Abstract

Classic toxicology studies often utilize in vivo animal models. Newer approaches employing in vitro organ-specific cellular models have been developed in recent years to help accelerate the speed and reduce the cost of traditional toxicology testing. Toward the goal of supporting in vitro cellular model research with a regulatory application in mind, we have developed a ‘designer’ human kidney cell line called HK2-Vi that can fluorescently measure the cytotoxicity of potential toxins on proximal tubule cell viability in a direct exposure in vitro model. HK2-Vi was designed to be a reagent-less kinetic assay that can yield data on short- or long-term cell viability after toxin exposure. To generate HK2-Vi, we used monocistronic lentiviral transduction methods to genetically engineer a human kidney cell line called HK-2 to stably co-express two transgenes. The first is Perceval HR, which encodes a fluorescent biosensor of both cytosolic ATP and ADP and the second is pHRed, which encodes a biosensor of cytosolic pH. Relative levels of cellular ATP and ADP effectively serve as a reliable and robust indicator of cell viability. Because the fluorescence Perceval HR is pH-dependent, we co-expressed the pHRed genetic biosensor to correct for variations in pH if necessary. Heterogenous populations of transduced renal cells were enriched by flow cytometry before monoclonal cellular populations were isolated by cell culture methods. A single clonal population of co-transduced cells expressing both Perceval HR and pHRed was selected to be HK2-Vi. This established cell line can now serve as a tool for in vitro toxicology testing and the methods described herein serve as a model for developing designer cell lines derived from other organs.

中文翻译：

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用于体外实时细胞毒性测试的基因工程人类肾细胞。

摘要

经典毒理学研究通常使用体内动物模型。近年来开发了采用体外器官特异性细胞模型的新方法，以帮助加快传统毒理学测试的速度并降低成本。为了支持体外细胞模型研究并考虑到监管应用的目标，我们开发了一种名为 HK2-Vi 的“设计”人肾细胞系，可以荧光测量潜在毒素在直接暴露下对近端小管细胞活力的细胞毒性体外模型。HK2-Vi 被设计为一种无需试剂的动力学分析，可以在毒素暴露后产生短期或长期细胞活力的数据。要生成 HK2-Vi，我们使用单顺反子慢病毒转导方法对称为 HK-2 的人肾细胞系进行基因工程，以稳定共表达两种转基因。第一个是 Perceval HR，它编码细胞溶质 ATP 和 ADP 的荧光生物传感器，第二个是 pHRed，它编码细胞溶质 pH 的生物传感器。细胞 ATP 和 ADP 的相对水平有效地作为细胞活力的可靠和稳健的指标。因为荧光 Perceval HR 是 pH 依赖性的，所以我们共同表达了 pHRed 基因生物传感器，以在必要时校正 pH 值的变化。在通过细胞培养方法分离单克隆细胞群之前，通过流式细胞术富集转导肾细胞的异质群。选择表达 Perceval HR 和 pHRed 的共转导细胞的单个克隆群作为 HK2-Vi。