Studying Nanotoxicity Using Bioprinted Human Liver Tissues

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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.