Generation of Three-Dimensional, Bioprinted, Functional Human Liver Tissue

三维、生物打印、功能性人类肝脏组织的生成

• 批准号: 8312779
• 负责人: Sharon Collins Presnell
• 金额: $ 29.01万
• 依托单位: ORGANOVO, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8312779
• 关键词: 3-Dimensional; Activities of Daily Living; Address; Admixture; Adult; Affect; Albumins; Architecture; Area; Biocompatible Materials; Biological Assay; Biology; Blood Coagulation Factor VII; Blood Vessel Tissue; Blood Vessels; Cardiac; Cardiac Myocytes; Cell Line; Cell Survival; Cell physiology; Cells; Cellular biology; Cytochrome P450; Deposition; Development; Dimensions; Drug Approval Processes; Drug Costs; Drug Industry; Drug toxicity; Endothelial Cells; Engineering; Enzymes; Extracellular Matrix; Failure; Fibroblasts; Fostering; Foundations; Generations; Goals; Hepatic; Hepatic Stellate Cell; Hepatocyte; Human; In Situ; In Vitro; Ink; Lead; Legal patent; Life; Liver; Liver diseases; Maintenance; Marketing; Measurable; Mesenchymal Stem Cells; Metabolic; Metabolism; Methods; Modeling; Molecular Conformation; Neurons; Pharmaceutical Preparations; Phase; Physiology; Play; Polymers; Positioning Attribute; Printing; Process; Production; Protocols documentation; Regimen; Research; Risk; Role; Screening procedure; Shapes; Smooth Muscle; Smooth Muscle Myocytes; Solutions; Source; Speed; Staging; Stem cells; Structure; System; Technology; Therapeutic; Time; Tissue Engineering; Tissue Microarray; Tissue Model; Tissues; Toxic effect; Toxicology; base; cell type; cost; density; drug development; drug discovery; drug metabolism; human embryonic stem cell; human tissue; improved; in vivo; induced pluripotent stem cell; innovation; instrument; liver function; liver-specific protein; metabolic abnormality assessment; novel; post-market; pressure; programs; prototype; safety testing; scaffold; stem; success; theories; tool

DESCRIPTION (provided by applicant): Ex vivo cultured human hepatocytes serve as highly useful models of human liver in studies of metabolism and toxicology. One of the first metabolic safety tests for a lead compound in the pharmaceutical industry is the examination of which liver enzymes are affected by its presence, an assay system that relies solely upon the availability of fresh primary (1o) hepatocytes. While these assays play a critical role in the drug development and approval process, there are two major obstacles: cell supply and maintenance of cell function ex vivo. This shortage has motivated the exploration of stem-cell derived hepatocytes generated from human embryonic stem (hES) cells or induced pluripotent stem (iPS) cells. In theory, these cell lines could provide a consistent and renewable source of hepatocytes. However, reproducible protocols to differentiate these cells into hepatocytes that acquire/ maintain mature hepatic functions are lacking. Although hES cells can be subjected to liver-specific differentiation regimens and acquire measurable expression of cytochrome P450 enzymes, levels of enzymatic activity are significantly lower than those in freshly isolated adult human hepatocytes. Thus, new strategies are needed that either unlock the replicative potential of 1o hepatocytes in vitro or render stem cell-derived hepatocytes functionally equivalent to 1o human hepatocytes. Recently, increased success has been achieved when culturing 1o cells in 3-dimensions (3D) as they typically retain more liver-associated functions. Organovo's 3D proprietary bioprinting platform is perfectly positioned to take full advantage of these findings b leveraging an automated precision instrument to build small-scale liver tissues layer-by-layer in vitro using micro-scale building blocks ('bio-ink') comprised of liver cells. Application of the technology to the in vitro engineering of cardiac tissue and blood vessels has shown that 3D bioprinted tissues generated by this platform mature in vitro to mimic the architecture of the native tissue in vivo, including the in situ deposition of native tissue-like matrix and the formaton of microvasculature. In the aims proposed herein, our goal is to extend the platform to generate in vitro human liver surrogates, first using 1o hepatocytes and then liver origin iPS-derived hepatocytes. Initially, we will develop the methods to generate and print liver bio-ink, to achieve the Milestone of bioprinting a 3D human liver tissue that remains viable and functional for at least 3-5 days in an in vitro culture system. Success in Aim 1 alone will lead to the development of a commercializable 1o hepatocyte-based tissue model, although multi-well product availability will be limited by hepatocyte supply. To address the shortage of 1o human hepatocytes, the second goal will be to substitute iPS-derived hepatocytes as a renewable cell source, leveraging the 3D conformation to foster more complete differentiation/maturation and achieve Milestone 2. The 3D human liver surrogates generated by this proposal will not only provide a valuable, commercializable tool for drug discovery and development, but can be extended to develop specific 3D human models of liver disease and implantable constructs for therapeutic augmentation of liver function in vivo. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop small-scale living 3-dimensional (3D) liver tissues, using a renewable source of functional liver cells, in a format compatible with current methods. Organovo's patent-pending bioprinting platform enables fabrication of 3D human tissues using cells as building blocks, without the use of synthetic or native tissue-derived biomaterials or scaffolding, yielding a tissue in vitro that recapitulates native tissue architecture and biology. The human liver tissues generated by this project have the potential to substantially improve the drug development process by providing a superior model of liver biology and liver toxicology. This effort could greatly reduce the cost of drug development by reducing the number of late stage drug failures thus the overall cost of drugs as well.

