New In Vitro Human Liver Toxicity Bioassay System

描述（由申请人提供）：摘要：本SBIR提案的总体目标是开发和商业化一种新的人体肝脏生物测定系统，用于体外化学试剂或药物的真实的长期毒性分析。目前，肝脏研究主要使用在合成或动物源性基质上培养的肝细胞进行。不幸的是，这些模型不能复制体内发现的真正的细胞-基质相互作用。此外，由于分离的肝细胞的表型不稳定性，迫切需要长期培养/测定模型。有趣的是，3D基质培养物中的球状体形成显示出改善细胞功能。我们最近创建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。采用这种生理相关的模型，我们建议开发新的3D或微型肝脏测定平台，其表现出功能益处并允许精确的毒性预测。第一阶段的具体目标是：1。使用原代人肝细胞和确定的HuBiogel共培养配置开发和优化3D生物测定系统;以及2.采用标准显微镜检查、生化和分子方案评价已知肝毒性药物的功能和代谢终点。HuBiogel研究和结果将与胶原蛋白I或基质胶测定系统进行比较。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，以使用人肝切片培养物进行验证。新的生物测定系统的高通量适应性将允许并行分析肝功能、药物代谢和毒性特征。我们相信这种先进的体外肝脏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：目前还没有可接受的商业肝脏生物测定模型，其利用了定义的人类生物基质系统。VBI将开发适用于HTS应用的新3D分析格式。作为研究和诊断工具，预包装培养试剂盒和包被板的销售将具有重要的全球市场。 公共卫生相关性：目前用于研究药物代谢和毒性的基于细胞的测定模型具有有限的实用性，因为肝细胞在其非生理的二维（2D）培养形式中快速死亡。我们公司最近创造了一种新的人类生物基质系统- HuBiogel“，它支持各种细胞类型的长期三维（3D）生长，存活和组织。我们建议采用独特的HuBiogel和NASA开发的旋转培养技术来开发一种新的3D人类肝脏生物测定法，该方法大大延长了肝细胞的存活和功能，超越了目前的培养方法，为体外药物代谢和毒性的实时分析提供了急需的工具。从供体人肝脏中分离的细胞是药物代谢特征和潜在肝毒性临床前评价的金标准。不幸的是，这些细胞在传统的细胞培养技术中失去功能并在数小时内死亡。支持肝细胞长期存活和功能的基于人类基质的3D生物测定系统的拟议开发将作为研究和诊断工具拥有全球市场。

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.