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08/03/2023

HaCaT Cells

HaCaT cells are derived from human epidermal keratinocytes and are commonly used to explore epidermal homeostasis and associated pathologies. These cells are considered spontaneously immortalized human keratinocytes, which is noteworthy because they were derived from normal skin keratinocytes without encountering crisis or growth retardation. This unique origin gives HaCaT cells a set of genetic and growth properties that differ from other cell lines. Despite exhibiting a transformed phenotype in vitro with clonogenic properties on agar or plastic, the HaCaT cell line remains non-tumorigenic. Being the first stable epithelial cell line from adult human skin that displays normal differentiation, HaCaT cells are a valuable tool for investigating the regulation of keratinization in human cells. These cells have a wide range of applications due to their ability to proliferate and differentiate effectively in vitro.

  1. Characteristics of HaCaT Cells
  2. Genetic Characteristics and Origin of HaCaT Cells
  3. How to Harvest HaCaT Cells in 5 Simple Steps
  4. Applications of HaCaT Cells
  5.   Featured Videos: Exploring the World of HaCaT Cells

1.       Characteristics of HaCaT Cells

HaCaT cells can be cultured under low calcium conditions and have a spindle-shaped appearance that lacks cell-to-cell tight junctions, like primary cells maintained under the same conditions. In the normal epidermis, keratinocytes develop from stem cells in the basal layer and progress through the spinous and granular layers before reaching the corneal layer and desquamating into the environment. This tightly regulated balance between proliferation and desquamation results in complete renewal or change every 28 days, a complex process known as keratinization. This process is highly regulated and involves marked changes in gene expression, structural design, and enzymatic activity. For in vitro and in vivo analysis, HaCaT cells must maintain the epidermal keratins characteristic of the differentiated layers of human epidermis cells, which are essential for maintaining the structural integrity and resilience of the skin. Although sequential chromosomal alterations can occur in HaCaT cells, they may not be linked to significant differentiation problems and instead demonstrate the spontaneous transformation of human adult keratinocytes in vitro.

HaCaT cells at high and low confluence

2.       Genetic Characteristics and Origin of HaCaT Cells

HaCaT cells are a spontaneously immortalized human keratinocyte cell line originating from adult skin and representing a unique evolutionary pathway. These cells possess mutations in both alleles of the p53 gene, which is typical for mutations induced by UV radiation [3,4]. Additionally, HaCaT cells are assumed to have been generated by p53 tumor suppressor gene mutations, followed by the loss of senescence genes [5].

The tumor suppressor gene p53, known for its role in DNA repair and as the guardian of the genome, induces the human skin's response to DNA damage [4]. It has been observed that HaCaT cells have partly lost their protection mechanism against DNA damage due to the in vivo mutation of the p53 gene, making them susceptible to accumulating cytogenetic changes in response to elevated culture temperatures. Another mechanism of immortalizing HaCaT cells involves increased telomerase enzyme activity [7]. In normal cells, the telomeres continuously shorten with each cell division until cellular senescence is reached. Telomerase is a specialized cellular enzyme complex with reverse transcriptase activity that maintains stable telomere length. In contrast, HaCaT cells show significantly increased telomerase activity, resulting in well-maintained telomere length. These observations confirm the role of telomerase in the immortalization process of HaCaT cells.

Three specific chromosomal translocations have been identified that result in losing one copy of chromosome arms 3p, 4p, and 9p, a gain of 9q, and isochromosome formation. The loss of the short arm of chromosome 3p may lead to the loss of senescence genes and the immortalization of HaCaT cells [8]. HaCaT cells are hypodiploid and possess distinct and stable marker chromosomes representing their monoclonal origin. The characteristics and head of the HaCaT cell line were confirmed using DNA fingerprinting with hypervariable minisatellite markers [3-6].

3.       How to Harvest HaCaT Cells in 5 Simple Steps

  1. Remove the culture medium and rinse the adherent cells using 3-5 mL of PBS without calcium and magnesium for T25 flasks or 5-10 mL for T75 flasks.
  2. Add 1-2 mL of freshly prepared 0.05% EDTA solution per T25 flask, or 2.5 mL per T75 flask, ensuring the entire cell sheet is covered, and incubate at 37°C for 10 minutes.
  3. Add 1 mL of freshly prepared trypsin/EDTA (0.05%/0.025%) solution per T25 flask, or 2.5 mL per T75 flask, again ensuring complete coverage of the cell sheet. The cells should detach within 1-2 minutes.
  4. Stop the trypsin activity by adding an FBS-containing cell culture medium.
  5. Dispense the cells into new flasks containing fresh cell culture medium.

