SBIR-STTR Award

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.

Public Health Relevance Statement:


Project narrative:
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.

NIH Spending Category:
Biotechnology; Chronic Liver Disease and Cirrhosis; Digestive Diseases; Liver Disease

Project Terms:
3-Dimensional; abstracting; Animals; Antineoplastic Agents; base; Biochemical; Biological Assay; Biological Markers; Cell Culture Techniques; Cell physiology; Cell Survival; cell type; Cells; Chemical Agents; Coculture Techniques; Collagen; design; Development; Diagnostics Research; drug discovery; drug metabolism; Drug toxicity; Environment; Exhibits; Extracellular Matrix; Future; Genomics; Goals; Gold; Grant; Growth; Growth Factor; Hepatocyte; Hepatotoxicity; Hour; Human; Human Development; Hydrogels; improved; In Vitro; in vivo; Laminin; Legal patent; Liver; liver function; Marketing; matrigel; Metabolic; Metabolism; Methods; Microscopy; Modeling; Molecular; Molecular Analysis; Molecular Profiling; Mus; Nature; NID gene; novel; Peptide Hydrolases; Pharmaceutical Preparations; Phase; Physiological; Polymers; pre-clinical; pre-clinical research; Property; Proteoglycan; Proteomics; Protocols documentation; public health relevance; Real-Time Systems; research clinical testing; Sales; scaffold; Slice; Small Business Innovation Research Grant; Study models; Support System; System; Technology; Time; tool; Toxic effect; Toxicogenomics; Tumor-Derived; tumorigenesis; two-dimensional; Validation

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.