New In Vitro Human Liver Toxicity Bioassay System

描述(由申请人提供)：这项SBIR提案的总体目标是开发和商业化一种适合对代谢功能和长期药物毒性情况进行实时分析的人体3D或迷你肝脏生物测定系统。目前，对肝功能的研究主要是利用HepG2细胞系和原代肝细胞在胶原或聚合物基质上培养的方法。然而，由于代谢功能的迅速丧失，这些2D单层模型仅允许进行部分或短期毒性分析。有趣的是，3D基质/支架培养中球体的形成被证明可以改善宿主细胞的功能。在第一阶段的研究中，我们成功地证明了一种新的人类生物支架-3D HuBiogel(高密度凝胶)支持原代肝细胞长期存活和维持的可行性。此外，旋转生物反应器系统的集成使得能够高效地生产/分析可存活的微型肝脏球体。我们现在建议完成新的3D-肝脏生物分析平台的开发，该平台具有更好的代谢功能，从而能够在体外进行精确的药物毒性测试。 第二阶段的具体目标是：1.通过优化3D HuBiogel支架和旋转生物反应器或灌流培养策略，建立高效的肝细胞培养方案；2.通过评估关键的生化和代谢终点来测试3D肝培养模型的功能；以及3.通过实时分析细胞色素P450的诱导和化学/药物毒性反应，验证微型肝检测系统的实用价值。比较对照研究将包括I型胶原或Matrigel的3D培养支架以及新鲜的人类肝脏切片培养。3D-肝脏生物分析的高通量适应性也将通过CYP-微阵列进行毒物基因组学检查。因此，新的商品化的3D-肝脏生物测定系统的生物学相关性将被证实，以改进对人体的药物毒性分析/预测。我们相信，这一先进的体外肝毒性模型将对基础、临床前和生物医学研究领域产生积极影响。商业重要性和意义：目前还没有使用人类生物支架培养系统的可接受的商业肝毒性检测方法。VBI将开发一种适应HTS应用的市场就绪的3D肝脏分析平台。作为一种研究和诊断工具，3D HuBiogel培养试剂盒或内部生物检测服务的销售将在全球范围内拥有巨大的市场。 与公共卫生相关：目前研究药物代谢和毒性的基于细胞的分析模型的实用性有限，因为分离的肝细胞在其非生理性的二维(2D)培养形式下迅速死亡。在第一阶段，我们已经证明了一种新型的人体三维(3D或高密度)生物基质支架-HuBiogel“支持原代人类肝细胞的长期培养、存活和功能。在第二阶段，我们将利用3D HuBiogel支架结合旋转或灌流培养技术完成新的微型肝脏生物分析平台的开发。这一强大的3D肝毒性分析方法的高通量应用将极大地推动生化、代谢和分子分析超越2D培养方法。因此，我们的研发工作为实时分析人体化学/药物代谢和肝脏毒性反应提供了亟需的体外工具。一个成功的结果将提供新的市场就绪的生物分析试剂盒和服务，在基础和药物研究领域都有世界范围的需求。

DESCRIPTION (provided by applicant): The overall objective of this SBIR proposal is to develop and commercialize a human 3D or mini- liver bioassay system suitable for real-time analysis of metabolic functions and long-term drug toxicity profiles. At present, liver functions are mostly studied using HepG2 cell line and primary hepatocytes cultured onto collagen or polymer matrices. However, these 2D monolayer models allow only partial or short-term toxicity analysis due to rapid loss of metabolic functions. Interestingly, spheroid formation in 3D matrix/scaffold cultures is shown to improve host cell functions. In Phase I studies, we successfully demonstrated the feasibility of a new human bioscaffold- 3D HuBiogel (high-density gel) for supporting long-term survival and maintenance of primary liver cells. Moreover, integration of rotary bioreactor system allowed efficient production/analysis of viable mini-liver spheroids. We now propose to complete the development of new 3D-liver bioassay platform that exhibits improved metabolic functions, and thus will enable precise drug toxicity testing in vitro. Phase II specific goals are: 1. Establish an efficient hepatic cells cultivation protocol by optimizing 3D HuBiogel scaffolds and rotary bioreactor or perfusion culture strategies; 2. Test functionality of 3D-liver culture model by evaluating key biochemical and metabolism endpoints; and 3. Validate the practical utility of mini-liver assay system via real- time analysis of CYP450 induction and chemical/drug toxicity responses. Comparative control studies will include 3D culture scaffolds of Type-I collagen or Matrigel as well as fresh human liver-slice cultures. High throughput adaptability of 3D-liver bioassay will also be examined for toxicogenomics via CYP-microarrays. Thus, biologic relevance of new marketable 3D-liver bioassay system will be confirmed for improved drug toxicity analysis/prediction in humans. We are confident this advanced in vitro hepatotoxicity model will positively impact basic, preclinical and biomedical research arenas. Commercial Importance & Significance: No acceptable commercial liver toxicity assay currently exists which utilizes a human bio-scaffold culture system. VBI will develop a market-ready 3D-liver assay platform adaptable to HTS applications. Sale of 3D HuBiogel culture kits or in-house bioassay services would have significant world-wide market, as a research & diagnostic tool. PUBLIC HEALTH RELEVANCE: Current cell-based assay models for studying drug metabolism and toxicity are of limited utility because isolated liver cells die rapidly in their no-physiologic, 2-dimensional (2D) culture formats. In Phase I, we have shown that a novel human 3-dimensional (3D or high- density) biomatrix scaffold- HuBiogel" supports long-term cultivation, survival and functions of primary human liver cells. In Phase II, we will complete the development of new mini-liver bioassay platform by employing 3D HuBiogel scaffolds combined with rotary or perfusion culture techniques. High throughput utility of this robust 3D hepatotoxicity assay will greatly advance biochemical, metabolic and molecular analysis beyond 2D culture methods. Thus, our R&D effort provides a much needed in vitro tool for real-time analysis of chemical/drug metabolism and liver toxicity responses in humans. A successful outcome will offer new market-ready bioassay kits and services with world-wide demand in both basic and pharmaceutical research areas.