4. Applications of HaCaT Cells

HaCaT cells are a valuable tool for studying keratinocytes [9]. These immortal cells function as preneoplastic cells and can provide insight into changes involved in malignant and neoplastic transformation [10]. Monolayer HaCaT cell cultures are essential for cellular toxicity and in vitro wound healing analysis applications. HaCaT cells can also be used to assess skin toxicity caused by various agents and neoplastic or inflammatory processes. They can be utilized to analyze different mechanisms of cutaneous allergic reactions, the effects of reactive oxygen species, and irradiation by UV. Upon stimulation, HaCaT cells can differentiate and express specific differentiation markers, such as involucrin, K14, and K10. HaCaT cells are also commonly used as a model for studying the pathophysiology of epidermal homeostasis [6].

Researcher conducting scientific studies to confirm the quality of cosmetics

HaCaT cells retain their capacity to reconstitute a structured epidermis in vivo after transplantation, resulting in a stratified epidermal structure that can be reverted between a basal and differentiated state by changes in calcium concentration in the medium. These cells also allow the characterization of several biological processes, such as their use as a vitamin D model system and metabolism in the skin. Because HaCaT cells are not genetically engineered, they present an unbiased view of the broad spectrum of initial genetic events in human skin.

5. Featured Videos: Exploring the World of HaCaT Cells

"HaCaT Cell Migration": This video showcases the process of cell migration in HaCaT cells. The migration of cells is an essential process for various biological processes, such as wound healing and cancer metastasis. The video demonstrates the movement of HaCaT cells under a microscope, providing a visual representation of how these cells migrate. The activity of the cells is observed as they move from one location to another, and the video provides a clear illustration of the changes that occur in the cells during this process.

"Scratch Assay carried out on HaCaT cells": This video showcases a Scratch Assay performed on HaCaT cells. The Scratch Assay is a widely used technique to study cell migration, and in this case, it is used to analyze the migration of HaCaT cells. The video demonstrates the process of creating a scratch on the surface of a cell culture dish, which is then observed under a microscope as HaCaT cells migrate and close the gap over time.

"Cell growth of HaCaT keratinocytes for wound healing experiments": This video demonstrates the process of cell growth of HaCaT keratinocytes for wound healing experiments. HaCaT keratinocytes are a commonly used cell line in wound healing studies.

"HaCaT Cell Differentiation": This video showcases the necessary steps to differentiate HaCaT cells. HaCaT cells can differentiate into different types of skin cells. The video demonstrates the changes in HaCaT cells as they differentiate, visually representing the various markers and characteristics of differentiation. The differentiation process is critical for normal skin functioning, and the video highlights the different stages of differentiation that HaCaT cells undergo.

References

  1. Angel P and Karin M: The role of Jun, Fos and the AP-1 complex in cell proliferation and transformation. Biochim Biophys Acta 1072:129-157, 1991 Argyris TS: The regulation of epidermal hyperplastic growth. Crit Rev Toxicol 9:151-200, 1981
  2. Baden HP, Kubilus J, Kvedar JC, Steinberg ML, Wolman SR: Isolation and characterization of a spontaneously arising long-lived line of human keratinocytes (NM-1). In Vitro Cell Dev Biol 23(3):205-13, 1987
  3. Lehmann TA, Modali R, Boukamp P, Stanek J, Bennett WP, Welsh JA, Metcalf RA, Stampfer MR, Fusenig NE, Rogan EM, Harriss CC: p53 mutations in human immortalized epithelial cell lines. Carcinogenesis 14:833-839, 1993
  4. Ziegler A-M, Leffell DJ, Kunala S, Sharma HW, Gailani M, Simon JA, Halperin AJ, Baden HP, Shapiro PE, Bale AE, Brash DE: Mutation hotspots due to sunlight in the p53 gene of nonmelanoma skin cancer. Proc Natl Acad Sci USA 90:4216-4220, 1993
  5. Fusenig NE, Boukamp P. Multiple stages and genetic alterations in immortalization, malignant transformation, and tumor progression of human skin keratinocytes. Mol Carcinog. 1998;23(3):144-158.
  6. Harle-Bachor C, Boukamp P: Telomerase activity in the regenerative basal layer of the epidermis in human skin and in immortal and carcinoma-derived skin keratinocytes. Proc Natl Acad Sci USA 93:6476-81, 1996
  7. 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.
  8. Boukamp, P. et al. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 106, 1996, 761–771.
  9. Gibbs, Graham: Analysing qualitative data. The Sage qualitative research kit. London: Sage  978-0-7619-4980-0.
  10. Hedrick TE, Bickman L, Rog DJ. 1993. Applied research design: a practical guide. Sage: London
  11. Boukamp P. Petrussevska R. T. Breitkreutz D. Hornung J. Markham A. Fusenig N. E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.  Cell Biol.(1988);106:761–771.

