基于细胞微球3D打印技术工程化肝组织构建及在药物毒性研究的应用

Detection of drug hepatotoxicity is an important link in the drug development. The current "gold standard" is the planar cultured human hepatocytes or animal model. But since the microenvironment of planar cultured cells is quite different from in vivo tissue, and the mebatolic regulation of animal liver is essentially different from human beings in many aspects, their detection results are often far from human clinical situation, which greatly limits the efficiency of new drug research and development. Due to the complexity of natural liver tissue and the limited means of tissue engineering research, the in vitro construction of 3D liver-like structure with good hepatic functions still facing great challenges...This research focuses on the development of cell microspheres 3D printing technology for construction of in vitro 3D functional human liver tissue and application in drug toxicity testing studies. Cell microsphere is the biomimicking structure of natural liver lobule. The carrier materials of cell mirospheres provide physical protection towards the cells inside from the damage of shear stress during the printing process. Bio-3D printing technology enables the deposition of cell microspheres, other supporting cells and matrix materials onto the pre-designed spatial location. Through the differentiation characteristics of stem cells and inductive medium, functional liver-like tissues with multicellular interactions, bile duct network and vascular pipeline structure, can be formed. The most widely used clinical drugs is chosen with as testing drugs for drug toxicity study on liver-like tissue structure to study the drug screening rules, and formulate promotion plan for drug hepatotoxicity screening of engineered human liver tissue.

药物毒性检测是药物研发的重要环节，目前的“黄金标准”是平面培养的人体肝细胞或动物模型，但平面细胞微环境与体内组织差别巨大，动物的肝脏代谢规律在许多方面都与人体有着本质的差别，极大限制了新药研发效率。仿生的体外三维人体肝组织可以克服上述平面细胞和动物模型的不足，为新药检测提供有效的实验模型。但由于肝组织生理结构复杂性和组织工程技术手段的局限性，体外功能三维肝组织的构建仍面临巨大挑战。.本研究旨在开发细胞微球3D打印技术构建体外三维功能肝组织并用于药物毒性检测。细胞微球用来体外仿生肝小叶微结构，细胞微球载体材料在打印时对球内细胞提供物理保护，保证打印后细胞成活；3D打印技术将细胞微球、其他支持细胞及基质材料打印在预先设计的仿生肝组织空间位置，通过干细胞分化和诱导培养得到具有多细胞相互作用、胆管管路和血管管路的功能化肝组织结构体，并以临床常用药为受试药物，研究所构建肝组织的药物毒性筛选规律。

药物肝毒性检测是药物安全性评价的重要环节。由于现有评价模型——平面培养的肝细胞和动物模型与人体真实情况的巨大差距，导致药物肝毒性检测的失真率高达70%，进而直接导致新药研发的成功率不足1%，极大限制了现代医疗的发展，也造成了严重的社会负担。.本项目旨在开发细胞微球3D打印技术以构建更加仿生、药物检测结果更接近人体真实情况的体外人体肝组织模型。细胞微球用来体外仿生肝小叶微结构，载体材料对球内细胞提供物理保护，保证打印后细胞成活；3D打印技术将细胞微球、其他支持细胞及基质材料打印在预先设计的仿生肝组织空间位置，通过干细胞分化和诱导培养得到具有多细胞相互作用和胆管管路的功能化肝组织结构体，并以临床常用药为受试药物，评价所构建肝组织用于药物毒性检测的可行性。.项目执行期按照计划进行，主要完成了以下五个方面的研究内容，并取得稳步进展。（1）系统优化非接触式高压静电场设备制备细胞微球的方法，基于多种载体材料，制备了形状完整、尺寸均匀，表面光滑的水凝胶微球；（2）实现了诱导多能干细胞（iPS）微球制备，细胞存活率达到94%以上，微球创造的三维培养环境可长期保持细胞的全能性和自我更新；（3）制备了人体肝细胞微球，肝细胞在微球中持续增殖，形成团簇结构并促进了肝脏相关功能的表达；（4）开发细胞微球3D打印技术并构建体外人体类肝组织，研究发现，生长因子的浓度和时间窗口可直接调控形成肝组织的生化特征和表型；（5）除了细胞学和组织学水平的生物学检测之外，本项目还系统评价了体外肝组织的生理功能和药物学检测特征：细胞打印的时间点、分化启动的时间点、分化时间窗口和生长因子浓度等因素都显著影响所得肝组织的功能水平和药物代谢效果，各种条件下3D打印得到的肝组织与二维培养的肝细胞相比都有显著提高，显示3D打印形成的肝组织在药物检测等研究中的重大潜力。.基于以上研究结果，本项目发表和接收带标注的SCI论文6篇，申请和获得授权国家发明专利6项。