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인공피부모델 KeraSkinTM을 이용한 유전독성 평가
이수현 ( Su-hyon Lee ),정행선 ( Haeng-sun Jung ),김설영 ( Seol-yeong Kim ),김혜수 ( Hye Soo Kim ),임경민 ( Kyung-min Lim ),정영신 ( Young-shin Chung ),최태부 ( Tae-boo Choe ) 대한화장품학회 2016 대한화장품학회지 Vol.42 No.3
소핵시험은 세포분열 단계 중 간기 세포의 세포질 내 소핵 유무를 조사함으로써 유전독성을 평가하는 시험법이다. 최근 화장품 안전성 평가에 동물실험을 금지하거나 최소화하려는 노력이 확산되고 있어 유전독성 평가에 있어서도 기존의 동물실험이 아닌 새로운 in vitro 시험법이 요구되고 있다. 본 연구에서는 3차원 배양인공피부모델인 KeraSkin<sup>TM</sup>을 이용하여 도포 처치된 물질의 유전독성을 평가하였다. 2종의 유전독성물질인 mitomycin C (MMC)와 methyl methanesulfonate (MMS)는 농도 의존적으로 세포독성과 소핵 형성이 유도된 반면, 대조물질인 4-nitrophenol (4-NP)와 trichloroethylene (TCE)에서는 농도 의존적으로 세포독성은 관찰되었으나 소핵은 형성되지 않았다. 따라서 인공피부모델을 이용한 소핵시험이 화장품과 같은 피부적용물질의 in vitro 유전독성 평가에 유용할 것으로 사료된다. Micronucleus test is genotoxicity assay for detection of micronuclei in the cytoplasm of interphase cells. The reduction and replacement of in vivo toxicity testing on animals require the development of in vitro models to predict the genotoxicity or other tests for cosmetic products. In this study, we evaluated a genotoxicity assay for topically applied chemicals using a three-dimensional human reconstructed skin model, KeraSkinTM. Two genotoxins, mitomycin C (MMC) and methyl methanesulfonate (MMS), induced significant dose-related increases in cytotoxicity and micronuclei induction in the skin model. In contrast, two non-genotoxins, 4-nitrophenol (4-NP) and trichloroethylene (TCE), induced cytotoxicity but not micronucleus formation. In conclusion, micronucleus test using human skin model may be useful for predicting in vitro genotoxic potentials of cosmetic products.
정행선 ( Haeng Sun Jung ),이수현 ( Su Hyon Lee ),류양환 ( Yang Hwan Ryu ),정경미 ( Kyoung Mi Jung ),김채욱 ( Chae Wook Kim ),임경민 ( Kyung Min Lim ),손영숙 ( Young Sook Son ) 한국동물실험대체법학회 2009 동물실험대체법학회지 Vol.3 No.1
Alternative methods to the Draize eye irritation test, such as the BCOP, HET-CAM, ICE, and IRE, are used to evaluate the ocular irritation potential of cosmetic, livelihood articles or industrial chemicals. In order to improve the sensitivity and specificity of alternative eye irritation test, we developed a three-dimensional human corneal model that uses normal human corneal epithelial cells. Our corneal tissue model consists of normal human corneal epithelial cells on cell culture inserts at the air liquid interface, differentiating to form a stratified, squamous epithelium similar to that found in the human cornea, exhibiting in vivo like morphological and histological characteristics. In this study, two laboratories have tested 20 reference chemicals using the same study protocol. The results were compared to in vivo data as well as previously published data obtained in the other three-dimensional corneal model test. Although an overall accuracy of 85% was obtained (sensitivity=100% and specificity=67%), further experiments are required to confirm and validate these preliminary results.
인공피부모델 KeraSkin(TM) 피부자극성 시험 대체시험법의 실험실간 검증
문지영 ( Ji Young Moon ),이수현 ( Su Hyon Lee ),정행선 ( Haeng Sun Jung ),류양환 ( Yang Hwan Ryu ),정경미 ( Kyoung Mi Jung ),임경민 ( Kyung Min Lim ),박철범 ( Cheol Beom Park ) 한국동물실험대체법학회 2010 동물실험대체법학회지 Vol.4 No.2
Reconstructed skin provides advantages to single cell culture testing and leads to promising results as shown by different validation studies. In this study we investigated the interlaboratory validation for the efficacy of “Keraskin(TM)” model. We conducted endpoint analysis including cell viability (MTT reduction) and IL-1α release. Eighteen item, which were ECVAM test meterial, were selected for this validation study. Results of MTT assay were not different between EPI-200 and Keraskin(TM), but IL-1α levels of some test items different between EPI-200 and Keraskin(TM). We concluded that this Keraskin(TM) model is useful for alternative skin irritation test, although other tests should be conducted for many other chemicals in international validation study.
