Measurements of Cell-Substratum Adhesions and Traction Force at the Negative Pressure Environment

Project: National Science and Technology CouncilNational Science and Technology Council Academic Grants

Project Details

Abstract

Both collective cell migration, referred as leading cells drive other less active cells to move toward one direction, and physical interactions between the cell- cell and cell- substratum play important roles in the wound-healing process. Although the negative-pressure wound therapy successfully treat difficult chronic wounds, the exact mechanisms are not fully understood. Electric cell-substrate impedance sensing (ECIS) is a technique to continuously monitor electric activities of cell migration on the exposed electrode. The optical phase and wide visual field microscope tracks spatial correlations between embedded fluorescent particles and cells to estimate the cell-substratum traction force. The purpose of this study was to use ECIS and traction force microscopy to quantify changes of the cell-cell adhesion, cell membrane morphologies, and cell-substratum traction force at the pressure of 635 mmHg. Relations between the above changes and the epithelial-mesenchymal transition in human keratinocytes (HaCaT cell line) were also investigated. First year: The electroporation will be given to wound the monolayer cells in the ECIS electrode array. The wound-healing process at the ambient pressure and the pressure of 635 mmHg will be continuously recorded in the frequency range of 22.5- 64 kHz from five hours before wounding to 12 hours after wound closure. The intercellular resistance (Rb), the cell-substratum resistance (α), and the membrane capacitance (Cm) will be studied during the wound-healing process. Second year: To observe the live cell images at different pressures, we planned to put the Lumascope 500 (Etaluma Inc, Carlsbad, CA, USA) into the negative-pressure incubating chamber to build the real-time live cell fluorescence imaging system. According to the temporal relations between cells and fluorescent embedded into the culture medium, we can estimate the cell- substratum traction and shear force. Third: Fluorescent stains for transcription factors of the slug, snail, β-catenin, and p-120 will be done in cells respectively at the ambient pressure and at the pressure of 635 mmHg for 12 hours. Western blots will be performed for those have different presentations. The wounded cells were incubated at the atmospheric pressure for 12 hours after the knockdown of slug and snail. We wish to compare the wound-healing process and E-cadherin expressions in cells after the above treatment with those at the pressure of 635 mmHg.

Project IDs

Project ID:PC10308-0981
External Project ID:MOST103-2314-B182-003
StatusFinished
Effective start/end date01/08/1431/07/15

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