Dejian, Jiang Xian, Ran Yuping et al. Effects of Candida albicans on the expression of β-defensin-2 mRNA in keratinocytes [J]. Chinese Journal of Dermatology,2005.
13. Cold Atmospheric Plasma Ameliorates Skin Diseases Involving Reactive Oxygen/Nitrogen Species-Mediated Functions. [PMID: 35720416]
14. The JAK–STAT pathway in keloid pathogenesis: a systematic review with qualitative synthesis.
15. Chen Xiangqi, Liu Xiangnong. Research progress on the mechanism of skin keratinocyte apoptosis induced by medium-wave ultraviolet radiation [J]. Chinese Journal of Leprosy and Dermatological Diseases,2006.
16. Sodium selenite improves the in vitro follicular development by reducing the reactive oxygen species level and increasing the total antioxidant capacity and glutathione peroxide activity. [PMID: 20139425]
17. Liu Sujun, Zhang Caiping, Zhou Wuqing et al. Expression of Toll-like receptors in epidermal keratinocytes [J]. Journal of Chinese Academy of Medical Sciences,2008.