Biofabrication of Multicompartment Human Liver Tissues for Chemical Screening

摘要/项目摘要 标题：用于化学筛选的多室人类肝脏组织的生物制造 药物性肝损伤 (DILI) 是临床前和临床药物损耗的主要原因，黑框警告 药品，以及从市场上撤回药品。不幸的是，动物模型并不总是足以 由于药物代谢途径存在显着的物种特异性差异，因此评估人类 DILI；因此，在 人类肝脏的体外模型越来越多地用于评估化合物（药物/化学品） 代谢和毒性。然而，目前的人类肝脏体外模型无法确定其影响 化合物对肝脏三个主要区室（即肝区、血管区和胆区）的影响，以及如何作用 对一个隔室的毒性可能会影响其他隔室。同样，虽然取得了一些进展 开发可植入肝组织替代物作为晚期患者的细胞疗法 肝功能衰竭，此类组织不包含上述具有生理功能的肝区室 互连。我们的研究表明，原代人肝细胞（PHH）和肝内皮细胞 (LEC) 当组织成 3 维 (3D) 时，在体外 4 周内表现出高水平的类体内功能 使用高通量液滴微流体平台生成的细胞外基质 (ECM) 微凝胶 （所谓的微组织）。这种微组织技术非常适合控制肝脏的微环境 细胞，并有可能保护细胞免受 3D 生物打印引起的剪切应力的影响。此外，我们还有 研究表明，胆管细胞在脱细胞肝脏 ECM (dECM) 中表现出萌芽行为，但在 I 型胶原蛋白中则不然 或单独使用基质胶，这种发芽行为可以通过 3D 生物打印来控制。在这个高风险/高回报的环境中 R21 提案中，我们将利用这些平台和研究结果来测试 3D 打印的新假设 含有肝微组织和肝dECM的生物材料支架可用于产生肝样功能 和综合室（血管、肝和胆管）。在目标 1 中，我们将制造并表征 3D- 包含肝微组织和 LEC 内衬血管通道的打印结构，而在目标 2 中，我们将 将胆管细胞纳入生物制造结构并研究控制和检测胆汁的能力 流动。如果成功，我们的努力将产生首个可扩展的 3D 打印人体肝脏组织，其中包含 整合的肝、血管和胆道室，显示出稳定水平的不同肝功能 体外几周。最终，我们的 3D 打印人体肝脏组织可用于研究 肝脏所有三个区室的化合物及其相互作用，以及植入动物体内 模型作为慢性肝病和急性肝衰竭的潜在细胞疗法。

ABSTRACT / PROJECT SUMMARY Title: Biofabrication of Multicompartment Human Liver Tissues for Chemical Screening Drug-induced liver injury (DILI) is a leading cause of preclinical and clinical drug attrition, black box warnings on drugs, and withdrawals of drugs from the marketplace. Unfortunately, animal models do not always suffice to evaluate human DILI due to significant species-specific differences in drug metabolism pathways; therefore, in vitro models of the human liver are being increasingly utilized to evaluate compound (drugs/chemicals) metabolism and toxicity. However, current in vitro models of the human liver are unable to determine the effects of compounds on the three major compartments of the liver, namely hepatic, vascular, and biliary, and how toxicity to one compartment may affect the other compartments. Similarly, while there has been some progress in developing implantable liver tissue surrogates as cell-based therapies for patients suffering from end-stage liver failure, such tissues do not contain the above-mentioned liver compartments with physiological interconnections. Our studies have shown that primary human hepatocytes (PHH) and liver endothelial cells (LEC) display high levels of in vivo-like functions for 4+ weeks in vitro when organized into 3-dimensional (3D) extracellular matrix (ECM) microgels that are generated using a high-throughput droplet microfluidics platform (so-called microtissues). This microtissue technology is uniquely suited to control the microenvironment of liver cells and could potentially protect cells from the shear stress induced via 3D bioprinting. Furthermore, we have shown that cholangiocytes display sprouting behavior in decellularized liver ECM (dECM) but not in collagen-I or Matrigel alone and such sprouting behavior can be directed via 3D bioprinting. In this high-risk/high-reward R21 proposal, we will leverage these platforms and findings to test the novel hypothesis that a 3D-printed biomaterial scaffold containing hepatic microtissues and liver dECM can be used to generate liver-like functional and integrated compartments (vascular, hepatic, and biliary). In aim 1, we will fabricate and characterize 3D- printed structures containing hepatic microtissues and LEC-lined vascular channels, while in aim 2, we will incorporate cholangiocytes into the biofabricated structures and investigate the ability to control and detect bile flow. If successful, our efforts will yield a first-of-its-kind scalable 3D-printed human liver tissue containing integrated hepatic, vascular, and biliary compartments that displays stable levels of diverse liver functions for several weeks in vitro. Ultimately, our 3D-printed human liver tissue can be used for investigating the effects of compounds on all three compartments of the liver and their interactions, as well as for implanting into animal models as potential cell-based therapy for chronic liver disease and acute liver failure.