同心凝胶系统来研究基质微环境的三维细胞迁移的生物物理作用
Summary
机械性能和细胞外基质的组织强烈地影响细胞的三维迁移。 的体外方法来研究在生物物理变量的环境中，在人口和单个细胞水平的时空细胞迁移行为，进行说明。
Abstract
细胞迁移的能力是在各种各样的细胞功能，在整个从胚胎发育寿命和伤口愈合肿瘤和肿瘤转移的关键。尽管强烈的研究努力，细胞迁移的基本生物化学和生物物理原理仍然没有完全了解，特别是在生理相关的三维（3D）的微环境。在这里，我们描述了在体外试验设计允许的三维细胞迁移行为定量检查。该方法利用了细胞的mechanosensing能力和倾向迁移到先前未被占据的细胞外基质（ECM）。我们使用高度侵袭性乳腺癌细胞的侵袭，MDA-MB-231，在胶原凝胶作为模型系统。细胞群的传播和单个细胞的迁移动力学数周的文化可以用活细胞成像进行监测和分析，以提取spatiotemporally分辨数据。此外中，该方法很容易适应于不同的细胞外基质，从而提供了一种简单而功能强大的方法来研究的生物物理因素对细胞迁移的微环境中的作用。
Introduction
细胞迁移起着各种生理反应，如胚胎发育，止血，和免疫反应，以及在病理过程，如血管疾病，炎症，癌症和1的关键作用。解剖底层细胞迁移的生物化学和生物物理因素，因此从根本上重要的是不仅要了解的细胞功能的基本原则，又能促进各种生物医学应用，例如在组织工程，抗转移和抗炎药物开发。由于体内观察是技术上的挑战，有很多的努力一直专注于体外再演细胞迁移。
体外方法来研究细胞迁移已经基本上被设计为检测在二维（2D）表面上，最显着的划伤或伤口愈合测定法2。这些试验提供了简单的实验装置，方便在线 -细胞成像，并提供有益的见解底层细胞迁移的各种生化机制。但是，这些测定法不占外基质（ECM）架构和重塑，这是在理解体内迁移关键方面。最近，人们已越来越多地认识到，3D培养模型，常常在基于胶原的基质3中，提供了更好的类似于体内情况的一个平台。事实上，细胞表现出的迁徙动力学是从那些在2D表面明显，特别是由于不同的维数的环境4。此外，基体的生物物理和机械性能灵敏影响细胞迁移5，包括在肿瘤细胞侵袭6的上下文中。
在这里，我们提出研究三维细胞迁移行为的ECM与生物物理特性，可以轻松地用不同的制备条件的方法。细胞是接种在“内凝胶”和被允许逃逸到并侵入最初无细胞“外凝胶”。该方法依赖于细胞的能力来识别的存在，并倾向成，无细胞迁移区域中的外凝胶，这是密切相关的细胞mechanosensing 7。在这项研究中，我们采用的胶原蛋白网络，通过高度侵袭性乳腺癌细胞，MD-MBA-231入侵的ECM。机械性能和两者的内部和外部的凝胶的微结构可以被调谐8和9，其特征实现生理学相关的条件。重建单元轨道和分析允许在两个群体水平和个体细胞水平的时空迁移行为的详细的定量检查。重要的是，同心凝胶系统的设置模仿面临迁移细胞，尤其是侵入癌细胞，从而提供了重要的见解的体内组织的拓扑对细胞迁移和转移的物理机制。
Protocol
Representative Results
Discussion
In this protocol we describe an in vitro assay to study the 3D migrational behavior of cells in matrix environments that topologically resemble ECMs encountered in vivo. There are three main strengths of this assay as compared to other currently available methods. First, this assay allows one to simultaneously examine the cell migration mechanisms at both population level and individual cell level. This opens up possibilities of studying collective cell migration13, which has to date been lar…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢W. Sun和K·詹森的批判性的讨论，并承认支持纳米生物力学实验室，在新加坡国立大学。 NAK承认支持由居里夫人IIF奖学金。
Materials
| Cell culture incubator | Fisher Scientific Pte Ltd | Model: 371, S/No 318854-6055 | |
| Confocal microscope | Nikon A1R | Inverted confocal laser scanning microscope equipped with incubator chamber | |
| Dulbecco's Modified Eagle's Medium (DMEM) | Life Technologies | 11965-092 | |
| Fetal Bovine Serum (FBS) | Life Technologies | 10082-147 | |
| Fluorescent CellTracker dye CMTMR | Life Technologies | C2927 | |
| Glass-bottom dish | IWAKI Cell Biology | 3931-035 | 35 mm diameter dish with 12 mm diameter glass-bottom well |
| Hemocytometer | iN CYTO | DHC-N01 (Neubauer Improved) | |
| Microprocessor pH meter | Hanna Instruments | pH 211 | |
| Nutragen Collagen | Advanced BioMatrix | #5010-D | Acid-solubilized bovine collagen type I (stock pH ~ 2) |
| Objective lens | Nikon | CFI Super Plan Fluor ELWD ADM 20XC, W.D. 8.2-6.9mm, NA 0.45. | |
| Penicillin-Streptomycin | Life Technologies | 15140-122 | |
| pH meter | Sartorius | S/No 29153352 | Basic pH Meter PB-11 |
| Trypsin-EDTA | Life Technologies | 15400-054 |
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