Studying Nanotoxicity Using Bioprinted Human Liver Tissues

总结 纳米颗粒（NP）在工业过程和生物医学应用中的使用，例如成像、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NP毒性研究主要有 对动物模型中的器官水平积累和人类肝细胞的传统二维培养进行了研究。 但动物模型通常成本高昂、通量低，并且在可靠预测方面受到限制 由于物种差异，NPs 对人类的肝毒性。使用人类肝细胞的传统二维培养是 仍不足以可靠地预测纳米颗粒的毒性。虽然一些毒理学研究使用基于人体的 3D 最近开发了模型，这些 3D 人体肝脏模型中的大多数都是通过混合均匀的 具有单一类型肝细胞的单一基质材料，因此不代表生理条件。 该提案的目标是开发高通量 3D 人类多细胞肝脏模型 将改善肝细胞功能，并对各种肝毒性进行更可靠的预测 NP。在具体目标 1 中，将使用快速 3D 生物打印方法来开发多细胞肝脏模型 封装人原代肝细胞或 hiPSC 衍生的肝祖细胞和其他非实质细胞 细胞转化为具有明确的肝脏特异性结构的天然细胞外基质成分。细胞活力， 将表征不同细胞群的增殖、形态和基因表达。肝脏 还将评估多细胞肝脏模型的功能。在具体目标 2 中，NP 引起的毒性 使用 CRISPR-Cas9 和生物打印 3D 多细胞肝脏模型的依赖性和机制将 调查了。几种常用的纳米颗粒，包括20 nm颗粒的Fe3O4、Mn3O4、MnO2、CuO、CuS和Ag 将研究柠檬酸盐、聚乙二醇和牛血清白蛋白等相关涂层的尺寸。 拟议的工作整合了研究生理学下纳米粒子毒性的几个创新方面 相关条件，包括a）新型3D生物打印系统，具有卓越的速度、分辨率和能力 打印多种材料和细胞，b) 具有多种细胞类型仿生排列的创新 3D 肝脏模型 以所需的几何形状和几种天然细胞外基质材料来概括天然微环境， c) 一种使用 CRISPR-Cas9 筛选来分析 3D 生物打印肝组织模型中的 NP 的新方法。安 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，肝脏病理学领域的领先专家。

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.