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Optimization of a Novel In Situ Hybridization Technology on 3D Organotypic Cell Cultures

机译：优化的新型原位杂交3 d Organotypic细胞培养技术

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Introduction: Developed by Advanced Cell Diagnostics, RNAscope?in situ hybridization technology enables detection of a target RNA in a cell-specific manner on formalin-fixed paraffin-embedded tissue sections and represents a good alternative to immunohistochemistry. The goal of this work is to illustrate an optimized protocol of the RNAscope technology to detect target genes in various human organotypic culture models (nasal, small airway, and gingival). These culture models retain the three-dimensional structure of native epithelium, mimic in vivo morphology and human physiology, and can be used as alternative sources to animal testing.Materials and Methods: After fixation and processing of five replicates of the three different organotypic cell cultures, the tissue morphology was checked by hematoxylin and eosin staining. The RNAscope protocols were optimized based on three crucial parameters: heat pretreatment, enzymatic digestion, and signal amplification. Digital images of the RNAscope stained slides were generated using the Hamamatsu NanoZoomer 2.0 slide scanner, and images were quantified using a custom-made plugin on Definiens Tissue Studio software (Definiens AG, Munich, Germany).Results: The tissue morphology demonstrates optimum fixation and processing for samples, while the optimized protocol for RNAscope shows preserved RNA with staining on the positive control probe with score ≥2 and no staining on the negative control probe with score <1.Discussion and Conclusion: RNAscope combined with organotypic cell cultures is a promising tool to better understand cell-specific RNA expression while implementing 3R (replace, reduce, and refine animal testing) principles.
机译：简介:由先进细胞技术公司开发的诊断,RNAscope ?技术使靶RNA的检测特异性的方式在formalin-fixed石蜡包埋组织切片和代表免疫组织化学的一个好的选择。这项工作的目的是为了说明一个优化协议RNAscope技术来检测目标基因在不同的人类器官型培养模型(鼻、小气道和牙龈)。文化保留三维模型本机上皮结构,模拟体内形态学和人体生理学,都可以使用作为动物的替代来源测试。处理五个复制的三个不同organotypic细胞培养、组织形态学检查了苏木精和伊红染色。基于三个重要参数:热量预处理、酶消化和信号放大。彩色幻灯片使用滨松生成NanoZoomer 2.0幻灯片扫描仪和图像量化使用定制的插件Definiens组织工作室(Definiens软件股份公司,德国慕尼黑)。演示了最佳固定和处理样本,而优化的协议RNAscope显示保存RNA与染色积极控制探针与分数≥2,没有染色负控制探针的分数< 1。organotypic细胞培养是一种很有前途的为了更好地理解特异性RNA的工具表达而实现3 r(替换,减少和改进动物试验)的原则。

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