정행선 ( Haeng Sun Jung ),이수현 ( Su Hyon Lee ),류양환 ( Yang Hwan Ryu ),정경미 ( Kyoung Mi Jung ),김채욱 ( Chae Wook Kim ),임경민 ( Kyung Min Lim ),손영숙 ( Young Sook Son ) 한국동물실험대체법학회 2009 동물실험대체법학회지 Vol.3 No.1
Alternative methods to the Draize eye irritation test, such as the BCOP, HET-CAM, ICE, and IRE, are used to evaluate the ocular irritation potential of cosmetic, livelihood articles or industrial chemicals. In order to improve the sensitivity and specificity of alternative eye irritation test, we developed a three-dimensional human corneal model that uses normal human corneal epithelial cells. Our corneal tissue model consists of normal human corneal epithelial cells on cell culture inserts at the air liquid interface, differentiating to form a stratified, squamous epithelium similar to that found in the human cornea, exhibiting in vivo like morphological and histological characteristics. In this study, two laboratories have tested 20 reference chemicals using the same study protocol. The results were compared to in vivo data as well as previously published data obtained in the other three-dimensional corneal model test. Although an overall accuracy of 85% was obtained (sensitivity=100% and specificity=67%), further experiments are required to confirm and validate these preliminary results.
연구논문 : 인공피부모델 KeraSkin(TM) 피부자극성 시험 대체시험법의 실험실간 검증
문지영 ( Ji Young Moon ),이수현 ( Su Hyon Lee ),정행선 ( Haeng Sun Jung ),류양환 ( Yang Hwan Ryu ),정경미 ( Kyoung Mi Jung ),임경민 ( Kyung Min Lim ),박철범 ( Cheol Beom Park ) 한국동물실험대체법학회 2010 동물실험대체법학회지 Vol.4 No.2
Reconstructed skin provides advantages to single cell culture testing and leads to promising results as shown by different validation studies. In this study we investigated the interlaboratory validation for the efficacy of “Keraskin(TM)” model. We conducted endpoint analysis including cell viability (MTT reduction) and IL-1α release. Eighteen item, which were ECVAM test meterial, were selected for this validation study. Results of MTT assay were not different between EPI-200 and Keraskin(TM), but IL-1α levels of some test items different between EPI-200 and Keraskin(TM). We concluded that this Keraskin(TM) model is useful for alternative skin irritation test, although other tests should be conducted for many other chemicals in international validation study.
미백 기능성 화장품 원료의 유효성 평가를 위한 In Vitro 색소화피부모델 개발
김설영 ( Seolyeong Kim ),이건희 ( Geonhee Lee ),곽은지 ( Eun Ji Gwak ),김수지 ( Su Ji Kim ),이수현 ( Su Hyon Lee ),임경민 ( Kyung-min Lim ) 대한화장품학회 2021 대한화장품학회지 Vol.47 No.4
본 연구에서는 미백 기능성 화장품 및 원료의 효능을 평가하는 동물대체시험법을 개발하기 위하여 세포수준과 색소화피부모델(KeraSkin-M<sup>TM</sup>)에서 기존에 잘 알려진 4종의 미백기능성원료(arbutin, ascorbic acid, kojic acid, niacinamide)의 효능을 평가하였다. 특히 기존 시험법의 보완을 위해 인체피부유래 케라틴세포와 멜라닌세포를 혼합하고 공배양하여 색소화피부모델을 제작하였다. 그 결과 색소화피부모델을 이용하여 미백효능을 평가함으로써 세포수준에서는 확인이 어려웠던 각 피부세포층에 따른 멜라닌 과립과 멜라닌캡(melanin cap)의 분포 등의 지표들을 추가로 확인할 수 있었으며 이미지분석을 통한 정량화로 음성대조군 대비 통계적 유의성을 확인할 수 있었다. 이러한 결과는 KeraSkin-M<sup>TM</sup>을 이용한 미백효능평가법이 기존에 사용하던 총멜라닌함량와 타이로시나아제 저해 평가를 보완할 수 있는 새로운 평가법으로 사용할 수 있음을 시사한다. In this study, we prepared a pigmented skin model, KeraSkin-M<sup>TM</sup> for the in vitro evaluation of whitening agents. For the purpose of complementing the existing mono-layer cell culture testing method, KeraSkin-M<sup>TM</sup> was produced through the co-culture of human skin-derived keratinocytes and melanocytes. The efficacy of four well-known whitening agents (arbutin, ascorbic acid, kojic acid, niacinamide) was evaluated in KeraSkin-M<sup>TM</sup> in order to assess its usefulness in assessing whitening efficacy. As a result, it was possible to observe additional details such as the distribution of melanin granules and melanin capping in each skin layer through KeraSkin-M<sup>TM</sup>, which was previously difficult to assess in the traditional 2D cell culture system. In addition, quantification through image analysis of KeraSkin-M<sup>TM</sup> allowed for a statistical analysis of the whitening effects. These results suggest that the KeraSkin-M<sup>TM</sup> can be used as a new evaluation method of evaluating whitening efficacy, as well as complement the traditional total melanin content and tyrosinase inhibition assays.