HaCaT cells are derived from human epidermal keratinocytes and are commonly used to explore epidermal homeostasis and associated pathologies. These cells are considered spontaneously immortalized human keratinocytes, which is noteworthy because they were derived from normal skin keratinocytes without encountering crisis or growth retardation. This unique origin gives HaCaT cells a set of genetic and growth properties that differ from other cell lines. Despite exhibiting a transformed phenotype in vitro with clonogenic properties on agar or plastic, the HaCaT cell line remains non-tumorigenic. Being the first stable epithelial cell line from adult human skin that displays normal differentiation, HaCaT cells are a valuable tool for investigating the regulation of keratinization in human cells. These cells have a wide range of applications due to their ability to proliferate and differentiate effectively in vitro.

  1. Characteristics of HaCaT Cells
  2. Genetic Characteristics and Origin of HaCaT Cells
  3. How to Harvest HaCaT Cells in 5 Simple Steps
  4. Applications of HaCaT Cells
  5.   Featured Videos: Exploring the World of HaCaT Cells

1.       Characteristics of HaCaT Cells

HaCaT cells can be cultured under low calcium conditions and have a spindle-shaped appearance that lacks cell-to-cell tight junctions, like primary cells maintained under the same conditions. In the normal epidermis, keratinocytes develop from stem cells in the basal layer and progress through the spinous and granular layers before reaching the corneal layer and desquamating into the environment. This tightly regulated balance between proliferation and desquamation results in complete renewal or change every 28 days, a complex process known as keratinization. This process is highly regulated and involves marked changes in gene expression, structural design, and enzymatic activity. For in vitro and in vivo analysis, HaCaT cells must maintain the epidermal keratins characteristic of the differentiated layers of human epidermis cells, which are essential for maintaining the structural integrity and resilience of the skin. Although sequential chromosomal alterations can occur in HaCaT cells, they may not be linked to significant differentiation problems and instead demonstrate the spontaneous transformation of human adult keratinocytes in vitro.

HaCaT cells at high and low confluence

2.       Genetic Characteristics and Origin of HaCaT Cells

HaCaT cells are a spontaneously immortalized human keratinocyte cell line originating from adult skin and representing a unique evolutionary pathway. These cells possess mutations in both alleles of the p53 gene, which is typical for mutations induced by UV radiation [3,4]. Additionally, HaCaT cells are assumed to have been generated by p53 tumor suppressor gene mutations, followed by the loss of senescence genes [5].

The tumor suppressor gene p53, known for its role in DNA repair and as the guardian of the genome, induces the human skin's response to DNA damage [4]. It has been observed that HaCaT cells have partly lost their protection mechanism against DNA damage due to the in vivo mutation of the p53 gene, making them susceptible to accumulating cytogenetic changes in response to elevated culture temperatures. Another mechanism of immortalizing HaCaT cells involves increased telomerase enzyme activity [7]. In normal cells, the telomeres continuously shorten with each cell division until cellular senescence is reached. Telomerase is a specialized cellular enzyme complex with reverse transcriptase activity that maintains stable telomere length. In contrast, HaCaT cells show significantly increased telomerase activity, resulting in well-maintained telomere length. These observations confirm the role of telomerase in the immortalization process of HaCaT cells.

Three specific chromosomal translocations have been identified that result in losing one copy of chromosome arms 3p, 4p, and 9p, a gain of 9q, and isochromosome formation. The loss of the short arm of chromosome 3p may lead to the loss of senescence genes and the immortalization of HaCaT cells [8]. HaCaT cells are hypodiploid and possess distinct and stable marker chromosomes representing their monoclonal origin. The characteristics and head of the HaCaT cell line were confirmed using DNA fingerprinting with hypervariable minisatellite markers [3-6].

3.       How to Harvest HaCaT Cells in 5 Simple Steps

  1. Remove the culture medium and rinse the adherent cells using 3-5 mL of PBS without calcium and magnesium for T25 flasks or 5-10 mL for T75 flasks.
  2. Add 1-2 mL of freshly prepared 0.05% EDTA solution per T25 flask, or 2.5 mL per T75 flask, ensuring the entire cell sheet is covered, and incubate at 37°C for 10 minutes.
  3. Add 1 mL of freshly prepared trypsin/EDTA (0.05%/0.025%) solution per T25 flask, or 2.5 mL per T75 flask, again ensuring complete coverage of the cell sheet. The cells should detach within 1-2 minutes.
  4. Stop the trypsin activity by adding an FBS-containing cell culture medium.
  5. Dispense the cells into new flasks containing fresh cell culture medium.

