HaCaT cells are human immortalized epidermal cells derived from keratinocytes, retaining the differentiation capacity of keratinocytes. They are a non-tumor-derived human normal skin immortalized keratinocyte cell line, with differentiation characteristics similar to normal human keratinocytes. These cells can proliferate for over 150 generations but are not tumor cells and do not possess tumor characteristics.

 

Cell Name	HaCaT (Human Immortalized Keratinocytes)
Cell Source	Normal skin from the peripheral area of a melanoma lesion in a 62-year-old male
Morphology	Epithelial cell-like
Growth Characteristics	Adherent growth
Nutrient System	89% MEM (abs9503) + 10% Fetal Bovine Serum (abs972) + 1% Penicillin-Streptomycin Solution (abs9244) + 0.2% Bovine Pituitary Extract (abs9119)
Passaging	1:3—1:5, passaging cycle 2-3 days
Cryopreservation	Serum-Free Cell Cryopreservation Solution Plus (abs9478) / 90% complete culture medium + 10% DMSO
Application	Skin biology and differentiation research

 

Morphology of well-grown HaCaT cells

 

These cells are relatively difficult to digest. Trypsin-EDTA solution (0.25%), containing phenol red (abs47014938), is used for digestion for 5 minutes, sometimes up to 10 minutes, depending on the degree of cell differentiation and confluence. Note that cells must be washed 2-3 times with PBS before digestion. When plating, ensure even distribution, as HaCaT cells grow in sheets. Uneven plating may result in overly dense growth in some areas, making digestion difficult, while sparse areas may become over-digested.

Figure a: Basic state of HaCaT cells under low Ca²⁺ conditions and Figure b: Differentiation state of HaCaT cells under high Ca²⁺ conditions

 

The Ca²⁺ in a 10% FBS culture system is sufficient to induce differentiation of these cells. In other words, under normal culture conditions, the cells will gradually differentiate. Before differentiation, the cells are spindle-shaped and loosely arranged. After differentiation, the cells become more cubic, tightly arranged, and grow in sheets.

Under low calcium ion conditions, HaCaT cells proliferate more rapidly. The expression of K14 is limited to the basal layer of the epidermis, while the presence of K10 and total keratin indicates a higher degree of phenotypic differentiation. Immunofluorescence experiments have confirmed the acquisition of differentiated phenotypes in HaCaT cells. Compared to day 6, the expression of the differentiation marker K10 in HaCaT cells significantly increased by day 14, with almost 100% of cells being positive, which may be related to cell density.

 

Bovine pituitary extract is commonly used as a growth supplement for serum-free cell culture, promoting the proliferation of epithelial cells, endothelial cells, and melanocytes. It is widely used as a supplement in the culture of various epithelial and melanocyte cells. Additionally, it has antioxidant properties, combating cell necrosis, protein oxidation, membrane disruption, and DNA damage induced by hydrogen peroxide.

 

References

[1] Wilson VG. Growth and differentiation of HaCaT keratinocytes. Methods Mol Biol. 2014;1195:33-41. doi: 10.1007/7651_2013_42. PMID: 24155234.
[2] Colombo I, Sangiovanni E, Maggio R, et al. HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes. Mediators Inflamm. 2017;2017:7435621. doi:10.1155/2017/7435621

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