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仿生微流体模型——研究血液动力学条件下内皮细胞和血管平滑肌细胞之间的信号传导

心血管疾病(CVD)是造成死亡最常见的原因之一,因此血管重塑,如动脉硬化中的冠状动脉重塑显得非常有必要。血管细胞以及血流力学信号传导在组织重塑和平衡中起到至关重要的作用。动脉血管壁呈多细胞结构,由内皮细胞(EC)层及其周围的血管平滑肌细胞(VSMC)组成。目前的3D血管壁构建体不能模拟天然组织的机械条件,也不能在相关的血液动力学条件下监测细胞间相互作用。

基于此,荷兰埃因霍芬理工大学的Cecilia M. Sahlgren、Nicole C. A. van Engeland团队建立了动脉内皮细胞和平滑肌细胞的3D微流控芯片模型,其模拟了细胞组成、细胞间相互作用以及动脉壁的机械环境。血液动力学EC-VSMC-芯片上信号传导由两个平行的聚二甲基硅氧烷(PDMS)细胞培养通道组成,由柔性多孔PDMS膜隔开,模仿内部弹性薄层的孔隙率(图 1A-E)。

血流动力学EC-VSMC-芯片上信号传导允许人主动脉内皮细胞(EC)和人主动脉血管平滑肌细胞(VSMC)的共培养,由多孔膜分离,这使得EC-VSMC相互作用和信号传导成为可能,这对血管壁的发育和稳态至关重要。该装置可以实现对细胞实时成像和对血液动力学条件的控制。培养通道两侧均被真空通道包围(图 1F),以通过施加循环抽吸诱导循环应变,导致细胞培养通道中膜的机械拉伸和松弛。另外,通过在EC侧产生介质流来模拟血流。

模拟血流 

Fig. 1 Schematic overview of the vessel wall on a chip device layer, consisting of two vacuum chambers and a cell culture chamber (A). The microfabricated vessel wall device uses compartmentalized PDMS microchannels to form an organized co-culture of ECs and VSMCs whereby physiological arterial strain and shear stress from the blood flow can be recreated (B). Schematic top view of the final microfluidic device with culture channels (red) and vacuum channels (blue) (C and D). Three PDMS layers are aligned and irreversibly bonded to form two sets of three parallel microchannels, separated by a thin PDMS membrane with pores (E). Selective etching of the membrane layers in the vacuum channels produces two large side chambers to which vacuum is applied, causing mechanical stretching of the membrane in the culture channel (F).

为了确定细胞确实通过基质涂覆的多孔膜接触,研究人员将EC和VSMC接种在常规装置和具有完整膜的对照装置中,并用对其进行免疫荧光染色。通过共聚焦显微镜成像显示EC和VSMC可以通过常规装置中的膜孔连接,而在对照装置中细胞不发生相互作用(图 2)。

细胞 

Fig. 2 Cross section of artery-on-a-chip. Immunohistochemistry staining of the device without pores (control, A) and with porous membrane (B). In green the VSMCs and in red the ECs. Number 1 depicts the VSMC side of the device, number 2 the membrane, and number 3 the ECs side. Scalebar represents 50 μm, n = 3–4.

为了研究血液动力学下EC和VSMC的行为,EC和VSMC分别用CellTracker Green和Orange标记,然后种到装置中。两种细胞类型在贴附到膜上后表现出随机排列和类似鹅卵石的形态(图3 A-D)。当细胞粘附到膜上后,装置的细胞培养通道连接到压力驱动的IBIDI系统以在膜的两侧分别保持细胞相应培养基流动。此外,真空通道连接到压力驱动的IBIDI系统以诱导应变,在脉动血流期间模拟循环周向应变。在动态培养后EC层没有看到明显的细胞排列,而VSMC的排列方向与给力方向垂直,且比静态条件下的细胞更趋于细长的纺锤形(图3E-H)。

纺锤形细胞 

Fig. 3 Live staining of vessel wall on a chip device. A–D. Images were taken directly after adhesion of VSMCs (B) and ECs (C) whereby A and D are merged images. E–H. Live cell stainings after 4 days of dynamic culturing, VSMCs (F), ECs (G) and merged images (E and H). ECs are stained in green and VSMCs in red. Scalebar represents 100 μm, n = 3.

 

本研究由荷兰埃因霍芬理工大学的Cecilia M. Sahlgren、Nicole C. A. van Engeland团队完成,于2018年5月发表于Lab on a Chip。论文链接:

http://pubs.rsc.org/-/content/articlelanding/2018/lc/c8lc00286j#!divAbstract(转载仅供参考学习及传递有用信息,版权归原作者所有,如侵犯权益,请联系删除)