具有液体输运功能的三维灌流血管网络的体外构建研究

Development of artificial tissue/organs is one of the major goals of tissue engineering. However one critical obstacle that facing tissue engineering is the development of functional vessel networks that can deliver oxygen and nutrients to the inner part of the engineered tissue as well as remove waste products. Due to the lack of functional vessel networks in vitro, the thickness of engineered tissue was limited to 1-2 mm. In our previous study, we developed 3-dimentional capillary networks by co-culture of endothelial cells (ECs) and fibroblasts, demonstrated that the capillary networks anastomosed with host vasculature after implantation and was perfused by host blood. Furthermore, tissue constructs with in vitro formed capillary networks was more viable post implantation than tissues without capillaries. However, it is undetermined whether the engineered capillaries can be functional in vitro since the EC formed capillaries were not connected to external fluid. The purpose of this proposal is to develop 3-dimentional perfused vessel networks and demonstrate whether the perfused vessel networks can be functional in vitro. We will focus on the following specific aims: formation of perfused microvessels by seeding endothelial cells in microfluid channels and formation of 3-D capillary networks by co-culture of endothelial cells and fibroblasts in hydrogel; Inducing anastomosis of the capillary networks with the perfused microvessels to form perfused vessel networks; Testify the in vitro function of the perfused vessel networks such as liquid transportation and mass exchange. Through this study, we will answer the question of how to induce the anastomosis of endothelial cells in vitro to form interconnected vessel networks. Our results will have wide applications in the fields of tissue engineering, disease models and drug discovery.

组织工程面临的一大难题是血管化，由于没有成熟的方法能够在体外构建功能性血管网络，人造组织的厚度只能达到1～2毫米。申请人在前期研究中已经在体外构建了三维毛细血管网络，证明了人造血管网植入动物体内后能够与动物血管联通，进行血液输送；并且有血管网络的人造组织植入体内后比没有血管网络的植入组织显示出更强的活性。然而在体外，由于这些血管网还没有与外界流体联通，无法验证其能否实现生理功能。本项目将在体外构建三维灌流血管网络并验证其功能：在微流管道内包被内皮细胞形成灌流小血管，在与微流管道相连的微腔体内培养三维毛细血管网络,通过诱导毛细血管网络与灌流小血管融合形成灌流血管网,验证灌流血管网的液体运输和物质交换等生理功能。通过本课题的研究将解决在体外如何诱导内皮细胞融合形成相互联通的血管网络的科学问题。本课题构建的灌流血管网在组织工程、疾病模型建立、药物筛选等领域都具有应用前景。

组织工程面临的一大难题是血管化，由于没有成熟的方法能够在体外构建功能性血管网络，人造组织的厚度只能达到1～2毫米。目前虽然有多种方法能够在体外构建灌流血管网，然而这些血管网缺乏自我更新和根据周围组织的需求进行重构的能力，无法满足组织工程和其它一些应用的需求。项目负责人在前期研究中已经在体外构建了三维毛细血管网络，证明了人造血管网植入动物体内后能够与动物血管联通，进行血液输送；并且有血管网络的人造组织植入体内后比没有血管网络的植入组织显示出更强的活性。然而在体外，由于这些血管网还没有与外界流体联通，无法实现液体运输的功能，因而无法达到增加人造组织尺寸的目的。. 本项目的研究目标是在体外构建具有液体输运功能的灌流血管网络，主要研究内容包括在微流管道内包被内皮细胞形成灌流小血管，在与微流管道相连的微腔体内培养三维毛细血管网络,诱导毛细血管网络与灌流小血管融合形成灌流血管网。目前本项目已经完成了微流控芯片的加工制作，在芯片上形成了灌流小血管和三维毛细血管网络，并通过施加间质流促进毛细血管网与灌流小血管融合，构建了灌流血管网络。初步探索了间质流对毛细血管新生和融合的影响。此外为了进一步将血管网应用于高通量药物筛选，本项目还开发了超疏水微孔阵列芯片，在微孔内能够培养包括血管内皮细胞在内的多种细胞，并且形成三维毛细血管网，通过点样和对准的方法能够向微孔内加入不同的药物。这一成果发表在Lab on a Chip上，为灌流毛细血管网进一步用于抗癌药物筛选等领域打下了良好基础。