New In Vitro Human Liver Toxicity Bioassay System

描述（由申请人提供）：该SBIR提案的总体目标是开发和商业化适合于实时分析代谢功能和长期药物毒性谱的人体3D或微型肝脏生物测定系统。目前，研究肝功能的方法主要是利用HepG2细胞系和原代肝细胞在胶原或聚合物基质上培养。然而，由于代谢功能的快速丧失，这些二维单层模型只能进行部分或短期毒性分析。有趣的是，在3D基质/支架培养中形成球体被证明可以改善宿主细胞功能。在I期研究中，我们成功证明了一种新的人类生物支架- 3D HuBiogel（高密度凝胶）的可行性，用于支持原代肝细胞的长期存活和维持。此外，旋转生物反应器系统的集成允许有效的生产/分析可行的微型肝球体。我们现在建议完成新的3d肝脏生物测定平台的开发，该平台具有改进的代谢功能，从而可以在体外进行精确的药物毒性测试。

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.