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3D CULTURE OF HaCaT KERATINOCYTE CELL LINE AS AN in vitro TOXICITY MODEL

Year 2022, Volume: 23 Issue: 2, 211 - 220, 15.10.2022
https://doi.org/10.23902/trkjnat.1158811

Abstract

Ex vivo dermal toxicology analyses are crucial for replacement of in vivo test methods and have been of interest in recent years for testing cosmetics, drugs, and chemicals. Development of an appropriate reconstructed epidermis model might overcome the limitations of monolayer culture systems. In the current study, we used the immortalized human keratinocyte cell line (HaCaT) to develop an ex vivo 3D cell culture system for keratinocyte-based toxicity analysis. Mouse embryonic fibroblast-conditioned medium and Matrigel matrix-based 3D HaCaT cell culture systems expressed skin-related genes and proteins in culture. The 3D HaCaT cultures demonstrated a skin-like phenotype and response against selected test compounds. Reliable results were obtained compared to monolayer HaCaT cells which were exposed to selected chemicals for 1 h and 24 h. Gene expression profiles of 3D HaCaT cell cultures and monolayer cultures were completely different after administration of the test compounds.
Overall, our results showed that a 3D HaCaT cell culture system generated in Matrigel matrix exerted a skin epidermis-like phenotype. Consequently, 3D HaCaT cell cultures may be an acceptable test method for conducting in vitro toxicology experiments. 

Thanks

This study was conducted in Yeditepe University. We would like to thank Yeditepe University Writing Center and Hakan Şentürk (İstanbul-TÜRKİYE) for language editing and proofreading support.

