Double-layered collagen gel hemisphere for cell invasion assay: Successful visualization and quantification of cell invasion activity
Double-layered collagen gel hemisphere for cell invasion assay: Successful visualization and quantification of cell invasion activity
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DOI：
10.1080/15419060701557859
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发表时间：
2007-01-01
影响因子：
--
通讯作者：
Okita, Yutaka
中科院分区：
文献类型：
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作者：
Takata, Masahiko;Maniwa, Yoshimasa;Okita, Yutaka
尽管已经描述了用于基于胶原凝胶的细胞侵袭测定的各种方法，但是仍然需要更简单和更客观的测定。在这里，我们描述了一种易于制备的双层胶原蛋白凝胶半球（DL-CGH）系统，满足这些要求，我们证明了这种新系统的优势，可视化细胞运动过程中的入侵。DL-CGH由一个中心核心胶原蛋白层和一个外包胶原蛋白层组成。一滴胶原蛋白I溶液（含有待检测细胞）在培养皿底部自然形成一个小半球。在该中心核心层凝胶化之后，将第二液滴放置在第一凝胶上，将其完全包封。将半球浸没在培养基中并培养。从内层向外层渗透的细胞的侵入活性可以通过光学方法进行评估。使用这种体外系统，我们测量了E-钙粘蛋白表达对癌细胞侵袭的抑制作用。DL-CGH还允许可视化入侵的癌细胞和基质之间的相互作用。缺乏直接进入三维胶原基质所需的蛋白酶的癌细胞被认为像变形虫一样滑过由成纤维细胞的细胞周蛋白水解活性产生的基质间隙。
Although various methods for collagen gel-based cell invasion assays have been described, there continues to be a need for a simpler and more objective assay. Here, we describe an easy-to-prepare double-layered collagen gel hemisphere (DL-CGH) system that satisfies these requirements, and we demonstrate the advantages of this new system for visualizing cell movements during invasion. DL-CGH consists of a central core collagen layer surrounded by an outer cover collagen layer. A droplet of collagen I solution ( containing cells to be examined) naturally forms a small hemisphere on the bottom of the culture dish. After this central core layer gels, a second droplet is placed atop the first gel, encapsulating it completely. The hemisphere is submerged in the medium and cultured. The invasive activity of cells that infiltrate from the inner to the outer layer can be evaluated optically. Using this in vitro system, we measured the inhibitory effect of E-cadherin expression on cancer cell invasion. DL-CGH also allowed visualization of interactions between invading cancer cells and the stroma. Cancer cells, which lack the proteases required for direct entrance into the three-dimensional collagen matrix, were seen to slip like amoebas through matrix gaps generated by the pericellular proteolytic activity of fibroblasts.