Studying Nanotoxicity Using Bioprinted Human Liver Tissues

摘要 将纳米粒子用于工业过程和生物医学应用，如成像、传感、 药物输送和治疗是纳米技术有望产生影响的领域之一。 然而，纳米材料的毒性是一个重大的健康问题。目前对NP毒性的研究主要是 在动物模型和传统的人肝细胞2D培养上进行器官水平的积累。 但动物模型往往昂贵，吞吐量低，而且在可靠预测方面受到限制。 由于物种差异，纳米颗粒对人体的肝脏毒性。使用人肝细胞的传统2D培养是 仍然不足以可靠地预测NPs的毒性。虽然一些毒理学研究使用的是基于人体的3D 最近开发了一些模型，这些3D人体肝脏模型中的大多数都是通过混合而均匀的 具有单一类型肝细胞的单一基质材料，因此不能代表生理条件。 本研究的目的是建立高通量的三维人体多细胞肝脏模型。 将提供改善的肝细胞功能，并产生更可靠的预测各种肝毒性 NPS。在具体目标1中，将使用快速3D生物打印方法来建立多细胞肝脏模型 包裹人原代肝细胞或HiPSC来源的肝祖细胞和其他非实质细胞 细胞转化为天然的细胞外基质成分，具有明确的肝脏特有结构。细胞存活率， 将描述不同细胞群体的增殖、形态和基因表达。肝病 多细胞肝脏模型的功能也将被评估。在特定目标2中，NP诱导的毒性 使用CRISPR-Cas9和生物打印的3D多细胞肝脏模型的依赖性和机制将是 调查过了。几种常用的纳米粒子，包括20 nm颗粒的Fe3O4、Mn3O4、MnO2、CuO、CuS和Ag 将研究相关涂层的尺寸，包括柠檬酸盐、聚乙二醇和牛血清白蛋白。 建议的工作集成了几个创新性的方面来研究NP在生理条件下的毒性。 相关条件，包括a)具有优越的速度、分辨率和能力的新型3D生物打印系统 打印多种材料和细胞，b)创新的3D肝脏模型，具有多种细胞类型的仿生排列 在所需的几何形状和几种天然细胞外基质材料中概括天然微环境， 以及c)使用CRISPR-Cas9筛选来分析3D生物打印肝组织模型中的NPs的新方法。一个 组建了跨学科团队，其中包括3D打印、生物打印、纳米材料和 纳米毒性，以及肝脏病理学方面的领先专家。

Summary The use of NPs (NPs) for industrial processes and biomedical applications such as imaging, sensing, drug delivery and treatment is one of areas where nanotechnology is expected to have an influential impact. However, the toxicity of nanomaterials is a significant health concern. Currently NP toxicity studies are mainly performed on organ level accumulation in animal models and on traditional 2D culture of human hepatocytes. But animal models are often costly, have a low throughput, and are limited in terms of reliably predicting hepatotoxicity of NPs on human due to species difference. Traditional 2D cultures using human liver cells are still insufficient to reliably predict the toxicity of NPs. While a few toxicology studies using human-based 3D models have been recently developed, the majority of these 3D human liver models are homogeneous by mixing a single matrix material with a single type of hepatic cells, therefore not representing the physiological conditions. The objective of this proposal is to develop high throughput 3D human multicellular liver models which will offer improved hepatocellular functions and generate more reliable prediction of hepatotoxicity of various NPs. In Specific Aim 1, a rapid 3D bioprinting method will be used to develop multicellular liver models by encapsulating human primary hepatocytes or hiPSC-derived hepatic progenitor cells and other non-parenchymal cells into native extracellular matrix components with a defined liver-specific structure. The cell viability, proliferation, morphology, and gene expression of different cell populations will be characterized. The hepatic function of the multicellular liver models will also be evaluated. In Specific Aim 2, the NP-induced toxicity dependencies and mechanisms using CRISPR-Cas9 and the bioprinted 3D multicellular liver models will be investigated. Several commonly used NPs, including Fe3O4, Mn3O4, MnO2, CuO, CuS, and Ag of 20 nm particle size with relevant coatings including citrate, polyethylene glycol, and bovine serum albumin will be studied. The proposed work integrates several innovative aspects for studying NP toxicity under physiologically- relevant conditions, including a) a novel 3D bioprinting system with a superior speed, resolution and ability to print muti-materials and cells, b) innovative 3D liver models with biomimetic arrangement of multiple cell types in desired geometry and several native extracellular matrix materials to recapitulate the native microenvironment, and c) a novel approach using CRISPR-Cas9 screening to analyze NPs in 3D bioprinted liver tissue models. An interdisciplinary team is assembled including a pioneer in 3D printing, bioprinting, nanomaterials and nanotoxicity, and a leading expert in liver pathology.