The Study Of In Vitro Skin Sensitization Test Based On Integrating 3D Model To H-CLAT

BackgroundSkin is an important barrier for the human body to block the external materials, and to provide an important guarantee for the human body. It mainly contains 4 kinds of cells, which is the keratinocytes, fibroblasts, Langerhans cells and melanocytes. Skin as the first barrier to interfere the external materials, it will defend against hazardous substances by special reactions. Allergic contact dermatitis is a type IV delayed hypersensitivity cutaneous immune reaction that is important for chemicals classification and identification as well as safe assessment and risk identification. Allergic contact dermatitis is a clinical symptom of skin sensitization, and it is estimated that 15-20% of the general population will be sensitized at some point in the course of their lives, whereas, it is common situation in our life. Because of that, it is essential to scan the potential nature of substances.Guinea pig methods(eg., guinea pig maximization test) have been the main methods used. More recently, the local lymph node assay(LLNA), as part of a quantitative risk assessment process, was proposed as an early evaluation tool. However, the 7th Amendment to the Cosmetics Directive(Directive 76/768/EEC) aims for the complete replacement of animal testing by 2013, someplace was forbidden to take the animal tests.As the situation, more and more non-animal testing was developed and be accepted by regulation. Due to the complicated of the mechanisms for skin sensitization, it is hard to all around describe the process of skin sensitization in one in vitro system, the development of an in vitro method should consider or address various aspects of the sensitization process. The availability of these methods has lead to the suggestion that a tiered approach using a battery of these methods could be used as an alternate to the in vivo skin sensitization method.During the induction phase of skin sensitization, Langerhans cells(LC) play a critical role because of their ability to initiate immune responses by processing and presenting antigens after exposure to chemical allergens. During the course, LC maturation is characterized by the upregulation of major histocompatibility complex(MHC) class II and expression of co-stimulatory molecules, such as CD86, CD54, CD80, and CD40 as well as cytokine production like interleukin(IL)-18. Many of the antigen-specific immune response methods have used dendritic cells(DC) including LC. Several investigators have published some promising data using these cells as surrogates for LC, but due to the technical issues are donor to donor variability and availability of human blood.To avoid the issues with obtaining DCs, some human cell lines, THP-1 cells, U937 cells, KG-1 cells and MUTZ-3, are used. At present study, based on the h-CLAT testing method integrated the Ha Ca T cells and EpiskinTM 3D skin models to discriminate the skin sensitization of substances.Objective Construction of h-CLAT testing method and respectively combined with Ha Ca T keratinocytes and EpiskinTM skin model to build a different integrated skin sensitization methods. By monitoring the cell viability, interleukin 18(IL-18) expression, cell surface marker CD54 /CD86 and relative fluorescence intensity of cells surface after the cells was exposures substances, to format a complex test strategies and assessment plans for skin sensitization powerful potency of chemicals and personal care products.MethodPart.1 The Study of human cell line activation test(h-CLAT)1. Cell cultureTHP-1 cells: THP-1 cells was cultured in RPMI1640 medium with 10% fetal bovine serum, and plus the 25 m M HEPES, 0.05 m M 2- mercaptoethanol as well as 1% of antibiotic-antimycotic.2. Test substanceNon-sensitization: SLS, lactic acid. Weak sensitization: cinnamyl alcohol, eugenol. Mild sensitization: citral, cinnamic aldehyde, phenylacetaldehyde. Strong sensitization: propyl gallate, maleic anhydride. Extreme sensitization: DNCB, p-benzoquinone. These substance be tested in h-CLAT. After construction of the h-CLAT, Microemulsion makeup remover, tricholoma matsutake extract, Angelica extract and Scutellaria baicalensis extract be tested and predicted for skin sensitization potency.Part.2 The Study for combination Ha Ca T cells with h-CLAT to assess skin sensitization1. Cell cultureHa Ca T cells: Ha Ca T cells was cultured in RPMI1640 medium with 10% fetal bovine serum, and plus the 25 m M HEPES, as well as 1% of antibiotic-antimycotic. THP-1 cells co-culture with Ha Ca T cells using the BD Falcon