Abhyankar Vinay V, Wu Meiye, Koh Chung-Yan, Hatch Anson V
Department of Biotechnology and Bioengineering, Sandia National Laboratories, Livermore, California, 94551, United States of America.
PLoS One. 2016 May 26;11(5):e0156341. doi: 10.1371/journal.pone.0156341. eCollection 2016.
Microfluidic barrier tissue models have emerged as advanced in vitro tools to explore interactions with external stimuli such as drug candidates, pathogens, or toxins. However, the procedures required to establish and maintain these systems can be challenging to implement for end users, particularly those without significant in-house engineering expertise. Here we present a module-based approach that provides an easy-to-use workflow to establish, maintain, and analyze microscale tissue constructs. Our approach begins with a removable culture insert that is magnetically coupled, decoupled, and transferred between standalone, prefabricated microfluidic modules for simplified cell seeding, culture, and downstream analysis. The modular approach allows several options for perfusion including standard syringe pumps or integration with a self-contained gravity-fed module for simple cell maintenance. As proof of concept, we establish a culture of primary human microvascular endothelial cells (HMVEC) and report combined surface protein imaging and gene expression after controlled apical stimulation with the bacterial endotoxin lipopolysaccharide (LPS). We also demonstrate the feasibility of incorporating hydrated biomaterial interfaces into the microfluidic architecture by integrating an ultra-thin (< 1 μm), self-assembled hyaluronic acid/peptide amphiphile culture membrane with brain-specific Young's modulus (~ 1kPa). To highlight the importance of including biomimetic interfaces into microscale models we report multi-tiered readouts from primary rat cortical cells cultured on the self-assembled membrane and compare a panel of mRNA targets with primary brain tissue signatures. We anticipate that the modular approach and simplified operational workflows presented here will enable a wide range of research groups to incorporate microfluidic barrier tissue models into their work.
微流控屏障组织模型已成为先进的体外工具,用于探索与外部刺激物(如候选药物、病原体或毒素)的相互作用。然而,对于终端用户,尤其是那些没有大量内部工程专业知识的用户来说,建立和维护这些系统所需的程序实施起来可能具有挑战性。在这里,我们提出了一种基于模块的方法,该方法提供了一个易于使用的工作流程,用于建立、维护和分析微观组织构建体。我们的方法始于一个可拆卸的培养插入物,该插入物通过磁力耦合、解耦,并在独立的预制微流控模块之间转移,以简化细胞接种、培养和下游分析。模块化方法提供了多种灌注选项,包括标准注射泵或与自给式重力供料模块集成,以实现简单的细胞维护。作为概念验证,我们建立了原代人微血管内皮细胞(HMVEC)培养物,并报告了在用细菌内毒素脂多糖(LPS)进行可控顶端刺激后的表面蛋白成像和基因表达情况。我们还通过将超薄(<1μm)、自组装的透明质酸/肽两亲物培养膜与脑特异性杨氏模量(~1kPa)集成,证明了将水合生物材料界面纳入微流控结构的可行性。为了强调在微观模型中纳入仿生界面的重要性,我们报告了在自组装膜上培养的原代大鼠皮质细胞的多层读数,并将一组mRNA靶点与原代脑组织特征进行了比较。我们预计,这里介绍的模块化方法和简化的操作工作流程将使广泛的研究小组能够将微流控屏障组织模型纳入他们的工作中。