Abstract
Considering the essential role of chemotaxis of adherent, slow-moving cells in processes such as tumor metastasis or wound healing, a detailed understanding of the mechanisms and cues that direct migration of cells through tissues is highly desirable. The state-of-the-art chemotaxis instruments (e.g. microfluidic-based devices, bridge assays) can generate well-defined, long-term stable chemical gradients, crucial for quantitative investigation of chemotaxis in slow-moving cells. However, the majority of chemotaxis tools are designed for the purpose of an in-depth, but labor-intensive analysis of migratory behavior of single cells. This is rather inefficient for applications requiring higher experimental throughput, as it is the case of e.g. clinical examinations, chemoattractant screening or studies of the chemotaxis-related signaling pathways based on subcellular perturbations. Here, we present an advanced migration assay for accelerated and facilitated evaluation of the chemotactic response of slow-moving cells. The revised chemotaxis chamber contains a hydrogel microstructure–the migration arena, designed to enable identification of chemotactic behavior of a cell population in respect to the end-point of the experiment. At the same time, the assay in form of a microscopy slide enables direct visualization of the cells in either 2D or 3D environment, and provides a stable and linear gradient of chemoattractant. We demonstrate the correctness of the assay on the model study of HT-1080 chemotaxis in 3D and on 2D surface. Finally, we apply the migration arena chemotaxis assay to screen for a chemoattractant of primary keratinocytes, cells that play a major role in wound healing, being responsible for skin re-epithelialization and a successful wound closure. In direction of new therapeutic strategies to promote wound repair, we identified the chemotactic activity of the epithelial growth factor receptor (EGFR) ligands EGF and TGFα (transforming growth factor α).
Highlights
The migration arena assay is based on the μ-Slide Chemotaxis, a microfluidic tool with the size of a microscopic slide (Fig 1A)
In order to verify the specificity of the detected chemotaxis response to epithelial growth factor (EGF) and transforming growth factor α (TGFα), we studied growth factors (GFs)-induced chemotaxis in presence of epithelial growth factor receptor (EGFR) inhibitors (Fig 6)
For some scientific questions a high experimental throughput may be more important than depth of detail
Summary
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The ibidi GmbH provided support in the form of salaries for two of the authors (LT and ZG), but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We declare that a European patent (No EP 1 741 487 A1) was filed by ibidi GmbH for the μ-Slide Chemotaxis. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Two of the authors are salaried employees of the ibidi GmbH, all experiments have been objectively performed such that all data are unbiased, accurate and realistic
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