Study on feasibility of HaCaT epidermal model as an alternative to skin irritation <i>in vitro</i> _Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIU Yang ¹ ,  LU Tao ¹ , ZHOU Zhoulin ¹ , LU Yongbo ² , ZHANG Yanyun ²

1. Department of Dermatology, Affiliated Hospital of Logistics College of Chinese Peolpe’s Armed Police Force, Tianjin, 300162, P.R.China;
2. Guangdong Biocell Biotechnology Co. Ltd, Dongguan Guangdong, 523808, P.R.China;

Corresponding author：

LU Tao, Email: 48144051@qq.com

Keywords：

Epidermal model; HaCaT; EpiKutis^® model; skin barrier; reference chemicals; skin irritation

DOI：

10.7507/1002-1892.201705031

Video：

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Objective To construct the epidermal model with HaCaT cells and evaluate the feasibility of this model as an in vitro skin irritation test tool. Methods The HaCaT model was reconstructed with HaCaT cells by adoption gas-liquid surface culture technique, and the EpiKutis^® model was reconstructed with human epidermal keratinocytes by the same techinique as control. Morphology changes of HaCaT and EpiKutis^® models were observed by HE staining. Barrier function assessment was performed with penetration test. According to Organization for Economic Cooperation and Development (OECD) Test Guideline 439, the surface of the HaCaT and the EpiKutis^® models were treated with 20 chemicals for 30 minutes, incubated for 42 hours, and determined tissue viability by MTT assay, to evaluate the irritation of chemicals. Then the results were compared with the irritation of chemicals with the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) system and validated reference method (VRM) for the classification of chemical, and evaluated the feasibility of this model as an in vitro skin irritation test tool. Results The results of HE staining showed that there was no complete stratified structure in the HaCaT model. The results of barrier function showed that the ET₅₀ was 0.99 hours. The results of skin irritation of chemicals showed that the sensitivity was 100% and 100%, the specificity was 50% and 70%, and the accuracy rate was 75% and 85% for HaCaT model and EpiKutis^® model respectively. Conclusion The epidermal model of HaCaT cells does not possess the complete epidermal physiological structure, the barrier function as ET₅₀ of the HaCaT model is lower than EpiKutis^® model, the chemicals in vitro skin irritation test results do not meet the OECD criteria for the determination of stimulants, so the HaCaT model is not suitable as a replacement tool in vitro to determine the chemicals skin irritation.

Citation： LIU Yang, LU Tao, ZHOU Zhoulin, LU Yongbo, ZHANG Yanyun. Study on feasibility of HaCaT epidermal model as an alternative to skin irritation in vitro . Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(10): 1262-1266. doi: 10.7507/1002-1892.201705031 Copy

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2.	程树军, 焦红. 实验动物替代方法原理及应用. 北京: 科学出版社, 2010: 9.
3.	Welss T, Basketter DA, Schröder KR. In vitro skin irritation: facts and future. State of the art review of mechanisms and models. Toxicology in Vitro, 2004, 18(3): 231-243.
4.	Portes P, Grandidier MH, Cohen C, et al. Refinement of the Episkin protocol for the assessment of acute skin irritation of chemicals: follow-up to the ECVAM prevalidation study. Toxicol In Vitro, 2002, 16(6): 765-770.
5.	Roguet R, Cohen C, Dossou KG, et al. Episkin, a reconstituted human epidermis for assessing in vitro the irritancy of topically applied compounds. Toxicol, 1994, 8(2): 283-291.
6.	Kandárová H, Liebsch M, Genschow E, et al. Optimisation of the EpiDerm Test Protocol for the Upcoming ECVAM Validation Study on In Vitro Skin Irritation Tests. ALTEX, 2004, 21(3): 107-114.
7.	Chatterjee A, Babu RJ, Klausner M, et al. In vitro and in vivo comparison of dermal irritancy of jet fuel exposure using EpiDerm^TM (EPI-200) cultured human skin and hairless rats. Toxicol Lett, 2006, 167(2): 85-94.
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9.	Netzlaff F, Lehr CM, Wertz PW, et al. The human epidermis models EpiSkin, SkinEthic and EpiDerm: an evaluation of morphology and their suitability for testing phototoxicity, irritancy, corrosivity, and substance transport. Eur J Pharm Biopharm, 2005, 60(2): 167-178.
10.	Tornier C, Amsellem C, Anne de Brugerolle de Fraissinette, et al. Assessment of the optimized SkinEthic^TM Reconstructed Human Epidermis (RHE) 42 bis skin irritation protocol over 39 test substances. Toxicol In Vitro, 2010, 24(1): 245-256.
11.	Katoh M, Hamajima F, Ogasawara T, et al. Assessment of Human Epidermal Model LabCyte EPI-MODEL for In Vitro Skin Irritation Testing According to European Centre for the Validation of Alternative Methods (ECVAM)-Validated Protocol. J Toxicol Sci, 2009, 34(3): 327-334.
12.	Hikima T, Kaneda N, Matsuo, K, et al. Prediction of percutaneous absorption in human using three-dimensional human culturedepidermis LabCyte EPI-MODEL. Biol Pharm Bull, 2012, 35(3): 362-368.
13.	卢永波, 张勇杰, 金岩. 组织工程皮肤技术及化妆品体外科学检测的发展. 北京日化, 2016, 124(3): 26-31.0.
14.	张广静, 卓金士, 金岩, 等. 人的原代上皮角质形成细胞和永生化的上皮角质形成细胞 HaCaT 的增殖能力的比较. 现代生物医学进展, 2011, 11(1): 68-70.0.
15.	OECD Guideline for the Testing of Chemicals. No.439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method. Paris: OECD, 2013.
16.	ECVAM SIVS: Performance standards for applying human skin models to in vitro skin irritation testing (2007)[S/OL]. http://ecvam.jrc.ec.europa.eu.
17.	Brohem CA, Cardeal LB, Tiago M, et al. Artificial skin in perspective: concepts and applications. Pigment Cell Melanoma Res, 2011, 24(1): 35-50.