References

  • 1. Almeida, A., Sarmento, B. & Rodrigues, F. 2017. Insights on in vitro models for safety and toxicity assessment of cosmetic ingredients. International Journal of Pharmaceutics, 519(1-2): 178-185.
  • 2. Boelsma, E., Verhoeven, M.C. & Ponec, M. 1999. Reconstruction of a human skin equivalent using a spontaneously transformed keratinocyte cell line (HaCaT). Journal of Investigative Dermatology, 112(4): 489-498.
  • 3. Capallere, C., Plaza, C., Meyrignac, C., Arcioni, M., Brulas, M., Busuttil, V., Garcia, I., Bauza, É. & Botto, J.-M. 2018. Property characterization of reconstructed human epidermis equivalents, and performance as a skin irritation model. Toxicology in Vitro, 53: 45-56.
  • 4. Colombo, I., Sangiovanni, E., Maggio, R., Mattozzi, C., Zava, S., Corbett, Y., Fumagalli, M., Carlino, C., Corsetto, P.A. & Scaccabarozzi, D. 2017. HaCaT cells as a reliable in vitro differentiation model to dissect the inflammatory/repair response of human keratinocytes. Mediators of Inflammation, 2017: 7435621.
  • 5. De Jong, W.H., Carraway, J.W., Liu, C., Fan, C., Liu, J., Turley, A.P., Rollins, T.S. & Coleman, K.P. 2020. The suitability of reconstructed human epidermis models for medical device irritation assessment: A comparison of in vitro and in vivo testing results. Toxicology in Vitro, 69: 104995.
  • 6. Demirci, S., Doğan, A., Aydın, S., Dülger, E.Ç. & Şahin, F. 2016. Boron promotes streptozotocin-induced diabetic wound healing: roles in cell proliferation and migration, growth factor expression, and inflammation. Molecular and Cellular Biochemistry, 417(1): 119-133.
  • 7. do Nascimento Pedrosa, T., Catarino, C.M., Pennacchi, P.C., de Assis, S.R., Gimenes, F., Consolaro, M.E.L., de Moraes Barros, S.B. & Maria-Engler, S.S. 2017. A new reconstructed human epidermis for in vitro skin irritation testing. Toxicology in Vitro, 42: 31-37.
  • 8. Gailit, J., Clark, R.A. & Welch, M.P. 1994. TGF-β1 stimulates expression of keratinocyte integrins during re-epithelialization of cutaneous wounds. Journal of Investigative Dermatology, 103(2): 221-227.
  • 9. Garach-Jehoshua, O., Ravid, A., Liberman, U., Reichrath, J., Glaser, T. & Koren, R. 1998. Upregulation of the calcium-dependent protease, calpain, during keratinocyte differentiation. The British Journal of Dermatology, 139(6): 950-957.
  • 10. Hamill, K.J., Hopkinson, S.B., Hoover, P., Todorović, V., Green, K.J. & Jones, J.C. 2012. Fibronectin expression determines skin cell motile behavior. Journal of Investigative Dermatology, 132(2): 448-457.
  • 11. Hänel, K.H., Cornelissen, C., Lüscher, B. & Baron, J.M. 2013. Cytokines and the skin barrier. International Journal of Molecular Sciences, 14(4): 6720-6745.
  • 12. Jackson, D.G., Cardwell, L.A., Oussedik, E. & Feldman, S.R. 2020. Utility of boron in dermatology. Journal of Dermatological Treatment, 31(1): 2-12.
  • 13. Kandarova, H., Willoughby, J.A., De Jong, W.H., Letasiova, S., Milasova, T., Bachelor, M.A., Breyfogle, B., Handa, Y., De la Fonteyne, L. & Coleman, K.P. 2018. Pre-validation of an in vitro skin irritation test for medical devices using the reconstructed human tissue model EpiDerm™. Toxicology in Vitro, 50: 407-417.
  • 14. Larjava, H., Koivisto, L. & Häkkinen, L. 2013. Keratinocyte interactions with fibronectin during wound healing, Madame Curie Bioscience Database [Internet], Landes Bioscience, https://www.ncbi.nlm.nih.gov/books/NBK6391/ (Date accessed: 08 May 2022)
  • 15. Micallef, L., Belaubre, F., Pinon, A., Jayat‐Vignoles, C., Delage, C., Charveron, M. & Simon, A. 2009. Effects of extracellular calcium on the growth‐differentiation switch in immortalized keratinocyte HaCaT cells compared with normal human keratinocytes. Experimental Dermatology, 18(2): 143-151.
  • 16. Miles, A., Berthet, A., Hopf, N.B., Gilliet, M., Raffoul, W., Vernez, D. & Spring, P. 2014. A new alternative method for testing skin irritation using a human skin model: a pilot study. Toxicology in Vitro, 28(2): 240-247.
  • 17. Mini, C., Dreossi, S., Abe, F., Maria-Engler, S. & Oliveira, D. 2021. Immortalized keratinocytes cells generates an effective model of Epidermal Human Equivalent for irritation and corrosion tests. Toxicology in Vitro, 71: 105069.
  • 18. OECD. 2021. Test No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris. https://doi.org/10.1787/9789264242845-en
  • 19. Pastar, I., Stojadinovic, O., Yin, N.C., Ramirez, H., Nusbaum, A.G., Sawaya, A., Patel, S.B., Khalid, L., Isseroff, R.R. & Tomic-Canic, M. 2014. Epithelialization in wound healing: a comprehensive review. Advances in Wound Care, 3(7): 445-464.
  • 20. Pellevoisin, C., Videau, C., Briotet, D., Grégoire, C., Tornier, C., Alonso, A., Rigaudeau, A.S., Bouez, C. & Seyler, N. 2018. SkinEthic™ RHE for in vitro evaluation of skin irritation of medical device extracts. Toxicology in Vitro, 50: 418-425.
  • 21. Reilly, D.M. & Lozano, J. 2021. Skin collagen through the lifestages: Importance for skin health and beauty. Plastic and Aesthetic Research, 8(2): 1-24.
  • 22. Schmidt, F.F., Nowakowski, S. & Kluger, P.J. 2020. Improvement of a three-layered in vitro skin model for topical application of irritating substances. Frontiers in Bioengineering and Biotechnology, 8: 388.
  • 23. Schürer, N., Köhne, A., Schliep, V., Barlag, K. & Goerz, G. 1993. Lipid composition and synthesis of HaCaT cells, an immortalized human keratinocyte line, in comparison with normal human adult keratinocytes. Experimental Dermatology, 2(4): 179-185.
  • 24. Şişli, H.B., Şenkal, S., Sağraç, D., Hayal, T.B. & Doğan, A. 2021. Feeder-Dependent/Independent Mouse Embryonic Stem Cell Culture Protocol. pp. 101-115. In: Turksen, K. (ed.) Embryonic Stem Cell Protocols. Methods in Molecular Biology, vol 2520. Humana, New York.
Year 2022, Volume: 23 Issue: 2, 211 - 220, 15.10.2022
https://doi.org/10.23902/trkjnat.1158811

