Functionalizing Non-Useable Cryopreserved Human Hepatocytes into Useable Hepatic

描述(由申请人提供)：1981年，第一个分离的人肝细胞2D培养提供了支持新分离的和立即培养的种群的基线方案。有了冷冻保存技术，人类肝细胞可以按需商业化获得，然而，世界上约75%的冷冻保存的人肝细胞低于行业质量控制标准，被认为是不可使用的。创造方法将可用的但不可用的冷冻保存的人肝细胞转变为可使用的细胞产品是一个主要目标。将稳定同位素(13C)分解代谢组学(SIRM)应用于2D培养，揭示了一个与代谢范式相反的重大发现：人肝细胞不代谢基础培养基配方中的两个主要碳源：葡萄糖和谷氨酰胺。与饥饿状态的大鼠肝细胞相比，人类肝细胞处于一种应激状态，这可能是由手术、死亡和肝脏获取过程所催化的。受压力的肝细胞消耗内部蛋白质和脂肪储存来产生葡萄糖。此外，非人细胞外基质是标准的2D培养条件。这些细胞的功能化基于两个假设。第一个假设：丙酮酸和丙酸是葡萄糖和谷氨酰胺以外的能量来源，葡萄糖和谷氨酰胺是克雷布斯循环的一个酶步骤，丙酮酸和丙酸是能量来源，它们将减少脂肪分解和蛋白质分解，允许氨基酸和脂肪储存转移到蛋白质和膜合成，以增加细胞的附着和活力。第二个假设是，一旦蛋白质分解被抑制，细胞外基质的蛋白质合成被优化，人类肝细胞遇到的自然生物材料将增加附着。这项研究创造了两种产品，(1)支持介质和细胞外基质，能够将不可用的人体肝细胞转变为可使用的冷冻保存肝细胞产品，(2)用于合同研究服务的有效13C SIRM方法，使用悬浮液或附着的人肝细胞，目前通过常规分析无法使用。这两个特定目标将：(1)确定添加TCA营养素(2-13C-丙酮酸和3-13C-丙酸)是否可以增加人肝细胞的存活率和/或贴壁；然后(2)使用特定目标1中确定的最佳培养基，使用新的基本培养基配方来测试三种人肝脏来源的细胞外基质混合物在提高存活率和贴壁方面的有效性。 公共卫生相关性：在这项题为“将不可用的冷冻保存的人肝细胞功能化”的拨款申请中，我们的研究将现成但目前无效的人肝细胞列为将低于标准的细胞产品转化为可在体外使用的人体肝脏毒理动力学和生物动力学模型的重要机会。这将通过使用稳定同位素(13C)分辨代谢组学(SIRM)验证不活跃和活跃的代谢组途径来实现，SIRM可以区分支持培养成功所需的细胞生态系统补充剂，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。这一提议将有助于提供丰富的细胞材料，同时降低开发成本和商业化时间。

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.