transwell in the 12 holes cell culture plate.2. Test substanceSLS, eugenol, DNCB and cinnamic aldehyde were tested by Ha Ca T cells to decide IC50, and then to construct method that THP-1 cells co-culture with Ha Ca T cells for discriminate the substances if skin sensitization. At the end, to test the culture medium from only Ha Ca T cells and co-culture system using the IL-18 test kit.Part.3 The Study for combination Episkin TM with h-CLAT to assess skin sensitization1. Cell cultureEpiskinTM 3D skin model : It was cultured in EME medium that is from a commercial package with skin model. The skin model co-culture with THP-1 in the 12 holes cell culture plate.2. Test substanceSLS, eugenol, DNCB and cinnamic aldehyde were tested by co-culture system that is combination EpiskinTM 3D skin model with THP-1 cells and to discriminate the substances if skin sensitization. As well as compare the results from Part.1 and Part.2 with the results from this part. At last, to test 6 Cleaning products whether contain potency of skin sensitization by the co-culture method.ResultPart.1 The Study of human cell line activation test(h-CLAT)The study on THP 1 cells to discriminate 9 kinds of skin sensitization positive substances and 2 kinds of skin sensitization negative materials, and to calculate the cell viability, as well as the cell surface molecule CD54 and CD86. At the end, to obtain the results of the lowest effected concentration EC150(CD86) and EC200(CD54). When the method be constructed, Microemulsion makeup remover, tricholoma matsutake extract, Angelica extract and Scutellaria baicalensis extract be tested and predicted for the skin sensitization potency. Therefore, Microemulsion makeup remover and tricholoma matsutake extract were decided to negative, and Angelica extract and Scutellaria baicalensis extract were recognized the positive.Part.2 The Study for combination Ha Ca T cells with h-CLAT to assess skin sensitizationDue to the keratinocyte is an essential part in the hypersensitivity reactions, hence the attach that to the h-CLAT, and detection of IL-18 by the kit. As a result, the IL-18 was significant increase in Ha Ca T cells only and co-culture system(P<0.05). Subsequently, THP-1 cells was exposed the non-toxic dose of IL-18 combined with DNCB, the results showed that there was no significant difference between the double substances treatment group and the DNCB group(P > 0.05). Following, the co-culture system was used to detect the SLS, eugenol, cinnamic aldehyde and DNCB, it was found that compared the h-CLAT to co-culture system, the CD86/CD54 fluorescence intensity did not significantly enhanced(P > 0.05). However, under the same conditions, the irritation and the sensitization of the materials can be detected in one in vitro test method, and to screen preliminarily the exposed dose of substance for the 3D skin model.Part.3 The Study for combination EpiskinTM with h-CLAT to assess skin sensitizationIn order to optimize the Ha Ca T keratinocyte and THP-1 cells co-culture model, the system method of co-culture of the biological activity of EpiskinTM skin model and THP-1 cells was constructed, and to screen four kinds of substances in the skin sensitization test. Eventually, it was found that to compare pure THP-1 cells and THP-1 cells and Ha Ca T keratinocytes co-culture system with EpiskinTM skin model and THP-1 cells co-culture system, the relative fluorescence intensity in the EpiskinTM skin model and THP-1 cells co-culture system was increased significantly(P < 0.05) by tested the 3 positive substances.Finally, 6 kinds of cleaning products were tested.ConclusionThrough the study of 9 kinds of known skin sensitization positive standard substances and 2 kinds of negative materials to establish h-CLAT, and 4 kinds of samples were distinguished. At the same time, in order to strengthen the single cell detection system, Ha Ca T cells was joined the h-CLAT, and discriminated 3 kinds of sensitive positive substance and 1 negative material accurately and a test method based on IL-18 to detect skin sensitivity and stimulation was formed. Finally, for more fit the action of the cosmetic products, the personal care products and other substances expose to human skin, the model h-CLAT combined with 3D skin model was built. Hence, the recognization and assessment of 3 kinds of skin sensitive positive materials, one negative materials and 6 kinds of cleaning product were active. The results show that the system could to make correct judgment for skin sensitization materials, and the physical type of substances became more choice than above mentioned methods.