The Frequent Sampling of Wound Scratch Assay Reveals the “Opportunity” Window for Quantitative Evaluation of Cell Motility-Impeding Drugs

Abstract

Wound healing assay performed with automated microscopy is widely used in drug testing, cancer cell analysis, and similar approaches. It is easy to perform, and the results are reproducible. However, it is usually used as a semi-quantitative approach because of inefficient image segmentation in transmitted light microscopy. Recently, several algorithms for wound healing quantification were suggested, but none of them was tested on a large dataset. In the current study, we develop a pipeline allowing to achieve correct segmentation of the wound edges in >95% of pictures and extended statistical data processing to eliminate errors of cell culture artifacts. Using this tool, we collected data on wound healing dynamics of 10 cell lines with 10 min time resolution. We determine that the overall kinetics of wound healing is non-linear; however, all cell lines demonstrate linear wound closure dynamics in a 6-h window between the fifth and 12th hours after scratching. We next analyzed microtubule-inhibiting drugs’, nocodazole, vinorelbine, and Taxol, action on the kinetics of wound healing in the drug concentration-dependent way. Within this time window, the measurements of velocity of the cell edge allow the detection of statistically significant data when changes did not exceed 10–15%. All cell lines show decrease in the wound healing velocity at millimolar concentrations of microtubule inhibitors. However, dose-dependent response was cell line specific and drug specific. Cell motility was completely inhibited (edge velocity decreased 100%), while in others, it decreased only slightly (not more than 50%). Nanomolar doses (10–100 nM) of microtubule inhibitors in some cases even elevated cell motility. We speculate that anti-microtubule drugs might have specific effects on cell motility not related to the inhibition of the dynamic instability of microtubules.

Single‐Particle Tracking and Trajectory Analysis of Fluorescent Nanodiamonds in Cell‐Free Environment and Live Cells

“…At the same time, the cell movement over the course of the short, 20‐min experiments is usually limited. An actively migrating HeLa cell on a glass surface moves at ≈12 µm h −1[ 36 ] (0.003 µm s −1 ), resulting in an expected displacement of 4 µm by the end of the experiment. The speed of the endosomal movement, driven by molecular motors, such as kinesins, myosins, and dynein, is ≈1 µm s −1[ 37 ] —two orders of magnitude higher than that of the entire cell.…”

Organic extract of Geodia cydonium induces cell cycle block in human mesothelioma cells

“…To the best of our knowledge, the present study was the first to describe the in vitro antiproliferative effect of GEOCYDO extract on mesothelioma, a rare but very aggressive cancer characterized by high chemoresistance (10,14). Mesothelioma displays a long latency period (30-40 years) and a generally unfavorable outcome (10).…”

“…Colony formation assay. Colony formation was assessed as previously reported by our group (14). For each cell line, 500 cells/well were plated in six-well plates, incubated for 7 days and then treated with 500 µg/ml GEOCYDO for 24 or 48 h under culture conditions before replacing the media.…”

“…Wound-healing assay. The wound-healing assay was performed as previously reported (14,15). For each cell line, ~3x10 5 cells/well were seeded in six-well plates.…”

3D Polymer Architectures for the Identification of Optimal Dimensions for Cellular Growth of 3D Cellular Models

“…We, therefore, apply N = 10 for further analyses, which is a value of 16.6 µm comparable to the average cell size, and slightly larger than the average cell motility of 10 µm/h found for A549 cells [ 29 ]. This value will optimize the number of cells analyzed since no two cells can occupy the same space in the scaffold, and give an individual cell motility range between 0 and 100 µm, in the time-lapse study.…”