Abstract

Ex vivo dermal toksikoloji analizleri, in vivo test yöntemlerinin yerini alabilmesi için çok önemlidir ve son yıllarda kozmetik, ilaç ve kimyasalların test edilmesi için ilgi çekici hale gelmiştir. Kültürde yapılandırılmış uygun bir epidermis modelinin geliştirilmesi, tek katmanlı kültür sistemlerinin sınırlamalarını ortadan kaldırabilir. Bu amaçla, mevcut çalışmada, keratinosit bazlı toksisite analizi amacı ile bir ex vivo 3D hücre kültürü sistemi geliştirmek için ölümsüzleştirilmiş insan keratinosit hücre hattı (HaCaT) kullandık. Fare embriyonik fibroblast kondisyonel besiyeri ve Matrigel matriksi tabanlı 3D HaCaT hücre kültürü sistemleri, kültürde deri ilgili genleri ve proteinleri ifade etti. 3D HaCaT kültürleri, deri benzeri bir fenotip gösterdi ve seçilen test bileşiklerine karşı tepki gösterdi. 1 saat ve 24 saat boyunca seçilen kimyasallara maruz bırakılan tek katmanlı HaCaT hücrelerine kıyasla güvenilir sonuçlar elde edildi. 3D HaCaT hücre kültürlerinin ve tek tabakalı kültürlerin gen ekspresyon profilleri, test bileşiklerinin uygulanmasından sonra tamamen farklıydı.
Özet olarak, sonuçlarımız Matrigel matrisinde oluşturulan bir 3D HaCaT hücre kültürü sisteminin cilt epidermisi benzeri bir fenotip uyguladığını gösterdi. Sonuç olarak, 3D HaCaT hücre kültürleri, in vitro toksikoloji deneylerinin yürütülmesi için kabul edilebilir bir test yöntemi olabilir.

