Functionalizing Non-Useable Cryopreserved Human Hepatocytes into Useable Hepatic

将不可使用的冷冻保存的人肝细胞功能化为可用的肝脏

基本信息

批准号：
8200956
负责人：
Rex E. Jeffries
金额：
$ 29.73万
依托单位：
SCIKON INNOVATION, INC.
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8200956
关键词：
Acute Amino Acids Applications Grants Biological Assay Carbon Cataloging Catalogs Cell Survival Cell-Matrix Junction Cells Cessation of life Chronic Chronic Toxicity Tests Citric Acid Cycle Collagen Type I Computer software Cryopreservation Development Dose Drug Formulations Ecosystem Energy-Generating Resources Enzymes Extracellular Matrix Glucose Glutamine Goals Health Hepatic Hepatocyte Human Image In Vitro Industry Label Lipids Lipolysis Liver Metabolic Methods Modeling Nutrient Octanoic Acids Operative Surgical Procedures Pathology Pathway interactions Phase Phenotype Population Procedures Process Production Propionates Protein Biosynthesis Proteins Proteolysis Protocols documentation Pyruvate Quality Control Rattus Recipe Research Contracts Science Services Solutions Source Standardization Sterilization Stress Suspension substance Suspensions System Systems Biology Technology Testing Time Tissue Donors Tissue Procurements Tissues Variant base commercialization cost demographics glucose metabolism improved matrigel membrane synthesis metabolomics miniaturize preconditioning research study response scale up stable isotope success toxicant

项目摘要

DESCRIPTION (provided by applicant): The first isolated human hepatoctyte 2D culture, in 1981, provided baseline protocols to support freshly isolated and immediately cultured populations. With cryopreservation technology, human hepatocytes are commercially-available on demand, however, about 75% of the world stores of cryopreserved human hepatocytes are below industry quality control standards and deemed non-useable. Creating methods to transition available but non-usable cryopreserved human hepatocytes into serviceable cell products is a primary goal. Using stable isotope (13C) resolved metabolomics (SIRM) applied to 2D cultures has revealed a major discovery that is contrary to metabolic paradigms: human hepatocytes do not metabolize the two primary carbon sources in basal media recipes, glucose and glutamine. When compared to rat hepatocytes in the starved state, it was concluded that human hepatocytes are in a stressed state that is probably catalyzed by surgery, death and the liver procurement process. The stressed hepatocytes consume internal proteins and lipid stores to generate glucose. In addition, nonhuman extracellular matrices are standard 2D culture conditions. Functionalizing these cells is based on two hypotheses. The first hypothesis: pyruvate and propionate are energy sources other than glucose and glutamine that are one enzyme step from the Krebs cycle, and will decrease lipolysis and proteolysis permitting amino acid and lipid stores to be diverted to proteins and membrane synthesis for increased cell attachment and viability. The second hypothesis is that natural biomatrices encountered by human hepatocytes will increase attachment once proteolysis is inhibited and protein synthesis of extracellular matrices is optimized. Two products are created from this study, (1) supporting media and extracellular matrices capable of transitioning non-usable human hepatocytes into useable cryopreserved hepatocyte products, and (2) a validated 13C SIRM method for contract research services using suspensions or attached human hepatocytes presently non-useable by conventional assays. The two specific aims will: (1) determine if addition of TCA nutrients (2-13C-pyruvate and 3-13C-propionate) increase human hepatocyte viability and/or attachment then (2) using the optimal media determined in specific aim 1, use the new basal media formulation to test three mixtures of human liver derived extracellular matrix for their efficacy at increasing viability and attachment. PUBLIC HEALTH RELEVANCE: In this grant application entitled "FUNCTIONALIZING NON-USEABLE CRYOPRESERVED HUMAN HEPATOCYTES", our studies catalog readily available, but currently ineffective, human hepatocytes as significant opportunities to transform subpar cellular products into serviceable in vitro human liver toxicodynamic and biokinetic models. This would be achieved by validating inactive and active metabolomic pathways using stable isotope (13C) resolved metabolomics (SIRM) that can demarcate needed cell eco-system supplements in support of culture success, and ultimately be applied in biokinetic models of human acute and chronic systems biology. This proposal will help to provide an abundance of cell material while decreasing developmental costs and time to commercialization.

描述（由申请人提供）：1981年，第一个孤立的人肝2D文化提供了基线方案，以支持新鲜隔离和立即培养的人群。借助冷冻保存技术，人类肝细胞在商业上可以根据需要进行商业上的需求，但是，约有75％的冷冻保存人肝细胞商店低于行业质量控制标准，并被认为不可用。创建可用的过渡方法，但不可用的冷冻保存的人肝细胞为可维修的细胞产品是一个主要目标。使用稳定的同位素（13C）分辨的代谢组学（SIRM）应用于2D培养物，发现了一个主要发现，与代谢范式相反：人肝细胞不会代谢基础培养基食谱中的两个主要碳源，葡萄糖和谷氨酰胺。与饥饿状态下的大鼠肝细胞相比，可以得出结论，人肝细胞处于压力状态，可能会因手术，死亡和肝脏采购过程而催化。应力的肝细胞消耗内部蛋白质和脂质储存量来产生葡萄糖。另外，非人类的细胞外矩阵是标准的2D培养条件。这些细胞功能化是基于两个假设。第一个假设：丙酮酸和丙酸酯是葡萄糖和谷氨酰胺以外的能源，这是距克雷布斯循环的酶步进的一个酶步进，它将减少脂解和蛋白水解允许氨基酸和脂质储存储存，以便将其转移到蛋白质和膜合成中，以增加细胞的附着和依然依恋。第二个假设是，一旦抑制蛋白水解并优化了蛋白质的蛋白质合成，人类肝细胞遇到的天然生物体将增加附着。这项研究创建了两种产品，（1）支持媒体和细胞外矩阵，能够将不可用的人肝细胞转变为可用的冷冻保存肝细胞产品，以及（2）使用悬架或附着的人肝细胞的合同研究服务的13C SIRM方法，目前是常规范围内的不可用的人。这两个具体目的将：（1）确定添加TCA养分（2-13C-丙酮酸和3-13C-丙酸）是否会增加人类肝细胞的生存能力和/或依恋，然后（2）使用特定目标1中确定的最佳培养基1，使用新的基础培养基制剂来测试人类乳细胞外的三种混合物，以提高其外皮上的效率和效率。公共卫生相关性：在此赠款申请中，标题为“功能化不可用的冷冻保存的人肝细胞”，我们的研究易于分类，但目前无效，人类肝细胞是将亚par蜂窝产物转化为可用的可用体外人肝脏毒性和生物动力学模型的重要机会。这将通过使用稳定的同位素（13C）分辨的代谢组学（SIRM）来验证非活性和主动代谢组学途径来实现这一目标，该途径可以划分所需的细胞生态系统补充剂以支持文化成功，并最终应用于人类急性和慢性系统生物学的生物动力学模型中。该提案将有助于提供大量的细胞材料，同时减少发育成本和商业化的时间。