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A novel scale-down cell culture and imaging design for the mechanistic insight of cell colonisation within porous substrate
Journal of Microscopy ( IF 1.5 ) Pub Date : 2017-03-15 , DOI: 10.1111/jmi.12555
C M Gabbott 1 , Z X Zhou 2 , G X Han 3 , T Sun 1
Affiliation  

At the core of translational challenges in tissue engineering is the mechanistic understanding of the underpinning biological processes and the complex relationships among components at different levels, which is a challenging task due to the limitations of current tissue culture and assessment methodologies. Therefore, we proposed a novel scale‐down strategy to deconstruct complex biomatrices into elementary building blocks, which were resembled by thin modular substrate and then evaluated separately in miniaturised bioreactors using various conventional microscopes. In order to investigate cell colonisation within porous substrate in this proof‐of‐concept study, TEM specimen supporters (10–30 μm thick) with fine controlled open pores (100∼600 μm) were selected as the modular porous substrate and suspended in 3D printed bioreactor systems. Noninvasive imaging of human dermal fibroblasts cultured on these free‐standing substrate using optical microscopes illustrated the complicated dynamic processes used by both individual and coordinated cells to bridge and segment porous structures. Further in situ analysis via SEM and TEM provided high‐quality micrographs of cell–cell and cell–scaffold interactions at microscale, depicted cytoskeletal structures in stretched and relaxed areas at nanoscale. Thus this novel scaled‐down design was able to improve our mechanistic understanding of tissue formation not only at single‐ and multiple‐cell levels, but also at micro‐ and nanoscales, which could be difficult to obtain using other methods.

中文翻译:

一种新型的按比例缩小的细胞培养和成像设计,用于多孔基质内细胞定植的机械洞察力

组织工程中转化挑战的核心是对基础生物过程和不同层次成分之间复杂关系的机械理解,由于当前组织培养和评估方法的局限性,这是一项具有挑战性的任务。因此,我们提出了一种新的按比例缩小的策略,将复杂的生物基质解构为基本构建块,这些构建块类似于薄的模块化基板,然后使用各种传统显微镜在微型生物反应器中分别进行评估。为了在该概念验证研究中研究多孔基质内的细胞定植,选择具有精细受控开孔(100~600 μm)的 TEM 样品支架(10-30 μm 厚)作为模块化多孔基质并悬浮在 3D 中印刷生物反应器系统。使用光学显微镜对在这些独立基质上培养的人类真皮成纤维细胞进行无创成像,说明了单个细胞和协同细胞用于桥接和分割多孔结构的复杂动态过程。通过 SEM 和 TEM 的进一步原位分析提供了高质量的微尺度细胞-细胞和细胞-支架相互作用的显微照片,描绘了纳米尺度拉伸和松弛区域的细胞骨架结构。因此,这种新颖的缩小设计不仅能够在单细胞和多细胞水平上,而且在微米和纳米尺度上都能够提高我们对组织形成的机制理解,而这可能是使用其他方法难以获得的。
更新日期:2017-03-15
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