Abstract
Impaired epithelial wound healing has significant pathophysiological implications in several conditions including gastrointestinal ulcers, anastomotic leakage and venous or diabetic skin ulcers. Promising drug candidates for accelerating wound closure are commonly evaluated in in vitro wound assays. However, staining procedures and discontinuous monitoring are major drawbacks hampering accurate assessment of wound assays. We therefore investigated digital holographic microscopy (DHM) to appropriately monitor wound healing in vitro and secondly, to provide multimodal quantitative information on morphological and functional cell alterations as well as on motility changes upon cytokine stimulation. Wound closure as reflected by proliferation and migration of Caco-2 cells in wound healing assays was studied and assessed in time-lapse series for 40 h in the presence of stimulating epidermal growth factor (EGF) and inhibiting mitomycin c. Therefore, digital holograms were recorded continuously every thirty minutes. Morphological changes including cell thickness, dry mass and tissue density were analyzed by data from quantitative digital holographic phase microscopy. Stimulation of Caco-2 cells with EGF or mitomycin c resulted in significant morphological changes during wound healing compared to control cells. In conclusion, DHM allows accurate, stain-free and continuous multimodal quantitative monitoring of wound healing in vitro and could be a promising new technique for assessment of wound healing.
Highlights
Epithelial wound healing is a common physiological process
To evaluate the potential of quantitative phase imaging with digital holographic microscopy (DHM) for monitoring of epithelial wound healing in vitro, Caco-2 cell wound assays were analyzed and results compared to microscope images acquired by white light illumination
We prove DHM to enable continuous, stain-free monitoring of intestinal epithelial wound healing in vitro and to provide simultaneous quantification of key cellular characteristics such as cell volume, cell thickness, dry mass and cell density which may help to characterize therapeutic effects of potential drug candidates
Summary
Epithelial wound healing is a common physiological process. Within the gastrointestinal tract, there is persistent regeneration of epithelial cells to compensate physiological exfoliation of surface cells [1]. Potential drug candidates are usually assessed in in vitro wound assays, such as the classical scratch assay established by Burk et al [7]. More sophisticated cell culture systems have been introduced, more precisely elucidating the extent of migration and proliferation in vitro [8]. One example includes a silicone cell cultureinserts onto the cell culture surface generating two reservoirs (see section ‘Cell layer wound assays’) that are separated by a 500 mm wall, which on removal leaves a well-defined border [9]. Valid determination of cell migration commonly requires cell staining, e.g. Giemsa staining [10,11] or transfection of the sample with fluorescent chromophores for cell tracking [12] which both require interaction with the sample
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