New In Vitro Human Liver Toxicity Bioassay System

描述（由申请人提供）：本SBIR提案的总体目标是开发和商业化适用于实时分析代谢功能和长期药物毒性特征的人体3D或微型肝脏生物测定系统。目前，肝功能的研究主要是使用HepG 2细胞系和原代肝细胞培养在胶原或聚合物基质上。然而，由于代谢功能的快速丧失，这些2D单层模型仅允许部分或短期毒性分析。有趣的是，3D基质/支架培养物中的球状体形成显示出改善宿主细胞功能。在I期研究中，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）用于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允许有效地生产/分析活的微型肝球状体。我们现在建议完成新的3D肝脏生物测定平台的开发，该平台具有改善的代谢功能，从而能够进行精确的体外药物毒性测试。 第二阶段的具体目标是：1。通过优化三维HuBiogel支架材料和旋转生物反应器或灌注培养策略，建立高效的肝细胞培养方案; 2.通过评估关键生化和代谢终点来测试3D肝脏培养模型的功能;以及3.通过对CYP 450诱导和化学/药物毒性反应的真实的实时分析，证实微型肝脏测定系统的实用性。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切片培养物。还将通过CYP微阵列检查3D肝脏生物测定的高通量适应性以用于毒理基因组学。因此，将确认新的市售3D肝脏生物测定系统的生物相关性，以改善人体药物毒性分析/预测。我们相信，这种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：目前还没有可接受的商业肝毒性试验，它利用了人类生物支架培养系统。VBI将开发一个市场就绪的3D肝脏检测平台，适用于HTS应用。销售3D HuBiogel培养试剂盒或内部生物测定服务将具有重要的全球市场，作为研究和诊断工具。 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）培养形式中快速死亡。在第一阶段，我们已经证明了一种新型的人类三维（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，我们将通过采用3D HuBiogel支架结合旋转或灌注培养技术完成新的微型肝脏生物测定平台的开发。这种强大的3D肝毒性测定的高通量实用性将大大推动生化，代谢和分子分析超越2D培养方法。因此，我们的研发工作提供了一个非常需要的体外工具，用于实时分析人类的化学/药物代谢和肝脏毒性反应。一个成功的结果将提供新的市场准备的生物测定试剂盒和服务，在基础和制药研究领域的全球需求。

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.