정경미 ( Kyoung Mi Jung ),문지영 ( Ji Young Moon ),이수현 ( Su Hyon Lee ),김채욱 ( Chae Wook Kim ),박철범 ( Cheol Beom Park ),김배환 ( Bae Hwan Kim ) 한국동물실험대체법학회 2008 동물실험대체법학회지 Vol.2 No.1
Several alternative in vitro methods for identifying skin irritants have been developed recently, the most promising of which use reconstituted human skin model. In this study, the two reconstituted human skin models, KeraskinTM and EPI-200, showed promising results in terms of interlaboratory variability and predictive capacity. The main endpoint measured for both KeraskinTM and EPI-200 was cell viability via MTT. In samples from chemicals which gave MTT assay results above the threshold of 50% viability, IL-1α release was also measured, to determine whether the additional endpoint would improve the predictive ability of the tests. In the interlaboratory validation study for KeraskinTM, sensitivity, specificity and accuracy of the KeraskinTM were 90%, 44% and 68%(MTT assay only), respectively. The KeraskinTM model was overly sensitive, resulting in the prediction of the non-irritant chemicals as irritant. The EPI-200 model had an accuracy of 63%, with a sensitivity rate of 70% and a specificity rate of 56%. Based on these results, although predictive capacity of KeraSkinTM model was considered insufficient, due to a low specificity, we concluded that this KeraSkinTM model is useful potential alternative skin irritation method. Further studies are in progress to improve the test protocols and prediction models for KeraSkinTM.
장원희 ( Won Hee Jang ),정경미 ( Kyoung Mi Jung ),최선경 ( Sun Kyung Choi ),정행선 ( Haeng Sun Jung ),이수현 ( Su Hyon Lee ),최영진 ( Young Jin Choi ),박영호 ( Young Ho Park ),임경민 ( Kyung Min Lim ) 한국동물실험대체법학회 2011 동물실험대체법학회지 Vol.5 No.1
Dry skin is induced by excessive transdermal water loss due to the damages of intracellular lipids. Moisturizer is safe and effective for improving dry skin, however, there is few efficient alternative method to evaluate moisturizing activity of new cosmetic ingredients. Here, we investigated the moisturizing activity of a face powder coated with mannosylerithritol lipids (MEL) that are similar to ceramides in structure and intermediates in the biosynthesis of intracellular lipids, without using animals or human but Keraskin™, a three-dimensional reconstructed human epidermis. After Keraskin™ was incubated, MEL-coated sericite or uncoated sericite was applied and then sodium lauryl sulphate (SLS) was treated with a nylon mesh covering to spread it uniformly. After incubation for 15 min, Keraskin™ was washed and it was post-incubated for 24 hr. At the end of post-incubation, Keraskin™ was incubated with MTT for 3 hr to measure viability. Formed purple formazan was extracted with isopropanol for 2 hr. Absorbance of extract(A590) was measured using spectrophotometer. Cell viability was calculated by dividing with the absorbance of negative control. The viability of reconstructed epidermis treated 10% SLS was determined to be 12.3 ± 4.87%. When SLS was co-treated with sericite or MEL-coated sericite, cell viability was increased to 23.7 ± 2.92% or 71.5 ± 20% (p<0.05), respectively. MEL-coated sericite inhibited SLS-induced cytotoxicity substantially in comparison with sericite alone. Based on these results, reconstructed human epidermis can be used for the evaluation of the efficacy of cosmetic ingredients or products.
