Scratch assay (cell monolayer)

The in vitro scratch assay is a well- developed method for measuring cell migration and can be used as a means of studying wound healing. A ‘scratch’ or ‘wound’ is made on a cell monolayer and images are captured at regular intervals observing the migration of cells across the ‘scratch/wound’. New cell-cell contacts are formed until the gap is closed. Images and computing software can be used to quantify the migration rate of the cells.

Traditional methods have used a pipette tip to scratch the cell monolayer manually. More recent methods use automated systems to not only scratch the cell monolayer, but to capture real-time images and generate quantitative data.

The IncucyteTM, manufactured by Essen Instruments, is a live imaging system and uses the WoundmakerTM and ImageLockTM plate to create wounds at precise locations across the cell monolayers in each individual well of the plate (as small as a 96-well plate may be used). This allows for consistent, quality wounds and assessment of multiple culture conditions simultaneously, without the need to move the cells out of the incubator. The IncucyteTM software uses the images captured (both phase-contrast and ‘woundmask’ – showing the border of the wound) to automatically produce cell confluence and wound width data over time.

Scratch assay (3-D human skin model)

Dermal wound healing involves interactions between dermal fibroblasts and epidermal keratinocytes, as well as cell and extracellular matrix interactions. 3-D human skin models consisting of both dermal and epidermal components, fibroblasts and proliferating keratinocytes, provide a more physiological test system for studying wound-healing as compared to cell monolayers of a single cell type. A dermal biopsy punch may be used to wound the skin model, which is in turn fixed at various time points and H&E stained sections prepared to evaluate the wound and the wound healing process.

ELISA

ELISA kits are readily available to assess levels of various matrix metalloproteinases and cytokines such as interleukins, tumour necrosis factor and interferon types on cell monolayers, when exposed to a chemical agent and compared to controls.

3-D human skin models consisting of both dermal and epidermal components, fibroblasts and proliferating keratinocytes, are also available to assess levels of interleukins such as IL-1α, IL-6 and IL-8.