New In Vitro Human Liver Toxicity Bioassay System

摘要：本SBIR提案的总体目标是开发一种新的人体肝脏生物测定系统，用于化学制剂或药物的体外实时、长期毒性分析。目前，肝脏研究大多是使用人工合成或动物源性基质培养的肝细胞进行的。不幸的是，这些模型不能复制在体内发现的真正的细胞-基质相互作用。此外，由于分离肝细胞的表型不稳定，迫切需要长期培养/测定模型。有趣的是，在三维基质培养中球体的形成被证明可以改善细胞功能。我们最近创造了一种新的人类生物基质培养系统- HuBiogel，它支持许多细胞类型的长期生长，存活和组织。利用这种生理相关模型，我们建议开发新的3D或迷你肝脏检测平台，展示功能优势并允许精确的毒性预测。第一阶段的具体目标是：开发和优化3D生物测定系统使用原代人肝细胞和定义HuBiogel共培养配置；和2。用标准显微镜、生化和分子方法评估已知肝毒性药物的功能和代谢终点。HuBiogel的研究和结果将与Collagen I或Matrigel检测系统进行比较。在该项目的第二阶段，肝毒性研究将扩大到包括基因组学和蛋白质组学分析，以利用人类肝脏切片培养进行验证。新生物测定系统的高通量适应性将允许并行分析肝功能，药物代谢和毒性分析。我们相信这种先进的体外肝毒性模型将对临床前研究和药物发现领域产生积极影响。商业重要性和意义：目前尚不存在可接受的商业肝脏生物测定模型，该模型使用已定义的人类生物基质系统。VBI将开发适应HTS应用的新的3D分析格式。作为研究和诊断工具，预包装培养试剂盒和涂布板的销售将具有重要的全球市场。

DESCRIPTION (provided by applicant): Abstract: The overall goal of this SBIR proposal is to develop and commercialize a new human liver bioassay system for real time, long-term toxicity analysis of chemical agents or drugs in vitro. At present, liver studies are mostly performed using hepatocytes cultured onto synthetic or animal-derived matrices. Unfortunately these models fail to replicate true cell- matrix interactions found in vivo. Moreover, due to phenotypic instability of isolated liver cells there is an urgent need for a long-term culture/assay model. Interestingly, spheroid formation in 3D matrix cultures is shown to improve cell functions. We have recently created a novel human biomatrix culture system- HuBiogel"which supports long-term growth, survival and organization of many cell types. Employing this physiologically-relevant model, we propose to develop new 3D or mini-liver assay platform that exhibit functional benefits and allow precise toxicity prediction. The Phase I specific goals are: 1. Develop and optimize 3D bioassay systems using primary human hepatocytes and defined HuBiogel co- culture configurations; and 2. Evaluate functional and metabolic endpoints with known hepatotoxic agents employing standard microscopy, biochemical and molecular protocols. HuBiogel studies and results will be compared with Collagen I or Matrigel assay systems. In Phase II of this project, hepatotoxicity studies will be expanded to include genomics and proteomics analysis for validation using human liver slice cultures. High throughput adaptability of new bioassay system would allow parallel analysis of liver function, drug metabolism and toxicity profiling. We are confident this advanced in vitro liver toxicity model will positively impact preclinical research and drug discovery arenas. Commercial Importance & Significance: No acceptable commercial liver bioassay model currently exists which utilizes a defined human biomatrix system. VBI will develop new 3D assay formats adaptable to HTS application. Sale of pre-packed culture kits and coated plates would have significant world-wide market, both as a research and diagnostic tool. PUBLIC HEALTH RELEVANCE: Current cell-based assay models for studying drug metabolism & toxicity are of limited utility because liver cells die rapidly in their non-physiologic, 2-dimensional (2D) culture formats. Our company has recently created a novel human biomatrix system- HuBiogel"which supports long term 3-dimensional (3D) growth, survival and organization of a variety of cell types. We propose to employ unique HuBiogel and a NASA-developed rotary culture technology to develop a new 3D human liver bioassay that greatly extends liver cell survival & function beyond current culture methods, providing a much needed tool for real-time analysis of drug metabolism & toxicity in vitro. Cells isolated from donor human livers are the gold standard for pre-clinical evaluation of drug metabolism profiles and potential hepatotoxicity. Unfortunately, these cells lose function and die within hours in traditional cell culturing techniques. The proposed development of human matrix-based 3D bioassay system supporting long-term survival & function of liver cells will have a world-wide market, both as a research and diagnostic tool.