4. Applications of HaCaT Cells

HaCaT cells are a valuable tool for studying keratinocytes [9]. These immortal cells function as preneoplastic cells and can provide insight into changes involved in malignant and neoplastic transformation [10]. Monolayer HaCaT cell cultures are essential for cellular toxicity and in vitro wound healing analysis applications. HaCaT cells can also be used to assess skin toxicity caused by various agents and neoplastic or inflammatory processes. They can be utilized to analyze different mechanisms of cutaneous allergic reactions, the effects of reactive oxygen species, and irradiation by UV. Upon stimulation, HaCaT cells can differentiate and express specific differentiation markers, such as involucrin, K14, and K10. HaCaT cells are also commonly used as a model for studying the pathophysiology of epidermal homeostasis [6].

Researcher conducting scientific studies to confirm the quality of cosmetics

HaCaT cells retain their capacity to reconstitute a structured epidermis in vivo after transplantation, resulting in a stratified epidermal structure that can be reverted between a basal and differentiated state by changes in calcium concentration in the medium. These cells also allow the characterization of several biological processes, such as their use as a vitamin D model system and metabolism in the skin. Because HaCaT cells are not genetically engineered, they present an unbiased view of the broad spectrum of initial genetic events in human skin.

5. Featured Videos: Exploring the World of HaCaT Cells

"HaCaT Cell Migration": This video showcases the process of cell migration in HaCaT cells. The migration of cells is an essential process for various biological processes, such as wound healing and cancer metastasis. The video demonstrates the movement of HaCaT cells under a microscope, providing a visual representation of how these cells migrate. The activity of the cells is observed as they move from one location to another, and the video provides a clear illustration of the changes that occur in the cells during this process.

"Scratch Assay carried out on HaCaT cells": This video showcases a Scratch Assay performed on HaCaT cells. The Scratch Assay is a widely used technique to study cell migration, and in this case, it is used to analyze the migration of HaCaT cells. The video demonstrates the process of creating a scratch on the surface of a cell culture dish, which is then observed under a microscope as HaCaT cells migrate and close the gap over time.

"Cell growth of HaCaT keratinocytes for wound healing experiments": This video demonstrates the process of cell growth of HaCaT keratinocytes for wound healing experiments. HaCaT keratinocytes are a commonly used cell line in wound healing studies.

"HaCaT Cell Differentiation": This video showcases the necessary steps to differentiate HaCaT cells. HaCaT cells can differentiate into different types of skin cells. The video demonstrates the changes in HaCaT cells as they differentiate, visually representing the various markers and characteristics of differentiation. The differentiation process is critical for normal skin functioning, and the video highlights the different stages of differentiation that HaCaT cells undergo.

References

  1. Angel P and Karin M: The role of Jun, Fos and the AP-1 complex in cell proliferation and transformation. Biochim Biophys Acta 1072:129-157, 1991 Argyris TS: The regulation of epidermal hyperplastic growth. Crit Rev Toxicol 9:151-200, 1981
  2. Baden HP, Kubilus J, Kvedar JC, Steinberg ML, Wolman SR: Isolation and characterization of a spontaneously arising long-lived line of human keratinocytes (NM-1). In Vitro Cell Dev Biol 23(3):205-13, 1987
  3. Lehmann TA, Modali R, Boukamp P, Stanek J, Bennett WP, Welsh JA, Metcalf RA, Stampfer MR, Fusenig NE, Rogan EM, Harriss CC: p53 mutations in human immortalized epithelial cell lines. Carcinogenesis 14:833-839, 1993
  4. Ziegler A-M, Leffell DJ, Kunala S, Sharma HW, Gailani M, Simon JA, Halperin AJ, Baden HP, Shapiro PE, Bale AE, Brash DE: Mutation hotspots due to sunlight in the p53 gene of nonmelanoma skin cancer. Proc Natl Acad Sci USA 90:4216-4220, 1993
  5. Fusenig NE, Boukamp P. Multiple stages and genetic alterations in immortalization, malignant transformation, and tumor progression of human skin keratinocytes. Mol Carcinog. 1998;23(3):144-158.
  6. Harle-Bachor C, Boukamp P: Telomerase activity in the regenerative basal layer of the epidermis in human skin and in immortal and carcinoma-derived skin keratinocytes. Proc Natl Acad Sci USA 93:6476-81, 1996
  7. 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.
  8. Boukamp, P. et al. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 106, 1996, 761–771.
  9. Gibbs, Graham: Analysing qualitative data. The Sage qualitative research kit. London: Sage  978-0-7619-4980-0.
  10. Hedrick TE, Bickman L, Rog DJ. 1993. Applied research design: a practical guide. Sage: London
  11. Boukamp P. Petrussevska R. T. Breitkreutz D. Hornung J. Markham A. Fusenig N. E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.  Cell Biol.(1988);106:761–771.