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'fences' a Cell Migration Assay System for 24-Well Tissue Culture Plates

Introduction
The introduced cell migration assay system consists of reusable 16 mm metal-silicone inserts placed in 24-well tissue culture plates. It allows cells only to adhere within a standard area. So a circle shaped cell monolayer of a defined diameter can be created (Fischer et al., 1990, J.Immunol. Meth. 128: 235-239). The assay is intended for studying the behaviour of soma cells like smooth muscle cells (SMC), fibroblasts, endothelial cells (like EPC or HUVEC) or fibroblasts. It may be not suitable for experiments with immune cells or granulocytes. It affords the opportunity of screening various parameters in parallel, since cell cultures can be developed under sterile conditions with relatively low expenditure of time, space and materials. The assay system has been used successfully in several laboratories all around the world to investigate drug effects as well as surface effects of biomaterials. It can be used with various 24-well tissue culture plates.

stainless steel - implantable silicone device
stainless steel / 'implantable' silicone devices ('fences').

Benefits
Similar assay techniques have been introduced by Pratt et al.(1984, Am.J.Path. 117: 349-354) and Hasson et al.(1986, Surgery 100: 384-391). The relatively large size of the barriers caused various problems such as cell layer injury, barrier displacement and insufficient sterility. These problems are being avoided in our assay system by reduction of the size of fences, use of new materials and use of 24-well culture plates. Furthermore, surface related problems as found in the wounded layer technique as well as a lot of problems that are inherent in techniques like the under-agarose assay or the Bøyden chamber system can be avoided.

human umbilical vein endothelial cells cultured 4 days

human umbilical vein endothelial cells cultured 4 days on
gelatine-coated 15mm cover glass circles in multiple well plates

left = unstimulated HUVEC,

right = HUVEC stimulated with
25 µg/ml ECGF and 90 µg/ml heparin.

Assay procedure - 'Methods'
The fences should be cleaned and sterilised before each assay. While not in use the fences should be kept up-side-down on the metal side to protect silicone washers. Use a detergent, ethanol or a short-time ultrasonic bath (1-2 min) to clean the fences. The optimum temperature for sterilisation is 121°C (2 atm, wet). Fences can also be sterilised at 180°C (1 atm, dry). However, this may cause damage to the silicone component in long-term use. Note: if used directly after sterilisation, the very adhesive silicone may lead to damage of surface coatings and the silicone washer itself. However, using freshly sterilised fences may help to minimise problems with leaky washers.

Before the experiment is started the sterilised supports are inserted into the wells and the plate is placed in an incubator for 30 min to adapt at 37°C and to allow the silicone washers to attach to the surface.

In the 'layer expansion' regime the internal chamber of each 16mm-device is charged with the cells suspended in 150 µl medium, e.g. with 30,000 HUVEC/well to obtain a confluent monolayer. In addition the external chamber of each support is to be charged with 650 µl of medium only, especially if there are problems with leaky washers. In the 'wound closure' regime the outer chamber is loaded with cells (e.g. 130,000 HUVEC in 650 µl) and the inner with 150 µl medium only. Similar cell interaction models may be developed placing one type of cells inside, another type outside the washer. It is important to introduce the tip of the pipette deeply into the internal chamber (2mm above the bottom) in order to avoid air bubbles.

After allowing the cells to attach (about 4-5 hours, depending on the cell type) the supports are removed using forceps and the cell layer is washed twice in order to remove non-adherent cells. Subsequently 1 ml of medium is applied with/without the drugs to be investigated. (Cell must not be cultured over days within the fences, as the liberation of metal ions may influence cell behaviour.)

In order to distinguish between proliferation and migration, at this time the cells can by exposed to irradiation with 15-25 Gy (1500-2500 rad) or to toxins like 5-fluorouracil (10-20 µg/ml; according to literature up to 100 µg/ml) to prevent proliferation.

The diameter at the beginning is about 6.7 mm, corresponding to an area of 35 mm2, depending on the type of cells. The cells are incubated for days (e.g. HUVECs for 5 to 7 days), while the incubation medium is changed every 2 days. After allowing the cell monolayer to expand for days the experiment is terminated by fixing the cells in ethanol:methanol (1:1) or in 5 to 10% paraformaldehyde in buffer for 3 to 10 minutes. After staining (e.g. with 10% Giemsa solution for 20 min followed by alcohol rinsing for 1 min) the cell-covered area is measured under a stereo-microscope (e.g. with help of a mm-graded foil) or is evaluated by a computer-driven video system. Assays should be done in triplicate or quadruplicate.

parts of a fence

the parts :

a = steel (function: weight)

b = silicone (function: washer-like fence)

c = channel (function: providing access to the outer chamber
and making the grasping of fences with forceps easier)

Annotations :   After a while silicone becomes 'yellow' ( amber ) that will not affect the material properties noticeable. But in case the silicone washers get detached or start to leak as a result of damage they should not be used anymore. The metal parts should be stored though, since they can be provided with new silicone washers.

Be aware that the media delivers metal ions from the fences. After some days the concentration in the media may reach a level influencing the behaviour of the cells.

Hoping the fences will work well also in your hands; feel free to ask, if you need any support. If you have any additional or contrary experiences, please inform us.


Purchase
You may purchase a migration assay kit (a set of 24 fences, for research purposes only)
for a price of € 440.oo (EURO) inclusive portage (world wide airmail), but exclusive tax.


Phone : +49 241 4500 358


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