Collaborative Research: High-throughput microliver platform for drug toxicity screening

人体肝脏的体外模型在评估药物和工业化学品在人类暴露之前的毒性方面发挥着关键作用。这些模型还有助于开发针对影响肝脏的全球疾病的治疗方法，如乙肝/丙型肝炎病毒感染、脂肪肝、疟疾、2型糖尿病和癌症。尽管在过去的几年里，在利用工程工具制造功能更高、寿命更长的人类肝脏模型方面取得了相当大的进展，但仍有一些关键的差距和限制需要解决，包括提高系统的吞吐能力，改善模型生产的重复性，以及在某些模型中包括癌细胞系。这项研究项目正在开发和优化一个高通量的“微型肝脏”平台，该平台由生物相容凝胶、人类肝细胞和支持细胞功能所需的额外生物分子组成。研究人员正在利用这一平台来测试这一假设，即该平台的微环境将在显著接近实际生理肝功能的水平上模拟肝功能。研究人员还在使用与临床相关的化合物来测试这些“微型肝脏”在药物代谢和毒性筛选中的效用。与这个项目相关的教育努力，利用这个项目的发现和新的装置，让高中教师和学生参与到研究中来。在高中早期引入尖端研究概念有望更好地为学生在大学水平上严格的工程课程做好准备。该研究项目致力于创建第一个高通量、三维(3D)的微肝平台，该平台具有可调节的细胞外基质(ECM)微环境，其中包含原代人肝细胞(PHHs)和复杂的肝基质细胞混合物。生成的数据将从根本上理解不同类型的人类肝细胞在3D环境中的相互作用，以及药物对这种相互作用的影响。这些发现将为组织工程和再生医学中更大规模的3D肝脏结构的生物制造提供设计标准。肝脏中细胞-细胞和细胞-ECM相互作用的微凝胶平台和系统探索也可以广泛适用于正在开发的其他组织类型，以整合到芯片上的器官系统中。该项目有两个目标：1)开发和测试一个高通量制造3D人肝微凝胶的微流控平台，用于研究细胞与细胞和细胞外基质之间的相互作用；2)研究药物对3D人肝微凝胶形态/功能的影响。目标1建立在最近开发的微流控流动聚焦装置的基础上，用于产生含有肝脏激发的ECM、PHHs和肝基质细胞类型的微凝胶。该设备将被用来测试这样的假设，即包含复杂的肝脏激发的ECM和关键的肝脏基质细胞类型的3D微环境将在几个月内增强并稳定PHH功能，其水平比现有系统可能的水平更接近生理结果。微流控系统的使用克服了使用块状胶原凝胶的3D培养方法的相关问题，这些胶原蛋白凝胶太耗费人力、时间和成本，不能用于高通量筛选，而且由于体积大，对营养物质和信号分子的扩散有很大限制。该系统能够在3D ECM微环境中系统地研究PHHs与基质细胞的相互作用，从而为构建更准确地概括人类肝功能和药物反应的人类肝脏模型提供设计原则。

In vitro models of the human liver play a critical role in assessing the toxicity of drugs and industrial chemicals prior to human exposure. Such models are also useful for developing therapeutics against global diseases that affect the liver, such as hepatitis B/C viral infections, fatty liver disease, malaria, type 2 diabetes, and cancer. While considerable progress has been made over the last few years in utilizing engineering tools to fabricate higher functioning and longer-lasting human liver models, some key gaps and limitations still need to be addressed including increasing the throughput capability of the system, improving reproducibility in the production of the model, and including cancer cell lines in certain models. This research project is developing and optimizing a high-throughput "micro-liver" platform comprised of a biologically compatible gel, human liver cells, and additional biological molecules needed to support the functions of the cells. The investigators are using this platform to test the hypothesis that the microenvironment of this platform will imitate liver functions at levels that are significantly closer to actual physiological liver function. The investigators are also using clinically-relevant compounds to test the utility of these "micro-livers" for drug metabolism and toxicity screening. The educational efforts associated with this project using the findings and novel devices of this project to engage high school teachers and students in research experiences. Introducing cutting-edge research concepts earlier in high school is expected to better prepare students for a rigorous engineering curriculum at the college level. This research project is focused on creating the first high-throughput, three-dimensional (3D) microliver platform with a tunable extracellular matrix (ECM) microenvironment containing primary human hepatocytes (PHHs) and a complex mixture of liver stromal cells. The data generated will yield a fundamental understanding of the interactions of different types of human liver cells in a 3D context and the effects of drugs on such interactions. These findings will provide design criteria for the biomanufacturing of larger-scale 3D liver constructs for tissue engineering and regenerative medicine. The microgel platform and systematic exploration of cell-cell and cell-ECM interactions in the liver can also be broadly applicable to other tissue types being developed for integration into organs-on-a-chip systems. The project has two objectives: 1) develop and test a microfluidic platform for the high-throughput fabrication of 3D human liver microgels for investigating cell-cell and cell-ECM interactions and 2) investigate the effects of drugs on the morphology/functions of 3D human liver microgels. Objective 1 builds on a recently developed microfluidic flow-focusing device for generating microgels containing liver-inspired ECM, PHHs, and liver stromal cell types. The device will be used to test the hypothesis that a 3D microenvironment, which contains the complex liver-inspired ECM coupled with key liver stromal cell types, will enhance and stabilize for several months PHH functions at levels that are significantly closer to physiological outcomes than possible with existing systems. Use of a microfluidic system overcomes the problems associated with 3D culture methods using bulk collagen gels that are too labor-, time-, and cost-intensive to use in high throughput screening and, due to large size, have significant diffusion limitations for nutrients and signaling molecules. The system enables systemic investigation of the interactions of PHHs with stromal cells within a 3D ECM microenvironment, thus providing design principles for the construction of a human liver model that more accurately recapitulates human liver functions and drug responses.