FOURTEENTH ANNUAL EUROPEAN PRESSURE ULCER ...
FOURTEENTH ANNUAL EUROPEAN PRESSURE ULCER ...
FOURTEENTH ANNUAL EUROPEAN PRESSURE ULCER ...
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Thursday September 1st<br />
Proceedings of the 14th Annual European Pressure Ulcer Meeting<br />
Oporto, Portugal<br />
Wound Models in Monolayer Cell Cultures,<br />
Quantitative Analysis of Cell Kinematics<br />
Amit Gefen * , Orna Shaharabany-Yosef, Gil Topman<br />
* Department of Biomedical Engineering, Faculty of Engineering, Tel Aviv University, Israel, gefen@eng.tau.ac.il<br />
Introduction<br />
Cell migration is a critical process in wound<br />
closure, including closure of pressure ulcers (PU).<br />
Wound healing assays are simple but effective<br />
means for studying cell migration in vitro, and<br />
such assays were found to represent the<br />
kinematics of in vivo migration to a reasonable<br />
extent. In wound healing assays, cells migrate<br />
from a populated area into a denuded area,<br />
created e.g. by local crush, scratch or ablation of<br />
the cells, and hence the "wound" is covered. One<br />
of the commonly used measures for the<br />
performances of the migrating cells is the area of<br />
the "wound" over time, and in particular how fast<br />
can that area be covered by cells post infliction of<br />
the damage. However, current methods that are<br />
available for measuring the area-time behavior of<br />
the "wound" are typically subjective and<br />
inaccurate, or costly, or cumbersome to apply.<br />
Here we present a new method, based on timelapse<br />
digital optical microscopy and image<br />
processing, for quantifying the kinematics of cell<br />
colonies migrating from populated areas into a<br />
denuded area, in the context of modeling cell<br />
migration in PU healing. In particular, we<br />
employed our new method for determining the<br />
migration kinematics of different cell types which<br />
can be potentially involved in PU healing<br />
(fibroblasts, preadipocytes and myoblasts) as well<br />
as for characterizing effects of ischemic factors<br />
associated with PUs (low glucose, low<br />
temperature and acidosis) on the migration.<br />
Methods<br />
NIH 3T3 fibroblasts, 3T3-L1 preadipocytes and<br />
C2C12 myoblasts were thawed from liquid<br />
nitrogen storage and cultured in standard media<br />
that are specific to each cell type. When cultures<br />
were near confluency, a micro-indentor (size<br />
0.46×0.38mm) was used to inflict localized<br />
crushing damage to the cultures. Time-lapse<br />
images of the cultures were then acquired every<br />
2 hours, using an Eclipse TS100 microscope<br />
(Nikon) and DS-Fi1 digital camera with a<br />
resolution of 2560×1920 pixels (3 pixels per<br />
micron). During image acquisition, cultures were<br />
kept at 37ºC using a temperature control system,<br />
and HEPES was supplemented to the media in<br />
order to control the pH level. The time-dependent<br />
micrographs were post-processed by a MATLAB<br />
46<br />
code, based on texture homogeneity measures.<br />
Specifically, denuded areas in the digital<br />
micrographs were characterized by a<br />
substantially lower standard deviation (SD) of<br />
pixel intensities, as opposed to cell-populated<br />
areas where the SD of pixel intensities was high.<br />
The SD distributions were mapped over the<br />
micrographs per each time point, using two<br />
window sizes for each cell type: the first being a<br />
window with a course resolution and the second<br />
with a fine resolution. An intersection of these SD<br />
distribution maps obtained when using the two<br />
window sizes resulted in an adequate<br />
measurement of the time-dependent area of the<br />
denuded region. The experimental area vs. time<br />
data were finally fitted to Richards nonsymmetrical<br />
sigmoid functions for calculating<br />
migration rates from the coefficients of these fits.<br />
Results<br />
Cells covered the damage area after ~24 hours,<br />
however there were cell-type-dependent<br />
differences in rates of coverage. Specifically,<br />
fibroblasts and the fibroblast-like preadipocytes<br />
(3T3-L1) were faster than the myoblasts in<br />
covering the damage area under control culture<br />
conditions (37ºC, glucose=4.5g/ml, pH=7.4). The<br />
migration rate of the NIH3T3 fibroblast cells was<br />
reduced by ~50% at an acidic environment<br />
(pH=6.7) but the other cell types were not<br />
significantly affected by the acidosis.<br />
Discussion<br />
Developing a reliable and reproducible wound<br />
healing model in vitro is essential for studies of<br />
the etiology of PU as well as for testing the<br />
performances of any medication or food<br />
supplement aimed at improving cell motility for<br />
wound healing. The present method meets these<br />
needs and is easy to implement in a cell lab.<br />
Clinical relevance<br />
A reliable, reproducible wound model system is<br />
essential for testing medications and food<br />
supplements claimed to improve healing of PU.<br />
Conflict of Interest: None<br />
References<br />
Topman, G., Shaharabany-Yosef, O., Gefen, A. A method<br />
for quantitative analysis of the kinematics of fibroblast<br />
migration in a monolayer wound model. Proceedings of the<br />
ASME 2011 Summer Bioengineering Conference,<br />
Farmington, PA, USA, June 22-25, 2011.<br />
Copyright © 2011 by EPUAP