Isolation of Highly Migratory and Invasive Cells in Three-Dimensional Gels.

We developed a modified invasion assay in three-dimensional (3D) gels that permits isolation of invading cells as living cells, termed an invading cell-trapping (iCT) assay. A small cell strainer consisting of nylon mesh with pores of 40-µm square is used in this assay. A layer of gel composed of extracellular-matrix components is formed on each side of the nylon mesh, which permits cell migration or invasion from one gel layer to the other. At the end of the assay, the two gel layers are removed from the apparatus and easily separated from each other. Invading cells from the primary gel are trapped in the secondary gel, which maintains the morphology and other properties of the invasive cells in a 3D matrix. The cells that have invaded are observed and counted with a standard light microscope without cell staining. There is no need for a specialized microscope, imaging analysis software, or advanced cell-biological technical knowledge in this assay. This assay can also reduce measurement of nonspecific cell invasion by monitoring the upward invasion of cells. The viability of both invading and non-invading cells trapped in the gels can be assessed by typical colorimetric assays, if desired. This assessment characterizes the total number of cells (invading and non-invading cells) and the ratio of invading cells to total cells. By repeating the iCT assay, further enrichment of invasive and noninvasive cells can be attained. Thus, this assay improves comparative analyses between invasive and noninvasive cells. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Measuring upward cell invasion into collagen gel Basic Protocol 2: Measuring cell invasion from Matrigel into collagen gel Basic Protocol 3: Isolation and enrichment of highly invasive cells.