基于三维仿生心肌微组织芯片的药物促干细胞分化活性筛选方法学研究

Screening of candidate drugs that inducing cardiomyocyte differentiation from embryonic stem cells (ESCs) has a great significance in cell therapy of myocardial infarction. Despite its importance, the standard bench-top technologies are difficult to mimic the 3D physiological environment of real myocardial tissue, which is a cause of late drug failure to be proven. Therefore, the goal of this proposed research is to develop a 3D microfluidic system of biomimetic myocardial microtissue for drug screening based on the model of differentiation of ESCs into cardiomyocytes in vitro. To accomplish the objective of this application, we will pursue the following specific aims: firstly, through μL droplet generation and automated manipulation, we aim to develop a new method for high-throughput and uniform-sized embryonic body (EBs) formation by using microneedle array. Afterwards, we will design a microfluidic micropillar array to allow for parallel single EBs traps for high-throughput imaging during EBs differentiation. The laminar flow will generate tunable gradients for precisely microenvironmental control of EBs. Finally, through the integration of above functions of EBs formation and single EBs trapping, the 3D biomimetic microfluidic system will be developed for screening of candidate drugs that inducing differentiation of ESCs into cardiomyocytes. The system constructs 3D biomimetic myocardial microtissue by EBs - cells coculture and PC porous membrane. The effectiveness of this system will be subsequently tested using a positive drug, and then the efficacy screening of chemical libraries using 2D and 3D microfluidic platforms will be compared. Consequently, a new drug candidate is desired to obtain then further explain its pharmacological mechanisms. The outcomes of this work are expected to offer an innovative 3D microfluidic system of biomimetic myocardial microtissue for drug screening, fundamentally advance microfluidic technology in the application of screening drugs that inducing differentiation of ESCs into cardiomyocytes.

药物促干细胞心肌分化活性筛选对于心肌梗死干细胞疗法的发展具有重要意义。传统筛选方法难以真实模拟心肌组织的三维生理环境，导致药物筛选后期淘汰率高。本项目以干细胞向心肌细胞分化为模型，提出一套基于微流控的三维仿生心肌微组织药物筛选系统。首先，利用微升级液滴生成和自动化操控技术，发展基于微针阵列的高通量单分散拟胚体（EBs）制备新方法；其次，将微柱阵列和层流操控技术结合，构建单个EBs高通量捕获及其微环境控制的微流控芯片；最后，将上述EBs制备和捕获芯片功能集成，利用三维细胞共培养和多孔膜技术，建立三维仿生心肌微组织芯片系统并用于药物促干细胞分化活性筛选。利用阳性药对该系统的有效性进行验证，比较三维和二维平台筛选结果的异同，以期获得具有较强活性的先导化合物并进行多参数药理评价。本项目拟搭建的三维仿生心肌微组织药物筛选系统，将为微流控技术在药物促干细胞分化活性筛选中的应用提供新的思路和技术借鉴。

构建心肌微组织芯片对于高效筛选治疗心肌梗死的药物具有重要意义。本项目成功构建了基于微流控技术的心肌微组织药物筛选系统。具体包括：用于高通量单分散EBs、三维肿瘤球制备的新技术及流体操控系统、用于单细胞高通量捕获及药物微环境精确控制的微流控芯片、多种仿生心肌微组织药物筛选芯片。多种仿生仿生心肌微组织药物筛选微流控芯片具体包括：基于细胞共培养技术的心肌炎症损伤模型、心肌梯度缺氧损伤模型、基于水凝胶材料的三维心肌组织共培养模型。本项目搭建的三维仿生心肌微组织药物筛选系统，将为微流控技术在活性筛选中的应用提供新的思路和技术借鉴，并为心肌梗死的发生、治疗、机理研究提供新的方法学平台，具有重要的学术价值和应用开发前景。依托项目培养硕士研究生4名、本科生1名（获北京市优秀毕业生称号），获优秀会议论文奖1次。项目负责人在项目执行期间获得北京大学医学部优秀青年人才、药学院青年科研人才培育计划支持。