FOURTEENTH ANNUAL EUROPEAN PRESSURE ULCER ...

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