References

  • 1. Almeida, A., Sarmento, B. & Rodrigues, F. 2017. Insights on in vitro models for safety and toxicity assessment of cosmetic ingredients. International Journal of Pharmaceutics, 519(1-2): 178-185.
  • 2. Boelsma, E., Verhoeven, M.C. & Ponec, M. 1999. Reconstruction of a human skin equivalent using a spontaneously transformed keratinocyte cell line (HaCaT). Journal of Investigative Dermatology, 112(4): 489-498.
  • 3. Capallere, C., Plaza, C., Meyrignac, C., Arcioni, M., Brulas, M., Busuttil, V., Garcia, I., Bauza, É. & Botto, J.-M. 2018. Property characterization of reconstructed human epidermis equivalents, and performance as a skin irritation model. Toxicology in Vitro, 53: 45-56.
  • 4. Colombo, I., Sangiovanni, E., Maggio, R., Mattozzi, C., Zava, S., Corbett, Y., Fumagalli, M., Carlino, C., Corsetto, P.A. & Scaccabarozzi, D. 2017. HaCaT cells as a reliable in vitro differentiation model to dissect the inflammatory/repair response of human keratinocytes. Mediators of Inflammation, 2017: 7435621.
  • 5. De Jong, W.H., Carraway, J.W., Liu, C., Fan, C., Liu, J., Turley, A.P., Rollins, T.S. & Coleman, K.P. 2020. The suitability of reconstructed human epidermis models for medical device irritation assessment: A comparison of in vitro and in vivo testing results. Toxicology in Vitro, 69: 104995.
  • 6. Demirci, S., Doğan, A., Aydın, S., Dülger, E.Ç. & Şahin, F. 2016. Boron promotes streptozotocin-induced diabetic wound healing: roles in cell proliferation and migration, growth factor expression, and inflammation. Molecular and Cellular Biochemistry, 417(1): 119-133.
  • 7. do Nascimento Pedrosa, T., Catarino, C.M., Pennacchi, P.C., de Assis, S.R., Gimenes, F., Consolaro, M.E.L., de Moraes Barros, S.B. & Maria-Engler, S.S. 2017. A new reconstructed human epidermis for in vitro skin irritation testing. Toxicology in Vitro, 42: 31-37.
  • 8. Gailit, J., Clark, R.A. & Welch, M.P. 1994. TGF-β1 stimulates expression of keratinocyte integrins during re-epithelialization of cutaneous wounds. Journal of Investigative Dermatology, 103(2): 221-227.
  • 9. Garach-Jehoshua, O., Ravid, A., Liberman, U., Reichrath, J., Glaser, T. & Koren, R. 1998. Upregulation of the calcium-dependent protease, calpain, during keratinocyte differentiation. The British Journal of Dermatology, 139(6): 950-957.
  • 10. Hamill, K.J., Hopkinson, S.B., Hoover, P., Todorović, V., Green, K.J. & Jones, J.C. 2012. Fibronectin expression determines skin cell motile behavior. Journal of Investigative Dermatology, 132(2): 448-457.
  • 11. Hänel, K.H., Cornelissen, C., Lüscher, B. & Baron, J.M. 2013. Cytokines and the skin barrier. International Journal of Molecular Sciences, 14(4): 6720-6745.
  • 12. Jackson, D.G., Cardwell, L.A., Oussedik, E. & Feldman, S.R. 2020. Utility of boron in dermatology. Journal of Dermatological Treatment, 31(1): 2-12.
  • 13. Kandarova, H., Willoughby, J.A., De Jong, W.H., Letasiova, S., Milasova, T., Bachelor, M.A., Breyfogle, B., Handa, Y., De la Fonteyne, L. & Coleman, K.P. 2018. Pre-validation of an in vitro skin irritation test for medical devices using the reconstructed human tissue model EpiDerm™. Toxicology in Vitro, 50: 407-417.
  • 14. Larjava, H., Koivisto, L. & Häkkinen, L. 2013. Keratinocyte interactions with fibronectin during wound healing, Madame Curie Bioscience Database [Internet], Landes Bioscience, https://www.ncbi.nlm.nih.gov/books/NBK6391/ (Date accessed: 08 May 2022)
  • 15. Micallef, L., Belaubre, F., Pinon, A., Jayat‐Vignoles, C., Delage, C., Charveron, M. & Simon, A. 2009. Effects of extracellular calcium on the growth‐differentiation switch in immortalized keratinocyte HaCaT cells compared with normal human keratinocytes. Experimental Dermatology, 18(2): 143-151.
  • 16. Miles, A., Berthet, A., Hopf, N.B., Gilliet, M., Raffoul, W., Vernez, D. & Spring, P. 2014. A new alternative method for testing skin irritation using a human skin model: a pilot study. Toxicology in Vitro, 28(2): 240-247.
  • 17. Mini, C., Dreossi, S., Abe, F., Maria-Engler, S. & Oliveira, D. 2021. Immortalized keratinocytes cells generates an effective model of Epidermal Human Equivalent for irritation and corrosion tests. Toxicology in Vitro, 71: 105069.
  • 18. OECD. 2021. Test No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris. https://doi.org/10.1787/9789264242845-en
  • 19. Pastar, I., Stojadinovic, O., Yin, N.C., Ramirez, H., Nusbaum, A.G., Sawaya, A., Patel, S.B., Khalid, L., Isseroff, R.R. & Tomic-Canic, M. 2014. Epithelialization in wound healing: a comprehensive review. Advances in Wound Care, 3(7): 445-464.
  • 20. Pellevoisin, C., Videau, C., Briotet, D., Grégoire, C., Tornier, C., Alonso, A., Rigaudeau, A.S., Bouez, C. & Seyler, N. 2018. SkinEthic™ RHE for in vitro evaluation of skin irritation of medical device extracts. Toxicology in Vitro, 50: 418-425.
  • 21. Reilly, D.M. & Lozano, J. 2021. Skin collagen through the lifestages: Importance for skin health and beauty. Plastic and Aesthetic Research, 8(2): 1-24.
  • 22. Schmidt, F.F., Nowakowski, S. & Kluger, P.J. 2020. Improvement of a three-layered in vitro skin model for topical application of irritating substances. Frontiers in Bioengineering and Biotechnology, 8: 388.
  • 23. Schürer, N., Köhne, A., Schliep, V., Barlag, K. & Goerz, G. 1993. Lipid composition and synthesis of HaCaT cells, an immortalized human keratinocyte line, in comparison with normal human adult keratinocytes. Experimental Dermatology, 2(4): 179-185.
  • 24. Şişli, H.B., Şenkal, S., Sağraç, D., Hayal, T.B. & Doğan, A. 2021. Feeder-Dependent/Independent Mouse Embryonic Stem Cell Culture Protocol. pp. 101-115. In: Turksen, K. (ed.) Embryonic Stem Cell Protocols. Methods in Molecular Biology, vol 2520. Humana, New York.
There are 24 citations in total.

