Leaf-Inspired Authentically Complex Microvascular Networks for Deciphering Biological Transport Process

The vascular transport of molecules, cells, and nanoconstructs is a fundamental biophysical process impacting tissue regeneration, delivery of nutrients and therapeutic agents, and the response of the immune system to external pathogens. This process is often studied in single-channel microfluidic devices lacking the complex tridimensional organization of vascular networks. Here, soft lithography is employed to replicate the vein system of a Hedera elix leaf on a polydimethilsiloxane (PDMS) template. The replica is then sealed and connected to an external pumping system to realize an authentically complex microvascular network. This satisfies energy minimization criteria by Murray’s law and comprises a network of channels ranging in size from capillaries (∼50 μm) to large arterioles and venules (∼400 μm). Micro-PIV (micro–particle image velocimetry) analysis is employed to characterize flow conditions in terms of streamlines, fluid velocity, and flow rates. To demonstrate the ability to reproduce physiologically relevant transport processes, two different applications are demonstrated: vascular deposition of tumor cells and lysis of blood clots. To this end, conditions are identified to culture cells within the microvasculature and realize a confluent endothelial monolayer. Then, the vascular deposition of circulating breast (MDA-MB 231) cancer cells is documented throughout the network under physiologically relevant flow conditions. Firm cell adhesion mostly occurs in channels with low mean blood velocity. As a second application, blood clots are formed within the chip by mixing whole blood with a thrombin solution. After demonstrating the blood clot stability, tissue plasminogen activator (tPA) and tPA-carrying nanoconstructs (tPA-DPNs) are employed as thrombolytics. In agreement with previous data, clot dissolution is equally induced by tPA and tPA-DPNs. The proposed leaf-inspired chip can be efficiently used to study a variety of vascular transport processes in complex microvascular networks, where geometry and flow conditions can be modulated and monitored throughout the experimental campaign.

分子、细胞与纳米构建体的血管运输是一类核心生物物理过程，其对组织再生、营养物质与治疗剂递送，以及免疫系统对外源病原体的应答均具有关键影响。当前该过程的研究多依托单通道微流控装置，此类装置缺乏血管网络的复杂三维结构。本研究采用软光刻技术，在聚二甲基硅氧烷（PDMS）模板上复刻常春藤（Hedera elix）叶片的脉管系统。将复刻结构封接并外接泵送系统，即可构建具有真实复杂结构的微血管网络。该网络遵循默里定律（Murray’s law）满足能量最小化准则，其通道尺寸覆盖毛细血管（约50 μm）至较大动静脉（约400 μm）的范围。本研究采用显微粒子图像测速法（Micro-PIV）对流动状态进行表征，分析参数包括流线、流体速度与流速。为验证该装置可复现生理相关的运输过程，我们展示了两类应用场景：肿瘤细胞的血管沉积与血凝块溶解。为此，我们首先确定了在微血管网络内培养细胞并形成融合内皮单层的条件；随后在生理相关流动条件下，观测到循环乳腺癌（MDA-MB 231）细胞在整个网络中的血管沉积过程，其中牢固的细胞黏附多发生于平均血流速度较低的通道内。作为第二类应用，我们通过将全血与凝血酶溶液混合，在芯片内形成血凝块；在验证血凝块稳定性后，分别采用组织型纤溶酶原激活剂（tPA）与负载tPA的纳米构建体（tPA-DPNs）作为溶栓剂进行实验。实验结果与既往研究一致：tPA与tPA-DPNs均可有效诱导血凝块溶解。本研究提出的仿叶芯片可高效用于复杂微血管网络中的各类血管运输过程研究，实验全程可对网络几何结构与流动状态进行调控与监测。