연구논문 : 인공피부모델을 이용한 보습파우더의 보습 효능 평가
장원희 ( Won Hee Jang ),정경미 ( Kyoung Mi Jung ),최선경 ( Sun Kyung Choi ),정행선 ( Haeng Sun Jung ),이수현 ( Su Hyon Lee ),최영진 ( Young Jin Choi ),박영호 ( Young Ho Park ),임경민 ( Kyung Min Lim ) 한국동물실험대체법학회 2011 동물실험대체법학회지 Vol.5 No.1
Dry skin is induced by excessive transdermal water loss due to the damages of intracellular lipids. Moisturizer is safe and effective for improving dry skin, however, there is few efficient alternative method to evaluate moisturizing activity of new cosmetic ingredients. Here, we investigated the moisturizing activity of a face powder coated with mannosylerithritol lipids (MEL) that are similar to ceramides in structure and intermediates in the biosynthesis of intracellular lipids, without using animals or human but Keraskin™, a three-dimensional reconstructed human epidermis. After Keraskin™ was incubated, MEL-coated sericite or uncoated sericite was applied and then sodium lauryl sulphate (SLS) was treated with a nylon mesh covering to spread it uniformly. After incubation for 15 min, Keraskin™ was washed and it was post-incubated for 24 hr. At the end of post-incubation, Keraskin™ was incubated with MTT for 3 hr to measure viability. Formed purple formazan was extracted with isopropanol for 2 hr. Absorbance of extract(A590) was measured using spectrophotometer. Cell viability was calculated by dividing with the absorbance of negative control. The viability of reconstructed epidermis treated 10% SLS was determined to be 12.3 ± 4.87%. When SLS was co-treated with sericite or MEL-coated sericite, cell viability was increased to 23.7 ± 2.92% or 71.5 ± 20% (p<0.05), respectively. MEL-coated sericite inhibited SLS-induced cytotoxicity substantially in comparison with sericite alone. Based on these results, reconstructed human epidermis can be used for the evaluation of the efficacy of cosmetic ingredients or products.
호흡기 3D 조직모델(SoluAirway<sup>TM</sup>)의 품질과 독성평가에 대한 국내 확립 연구
정미호 ( Mi-ho Jeong ),김설영 ( Seol-yeong Kim ),김준우 ( Jun-woo Kim ),방인재 ( In-jae Bang ),김가은 ( Ga-eun Kim ),김혜수 ( Hye-soo Kim ),이수현 ( Su-hyon Lee ),김하룡 ( Ha-ryong Kim ) 한국동물실험대체법학회 2019 동물실험대체법학회지 Vol.13 No.1
The concern of health and safety is growing due to increasing exposure of inhalable chemicals. However, there is no appropriate alternative testing method for assessing the hazard caused by inhalation exposure. The aim of this study is to establish respiratory 3-dimentional (3D) tissue model which can be applied for assessing the toxicity testing in Korea. First of all, respiratory 3D tissue model, SoluAirway™ was reconstituted by using human nasal mucosa. For evaluating its stability, the viability, Trans-Epithelial Electric Resistance (TEER), and histology was analyzed after delivered to 3 areas in Korea. Then, bleomycin (BLM) and polyhexamethylene guanidine phosphate (PHMG) which were known as toxic materials in pulmonary system were treated to various 3D tissue models to demonstrate applicability in evaluating chemical toxicity. The cytotoxicity and secretion of inflammatory cytokines was acquired with MTT and ELISA, respectively. The cell viability, TEER and integrity of the 3D tissue model which quality was controlled between products, had no significantly different values after delivery. The cytotoxicity of BLM and PHMG showed dose-dependently. Furthermore, the secretion of IL-8 was shown at the highest concentration of BLM and PHMG-p in SoluAirway™, not in EpiAirway™. When applied to KeraSkin™ which was a skin model, the secretion of IL-8 was declined. Taken together, these data showed that SoluAirway™ could be used as toxicity testing model for inhalable substances in Korea.