基于iPSCs和3D打印集成制造技术构建个性化肝脏芯片研究

The occurrence rate of liver disease in China is very high, with more than 100 million patients in total. Due to the limitations of planar cultured cell models and animal models, there poses a significant demand for the development of physiological functional in vitro models with the patient's own characteristics to assist pathogenesis study and drug development research. Organ-on-a-chip technology provides a powerful tool in simulating the complex human microenvironments, and recapitulating the physiological functions and pathological features from tissue to organ level. The research level of organ-on-a-chip is strongly related to its manufacturing technology and choice of seeding cells. This project is aimed at human liver tissue, which poses the most functions and the most complex microstructures. We proposed the fabrication of liver-on-a-chip with highly relevant physiological and pathological process to human liver through iPSC-based 3D cell integrated printing technology. Key research aspects includes: 1) basic theory of integrated liver-on-a-chip 3D printing technology; 2) key technologies for the development of integrated and personalized liver-on-a-chip 3D printing based on iPSCs characteristics; 3) the influence and regulation of fabrication process on stem cell phenotype and differentiation; 4) influence of engineering factors on the stem cell differentiation, tissue maturation, function reoccurring/maintenance and application of the liver-on-a-chip. With the success of this project, the research level of organ-on-chip technology, especially liver-on-a-chip will be greatly enhanced, thus contributing to the research of new drug discovery and personalized therapy development.

我国是肝病发生大国，各类肝病患者数量过亿。由于常用细胞平面培养模型和动物模型的局限性，目前亟待开发具有生理功能并携带供体自身特征的体外模型辅助发病机制和药物开发等研究。器官芯片为模拟人体复杂的微环境，从组织和器官水平再现人体的生理功能和病理特征提供了强有力手段，其研究水平与芯片制造工艺和种子细胞密切相关。本项目针对人体功能最多、微结构最复杂的肝脏组织，提出基于诱导性多能干细胞（iPSCs）和3D打印技术，集成制造与肝脏功能和药物代谢过程高度相关的个性化器官芯片。重点研究包括：1）3D打印集成制造肝脏芯片技术的基础理论；2）适用于iPSCs特征的3D打印集成制造个性化肝脏芯片关键技术；3）工艺过程对干细胞表型和分化的影响；4）工程化因素对细胞分化、组织成熟、功能形成/维持和肝脏芯片下游应用的影响。本项目如成功，将大大提高器官芯片，特别是肝脏芯片的研究水平，为新药研发和个性化疗法开发做出贡献。

本项目围绕基于iPSCs和3D打印的集成制造个性化肝脏芯片技术的基础理论及关键技术展开，通过开发适用于iPSCs细胞3D打印集成制造肝脏芯片技术，构建多种细胞可控三维排列并具有多套内通道管路系统的高度仿生个性化肝脏芯片，结合药物肝毒性和肝脏发育学研究，建立完备的复杂组织和器官芯片的集成制造实验平台，研究多种细胞三维空间排列、诱导条件、基质微环境等因素对细胞生长和组织发育的影响规律及科学实质。项目执行期主要完成了以下四个方面的研究目标：（1）基于血液单核细胞重编程制备了个性化的iPSCs细胞，建立了稳定的iPSCs向肝细胞和内皮细胞谱系定向分化的流程和类器官制备技术，多种细胞在打印形成的水凝胶结构中协同发育成熟并自组装形成具有肝脏特征的均匀类器官；（2）针对3D打印组织的结构和尺寸要求，开发了新型整体式器官芯片，动态培养的人工组织可长期存活并保持打印构建的几何微结构；（3）通过具有多层级结构的大孔支架批量制备肝胆细胞团簇，再基于挤出式生物3D打印技术，以批量制备的肝胆细胞团簇作为种子细胞，构建体外肝脏模型并予以生物因子诱导培养，指导肝脏模型中胆管的形态发生，成功构建肝胆双系统肝脏模型，该模型高表达肝细胞分泌蛋白、解毒、药物代谢、胆汁酸合成以及胆汁酸转运等肝胆功能性基因和蛋白，并具有卓越的白蛋白分泌和氨代谢功能；（4）肝胆双系统肝脏模型可以准确检测药物肝毒性，且对药物毒性的反应灵敏度可以与金标准媲美，显示出肝胆双系统肝脏模型作为金标准替代模型应用于药物毒性预测和筛选的潜力。基于以上研究结果，本项目形成成果31项：发表和接收带标注的SCI论文5篇（含SCI收录的学术专著一篇），申请和授权专利14项（含PCT国际专利5项），在国际和国内会议上宣读报告6次（含邀请报告3次），参与组织国际会议2次（含中方会议召集人1次），项目执行期间培养博士毕业4名同学。