Planta Med 2015; 81 - PM_244
DOI: 10.1055/s-0035-1565621

Comparison of different wound healing assays performed with lupeol-treated human keratinocytes

P Werner 1, T Wardecki 1, J Madl 2, J Nascimento 3, 4, W Römer 2, M Börries 3, 4, 5, I Merfort 1
  • 1Institute of Pharmaceutical Science, Department of Pharmaceutical Biology and Biotechnology, University of Freiburg, Freiburg, Germany
  • 2Faculty of Biology, BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany
  • 3Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany
  • 4German Cancer Consortium (DKTK), Heidelberg, Germany
  • 5German Cancer Research Centre (DKFZ), Heidelberg, Germany

Wound healing is a complex process comprising three different phases: inflammation, new tissue formation, and remodeling [1]. To avoid expensive animal models, where also ethical aspects have to be considered, in vitro assays with monocultures of human skin cells represent an alternative. There are several assays described in the literature focusing on the tissue formation phase where keratinocyte and fibroblast migration is essential [2]. In our studies, we tested five in vitro wound-healing assays and evaluated their advantages and limitations. We used human keratinocytes that were treated with HGF and the natural compound lupeol. HGF is known to promote the migration and lupeol is one of the main components of a triterpene enriched birch bark extract (TE1) that is known to improve wound healing [3, 4]. We tested the “classical” scratch assay where a confluent cell monolayer is disrupted with a pipette tip and subsequently “wound” closure is observed over time. Additionally, the OrisTM-Cell Migration Assay and experiments with ibidi culture-inserts were carried out. For these assays keratinocytes were grown in a special chamber with a space holder that creates a defined cell-free gap in which the cells start to migrate after removal of the space holder. As a further assay the xCelligence method is described where cells have to migrate through a membrane in a modified Boyden chamber and the migration is measured by impedance. A more laborious approach was to track single cell movement using a fluorescence microscope. All these methods have their advantages but also their limitations; nevertheless, the simple “scratch assay” seems to be the one where the advantages predominate.

Acknowledgement: Financial support by Aif is gratefully acknowledged.

References:

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[2] Pollok S et al. Cell Mol Med 2011; 15: 861 – 873

[3] Matsumoto K et al. Cell Res 1991; 196: 114 – 120

[4] Ebeling S et al. PLoS One 2014; 9: e86147