Details

Primary Language English
Subjects Structural Biology
Journal Section Research Article/Araştırma Makalesi
Authors

Selinay Şenkal 0000-0002-6897-7307

Derya Burukçu This is me 0000-0002-5178-0620

Taha Bartu Hayal 0000-0003-1369-2715

Binnur Kıratlı This is me 0000-0002-5234-5911

Hatice Burcu Şişli This is me 0000-0001-6528-8365

Derya Sağraç 0000-0002-3582-1103

Burçin Asutay 0000-0002-0253-8336

Engin Sümer This is me 0000-0002-9228-7963

Fikrettin Şahin 0000-0003-1503-5567

Ayşegül Doğan 0000-0003-4160-2270

Publication Date October 15, 2022
Submission Date August 8, 2022
Acceptance Date October 11, 2022
Published in Issue Year 2022 Volume: 23 Issue: 2

Cite

APA Şenkal, S., Burukçu, D., Hayal, T. B., Kıratlı, B., et al. (2022). 3D CULTURE OF HaCaT KERATINOCYTE CELL LINE AS AN in vitro TOXICITY MODEL. Trakya University Journal of Natural Sciences, 23(2), 211-220. https://doi.org/10.23902/trkjnat.1158811
AMA Şenkal S, Burukçu D, Hayal TB, Kıratlı B, Şişli HB, Sağraç D, Asutay B, Sümer E, Şahin F, Doğan A. 3D CULTURE OF HaCaT KERATINOCYTE CELL LINE AS AN in vitro TOXICITY MODEL. Trakya Univ J Nat Sci. October 2022;23(2):211-220. doi:10.23902/trkjnat.1158811
Chicago Şenkal, Selinay, Derya Burukçu, Taha Bartu Hayal, Binnur Kıratlı, Hatice Burcu Şişli, Derya Sağraç, Burçin Asutay, Engin Sümer, Fikrettin Şahin, and Ayşegül Doğan. “3D CULTURE OF HaCaT KERATINOCYTE CELL LINE AS AN in Vitro TOXICITY MODEL”. Trakya University Journal of Natural Sciences 23, no. 2 (October 2022): 211-20. https://doi.org/10.23902/trkjnat.1158811.
EndNote Şenkal S, Burukçu D, Hayal TB, Kıratlı B, Şişli HB, Sağraç D, Asutay B, Sümer E, Şahin F, Doğan A (October 1, 2022) 3D CULTURE OF HaCaT KERATINOCYTE CELL LINE AS AN in vitro TOXICITY MODEL. Trakya University Journal of Natural Sciences 23 2 211–220.
IEEE S. Şenkal, “3D CULTURE OF HaCaT KERATINOCYTE CELL LINE AS AN in vitro TOXICITY MODEL”, Trakya Univ J Nat Sci, vol. 23, no. 2, pp. 211–220, 2022, doi: 10.23902/trkjnat.1158811.
ISNAD Şenkal, Selinay et al. “3D CULTURE OF HaCaT KERATINOCYTE CELL LINE AS AN in Vitro TOXICITY MODEL”. Trakya University Journal of Natural Sciences 23/2 (October 2022), 211-220. https://doi.org/10.23902/trkjnat.1158811.
JAMA Şenkal S, Burukçu D, Hayal TB, Kıratlı B, Şişli HB, Sağraç D, Asutay B, Sümer E, Şahin F, Doğan A. 3D CULTURE OF HaCaT KERATINOCYTE CELL LINE AS AN in vitro TOXICITY MODEL. Trakya Univ J Nat Sci. 2022;23:211–220.
MLA Şenkal, Selinay et al. “3D CULTURE OF HaCaT KERATINOCYTE CELL LINE AS AN in Vitro TOXICITY MODEL”. Trakya University Journal of Natural Sciences, vol. 23, no. 2, 2022, pp. 211-20, doi:10.23902/trkjnat.1158811.
Vancouver Şenkal S, Burukçu D, Hayal TB, Kıratlı B, Şişli HB, Sağraç D, Asutay B, Sümer E, Şahin F, Doğan A. 3D CULTURE OF HaCaT KERATINOCYTE CELL LINE AS AN in vitro TOXICITY MODEL. Trakya Univ J Nat Sci. 2022;23(2):211-20.

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