A novel wounding device suitable for quantitative biochemical analysis of wound healing and regeneration of cultured epithelium.

摘要

We describe the fabrication and use of an in vitro wounding device that denudes cultured epithelium in patterns designed to leave behind strips or islands of cells sufficiently narrow or small to ensure that all the remaining cells become rapidly activated and then migrate, dedifferentiate, and proliferate in near synchrony. The design ensures that signals specific to regenerating cells do not become diluted by quiescent differentiated cells that are not affected by wound-induced activation. The device consists of a flat circular disk of rubber, engraved to produce alternating ridges and grooves in patterns of concentric circles or parallel lines. The disk is mounted at the end of a pneumatically controlled piston assembly. Application of controlled pressure and circular or linear movement of the disk on cultures produced highly reproducible wounding patterns. The near-synchronous regenerative activity of cell bands or islands allowed the collection of samples large enough for biochemical studies to sensitively detect alterations involving mRNA for several early response genes and protein phosphorylation in major signaling pathways. The method is versatile, easy to use and reproducible, and should facilitate biochemical, proteomic, and genomic studies of wound-induced regeneration of cultured epithelium.