Spatially patterned vascular cell co-cultures for combined electrical-optical monitoring of cell-cell communication

生物细胞之间的局部通讯，通过细胞间的直接相互作用和旁分泌信号，是正常组织生理学的核心。这种交流的中断在包括心血管疾病在内的许多疾病过程中起着核心作用。该项目将开发一种全新的动态监测血管细胞通讯的方法，通过结合微制造技术和表面功能化来创建具有空间细胞图案化能力的阻抗分析微设备。这将为心血管疾病提供新的机械洞察力，为改进治疗方法的发展开辟机会。背景心血管疾病是由复杂的血管壁重建引起的。这一过程的两个关键方面是初始内皮细胞(EC)功能障碍和随后的表型变化和平滑肌细胞(SMC)的积累，并固有地破坏局部细胞与细胞之间的通讯。内皮细胞损伤和SMC增殖也是再狭窄的原因，这仍然是支架治疗的阿喀琉斯之踵。推动这些变化的过程仍然知之甚少，极大地阻碍了开发新疗法的努力。因此，需要模拟心血管重构的新系统来梳理相关的复杂信号机制，并建立药物筛选模型。桑迪森博士和麦考密克博士的联合研究经验使他们能够很好地满足这一需求。Sandison博士最近的出版物集中于对SMC表型变化的详细研究，包括开发分离、表征和成像血管群体的方法。McCormick博士开发了一种用于近乎实时监测细胞反应的全自动阻抗谱系统，表明EC和SMC共培养会产生明显的阻抗谱，这些阻抗谱受到细胞间通信机制的强烈影响。再加上桑迪森博士在微系统技术方面的丰富经验，这个研究基地将确保该项目的成功实施。拟议的研究将开发一种新的设备，它结合了透明、薄膜、微制造的电极，能够同时进行电光细胞监测。通过将时间推移成像与阻抗测量相关联，它将能够详细描述细胞群体之间的通信，为不同的共培养环境提供一系列有效的阻抗曲线。将使用硅烷化学和电化学解吸的组合来顺序地创建粘附区，用于在特定的微电极区域上图案化不同类型的细胞。值得注意的是，通过系统地改变微电极的几何形状，细胞通讯的模式将被区分开来。为了更好地模拟活体条件，将通过集成到微流体系统中应用受控剪应力。目的：(1)制造一种新的阻抗分析微设备，开发多种细胞类型的顺序细胞构型的协议。(2)获得单个血管群体(如内皮细胞、平滑肌细胞)向融合方向增殖时的控制谱。(3)对空间图案化的共培养进行阻抗分析(仅限于细胞之间的生化通讯)和不同种群之间的小差距(通过细胞过程进行物理通讯)。(4)开发一种集成的微流控系统，用于连续/脉动流动下的分析和受控药物输送，检测支架涂层中使用的药物的效果。除了产生一个具有良好特征的血管细胞-细胞通讯的体外模型外，所产生的微系统将适合于扩大到阵列形式，用于多路、无标记的药物筛选。

OverviewLocal communication between biological cells, through direct cell-cell interactions and paracrine signalling, is central to normal tissue physiology. Disruption of this communication plays a central role in numerous disease processes, including cardiovascular disease. This project will develop a completely new approach to dynamically monitoring vascular cell communication, by combining microfabrication techniques and surface functionalisation to create an impedance analysis microdevice with spatial cell patterning capability. This will provide novel mechanistic insights into cardiovascular disease that will open up opportunities for the development of improved treatments. BackgroundCardiovascular disease results from a complex remodelling of the blood vessel wall. Two key aspects of this process are initial endothelial cell (EC) dysfunction and subsequent phenotypic change and accumulation of smooth muscle cells (SMCs), with inherent disruption in local cell-cell communication. EC damage and SMC proliferation also underlie restenosis, which remains the Achilles heel of stent treatments. The processes driving these changes remain poorly understood, greatly hampering efforts to develop new treatments. Novel systems that mimic cardiovascular remodelling are therefore required both to tease apart the complex signalling mechanisms involved and for drug screening models.Dr Sandison and Dr McCormick's combined research experience leaves them well placed to address this need. Dr Sandison's recent publications have focussed on detailed investigations into phenotypic changes in SMCs, including developing methods for isolating, characterising and imaging vascular populations. Dr McCormick has developed a fully automated impedance spectroscopy system for monitoring cell responses in near real-time, showing that EC and SMC co-cultures give rise to distinct impedance spectra that are strongly influenced by cell-cell communication mechanisms. Together with Dr Sandison's broad experience in microsystems technology, this research base will ensure successful delivery of the project.Proposed ResearchA novel device incorporating transparent, thin-film, microfabricated electrodes, enabling simultaneously electrical-optical cell monitoring, will be developed. By correlating time-lapse imaging with impedance measurements, it will enable detailed characterisation of communication between cell populations, providing a series of validated impedance profiles for different co-culture environments. A combination of silane chemistry and electrochemical desorption will be employed to sequentially create adherent regions for patterning different cell types upon specific microelectrode regions. Significantly, modes of cellular communication will be teased apart by systematically modifying microelectrode geometries. To better mimic in vivo conditions, controlled shear stress will be applied by integration into a microfluidic system. Objectives:(1) Produce a new impedance analysis microdevice, developing protocols for sequential cellular patterning of multiple cell types.(2) Acquire control spectra for single vascular populations (e.g. ECs, SMCs) as they proliferate towards confluency.(3) Perform impedance analysis of spatially patterned co-cultures with both large (biochemical communication between cells only) and small (enabling physical communication via cellular processes) gaps between different populations.(4) Develop an integrated microfluidic system for analysis under continuous/pulsatile flow and for controlled drug delivery, examining the effect of drugs used in stent coatings.As well as producing a well-characterised in vitro model of vascular cell-cell communication, the resulting microsystem will be suitable for upscaling into an array format for multiplexed, label-free drug screening.