A novel cell exclusion zone assay with a barrier made from room temperature vulcanizing silicone rubber

Objective
To examine the usefulness of room temperature vulcanizing (RTV) silicone rubber as a barrier material for cell exclusion zone assays.
Methods
We created barriers using three types of RTV silicone rubber with differing viscosities. We then assessed the adherence of these barriers to culture dishes and their ease of removal from the dishes. We tested the effect of the newly created barriers on the extracellular matrix (ECM) protein fibronectin by attaching and then removing them from fibronectin-coated culture dishes. We also conducted cell exclusion zone assays with MIO-M1 cells using this new barrier in order to measure cell migration. We used real time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining to measure the effect of fibronectin on MIO-M1 cell migration and the effect of migration (with fibronectin coating) on basic fibroblast growth factor (bFGF) expression in MIO-M1 cells.
Results
Of the three types of RTV silicon rubber tested, KE-3495-T was the best in terms of adherence to the dish and ease of removal from the dish. When barrier attachment and removal tests were performed, this rubber type did not have an effect on the fibronectin that coated the dish. In the cell exclusion assay, removal of the barrier revealed that a cell-free area with a distinct margin had been created, which allowed us to conduct a quantitative assessment of migration. Fibronectin significantly promoted the migration of MIO-M1 cells (P = 0.02). In addition, both real time RT-PCR and immunohistological staining indicated that bFGF expression in migrating MIO-M1 cells was significantly higher than that in non-migrating cells (P = 0.03).
Conclusions
RTV silicone rubber can be used to create an effective barrier in cell exclusion zone assays and allows simple and low-cost multi-parametric analysis of cell migration.

研究目的：评估室温硫化（room temperature vulcanizing, RTV）硅橡胶作为细胞排除区实验屏障材料的实用性。

研究方法：我们采用三种不同黏度的RTV硅橡胶制备细胞屏障。随后，我们评估了各屏障对培养皿的附着性能及从培养皿上移除的便捷程度。我们通过在包被细胞外基质（extracellular matrix, ECM）纤连蛋白的培养皿上安装并移除屏障，测试了新型屏障对纤连蛋白的影响。此外，我们使用MIO-M1细胞开展细胞排除区实验，借助该新型屏障检测细胞迁移能力。我们采用实时逆转录聚合酶链反应（real time reverse transcription polymerase chain reaction, RT-PCR）与免疫组织化学染色，分别检测纤连蛋白对MIO-M1细胞迁移的影响，以及经纤连蛋白包被的迁移过程对MIO-M1细胞碱性成纤维细胞生长因子（basic fibroblast growth factor, bFGF）表达的影响。

研究结果：在测试的三种RTV硅橡胶中，KE-3495-T在培养皿附着性能与移除便捷性方面表现最优。屏障安装与移除实验显示，该型号硅橡胶对培养皿表面包被的纤连蛋白无不良影响。在细胞排除区实验中，移除屏障后可观察到边界清晰的无细胞区域，为定量检测细胞迁移提供了可行条件。纤连蛋白可显著促进MIO-M1细胞迁移（P=0.02）。此外，实时RT-PCR与免疫组织化学染色结果均表明，迁移中的MIO-M1细胞的bFGF表达水平显著高于未迁移细胞（P=0.03）。

研究结论：RTV硅橡胶可用于细胞排除区实验中构建高效细胞屏障，且能够实现简便、低成本的细胞迁移多参数分析。