1. Rosholt AP. The Seventh Amendment directive—an unnecessary measure to a necessary end-possible legal challenges to directive 2003/15/EC of the European Parliament and of the Council Amending Council Directive 76/768/EEC under European Union law. Food Drug Law J, 2005, 60(3): 421-446.
2. 程树军, 焦红. 实验动物替代方法原理及应用. 北京: 科学出版社, 2010: 9.
3. Welss T, Basketter DA, Schröder KR. In vitro skin irritation: facts and future. State of the art review of mechanisms and models. Toxicology in Vitro, 2004, 18(3): 231-243.
4. Portes P, Grandidier MH, Cohen C, et al. Refinement of the Episkin protocol for the assessment of acute skin irritation of chemicals: follow-up to the ECVAM prevalidation study. Toxicol In Vitro, 2002, 16(6): 765-770.
5. Roguet R, Cohen C, Dossou KG, et al. Episkin, a reconstituted human epidermis for assessing in vitro the irritancy of topically applied compounds. Toxicol, 1994, 8(2): 283-291.
6. Kandárová H, Liebsch M, Genschow E, et al. Optimisation of the EpiDerm Test Protocol for the Upcoming ECVAM Validation Study on In Vitro Skin Irritation Tests. ALTEX, 2004, 21(3): 107-114.
7. Chatterjee A, Babu RJ, Klausner M, et al. In vitro and in vivo comparison of dermal irritancy of jet fuel exposure using EpiDerm^TM (EPI-200) cultured human skin and hairless rats. Toxicol Lett, 2006, 167(2): 85-94.
8. European Centre for the Validation of Alternative Methods. Statement on the application of the EPIDERM^TM humanskin model for skin corrosivity testing. ATLA, 2001, 28: 365-367.
9. Netzlaff F, Lehr CM, Wertz PW, et al. The human epidermis models EpiSkin, SkinEthic and EpiDerm: an evaluation of morphology and their suitability for testing phototoxicity, irritancy, corrosivity, and substance transport. Eur J Pharm Biopharm, 2005, 60(2): 167-178.
10. Tornier C, Amsellem C, Anne de Brugerolle de Fraissinette, et al. Assessment of the optimized SkinEthic^TM Reconstructed Human Epidermis (RHE) 42 bis skin irritation protocol over 39 test substances. Toxicol In Vitro, 2010, 24(1): 245-256.
11. Katoh M, Hamajima F, Ogasawara T, et al. Assessment of Human Epidermal Model LabCyte EPI-MODEL for In Vitro Skin Irritation Testing According to European Centre for the Validation of Alternative Methods (ECVAM)-Validated Protocol. J Toxicol Sci, 2009, 34(3): 327-334.
12. Hikima T, Kaneda N, Matsuo, K, et al. Prediction of percutaneous absorption in human using three-dimensional human culturedepidermis LabCyte EPI-MODEL. Biol Pharm Bull, 2012, 35(3): 362-368.
13. 卢永波, 张勇杰, 金岩. 组织工程皮肤技术及化妆品体外科学检测的发展. 北京日化, 2016, 124(3): 26-31.0.
14. 张广静, 卓金士, 金岩, 等. 人的原代上皮角质形成细胞和永生化的上皮角质形成细胞 HaCaT 的增殖能力的比较. 现代生物医学进展, 2011, 11(1): 68-70.0.
15. OECD Guideline for the Testing of Chemicals. No.439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method. Paris: OECD, 2013.
16. ECVAM SIVS: Performance standards for applying human skin models to in vitro skin irritation testing (2007)[S/OL]. http://ecvam.jrc.ec.europa.eu.
17. Brohem CA, Cardeal LB, Tiago M, et al. Artificial skin in perspective: concepts and applications. Pigment Cell Melanoma Res, 2011, 24(1): 35-50.

Chinese Journal of Reparative and Reconstructive Surgery

Study on feasibility of HaCaT epidermal model as an alternative to skin irritation in vitro

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