描述（由申请人提供）：在代谢和毒理学研究中，体内培养的人肝细胞是人类肝脏的非常有用的模型。制药行业中铅化合物的首批代谢安全测试之一是对肝酶受其存在影响的检查，该测定系统仅依赖于新鲜的原发性（1O）肝细胞的可用性。尽管这些测定在药物开发和批准过程中起着至关重要的作用，但存在两个主要障碍：细胞供应和维护细胞功能的维护。这种短缺促使探索由人类胚胎（HES）细胞或诱导多能茎（IPS）细胞产生的干细胞衍生的肝细胞。从理论上讲，这些细胞系可以提供肝细胞的一致且可再生的来源。但是，将这些细胞区分为获取/维持成熟肝功能的肝细胞的可再现方案。尽管HES细胞可以接受肝特异性分化方案并获得可测量的细胞色素P450酶，但酶活性的水平显着低于新鲜分离的成年人类肝细胞中的酶活性。因此，需要新的策略，即可以在体外或渲染干细胞衍生的肝细胞功能等同于1O人类肝细胞的体外或渲染干细胞衍生的肝细胞中解锁1O肝细胞的复制潜力。 最近，在三维（3D）中培养1O细胞时，成功的成功增加，因为它们通常保留更多与肝相关的功能。 Organovo的3D专有生物打印平台非常适合这些发现B充分利用B.利用自​​动精密仪器，以使用由肝细胞组成的微型构建块（“ Beio-Ink”）在体外构建小规模的肝组织。该技术在心脏组织和血管的体外工程中的应用表明，该平台在体外成熟生成的3D生物打印组织，以模仿体内天然组织的结构，包括天然组织样基质的原位沉积和微举性的形式。在本文提出的目的中，我们的目标是扩展平台以生成体外人肝替代物，首先使用1O肝细胞，然后使用肝脏来源IPS来源的肝细胞。最初，我们将开发生成和打印肝脏生物墨水的方法，以实现 在体外培养系统中，生物打印的3D人肝组织的里程碑至少可行3-5天。仅在AIM 1中的成功将导致开发可商业化的1O基于肝细胞的组织模型，尽管多孔产品的可用性将受肝细胞供应的限制。为了解决1O人肝细胞的短缺，第二个目标是将IPS来源的肝细胞代替为可再生的细胞来源，利用3D构象来促进更完整的差异/成熟/成熟并实现里程碑2。肝病的模型和可植入的构建体，用于体内肝功能的治疗性增强。 公共卫生相关性：该项目的目的是使用与当前方法兼容的功能性肝细胞的可再生能源来开发小规模的生存3维（3D）肝组织。 Organovo的申请专利生物打印平台可以使用细胞作为构建块制造3D人体组织，而无需使用合成或天然组织衍生的生物材料或脚手架，从而在体外产生了组织的组织，从而缩减了天然组织结构和生物学。该项目产生的人肝组织具有通过提供肝生物学和肝毒理学的卓越模型来大大改善药物发育过程的潜力。这种努力可以通过减少晚期药物衰竭的数量，从而大大降低药物开发的成本，从而总体上的药物成本。