Institute of Clinical Pharmacology and Toxicology, Charité, Berlin, Germany.
Toxicol Appl Pharmacol. 2010 Jun 1;245(2):211-8. doi: 10.1016/j.taap.2010.02.019. Epub 2010 Mar 8.
We have developed a coculture system which in parallel indicates the sensitizing and irritative potential of xenobiotics. The assay is named loose-fit coculture-based sensitization assay (LCSA) and may be performed within 5 days. The system is composed of human monocytes that differentiate to a kind of dendritic cells by 2-day culturing in the presence of allogenic keratinocytes. The culture medium is enriched by a cocktail of recombinant cytokines. On day 3, concentration series of probes are added. On day 5, cells are harvested and analyzed for expression range of CD86 as a marker of sensitizing potential and for uptake of the viability stain 7-AAD as a marker of irritative potential. Estimation of the concentration required to cause a half-maximal increase in CD86 expression allowed quantification of sensitizing potential, and estimation of the concentration required to reduce viability to 50% allowed quantification of irritative potential. Examination of substances with known potential resulted in categorization of test scores. To evaluate our data, we have compared results with those of the validated animal-based sensitization test, the murine local lymph node assay (LLNA, OECD TG 429). To a large extent, results from LCSA and from LLNA achieved analogous grouping of allergens into categories like weak-moderate-strong. However, the new assay showed an improved capacity to distinguish sensitizers from non-sensitizers and irritants. In conclusion, the LCSA contains potential to fulfil the requirements of the EU's programme for the safety of chemicals "Registration, Evaluation, Authorization and Restriction of chemical substances" (REACH, 2006) to replace animal models.
我们开发了一种共培养系统，该系统可同时指示外源性物质的致敏和刺激性潜能。该测定方法命名为松配合共培养敏化测定法（LCSA），可在 5 天内完成。该系统由人类单核细胞组成，这些单核细胞在存在同种异体角质形成细胞的情况下培养 2 天，分化为一种树突状细胞。培养基富含重组细胞因子的混合物。第 3 天，加入浓度系列探针。第 5 天，收获细胞并分析 CD86 的表达范围，作为致敏潜能的标志物，以及摄取活细胞染色 7-AAD，作为刺激性潜能的标志物。估计引起 CD86 表达增加半最大值所需的浓度允许定量致敏潜能，并且估计将存活率降低至 50%所需的浓度允许定量刺激性潜能。对具有已知潜能的物质进行检查，结果对测试分数进行了分类。为了评估我们的数据，我们将结果与经过验证的动物致敏测试（OECD TG 429 中的小鼠局部淋巴结测定法）进行了比较。在很大程度上，LCSA 和 LLNA 的结果将过敏原分为弱-中-强等类别。然而，新的测定法显示出更好的能力来区分致敏剂和非致敏剂以及刺激性物质。总之，LCSA 具有满足欧盟化学品安全计划“注册、评估、授权和限制化学物质”（REACH，2006 年）的要求的潜力，以替代动物模型。
