A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

Podsumowanie

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

Streszczenie

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Wprowadzenie

Cell migration plays an important role in cellular processes, such as embryogenesis, neurogenesis, angiogenesis, wound healing, mucosal repairing, epithelial-mesenchymal-transitions under normal development and disease situation¹. It is a complicated process which involves the coordination of numerous inter- and intra-cellular events including signaling molecule interactions, cell polarization, cytoskeletal reorganization, matrix remodeling, membrane protrusion and dynamic cell-cell adhesion modulation². By studying cell migration, the discovery and validation of chemicals or biomolecules leading to cell movement and its related biochemical pathways can be determined. This fundamental process can be used beneficially as a proxy for various applications, such as disease treatment and drug targeting.

The wounding assay is one of the cell migration assays³. Here, an individual sample of cells will be partially removed by mechanical means to produce a denuded area where cells will migrate to cover the area. The percentage of recovery at a particular time point will be monitored. When handling large number of samples, issues such as the experimental cost and cross-contamination within samples can be problematic. Although many commercial tools and assays are available for studying cell migration^4,5,6, many of them required expensive and sophisticated equipment and improvements are still needed. For these reasons, an 8-channel mechanical cell wounder (Figure 1) is developed.

Our 8-channel mechanical cell wounder has incorporated several unique features in solving the above-mentioned problems. It provides the flexibility to perform cell migration/wounding assays in the 96-well culture plate format. The adjustable guiding bar design ensures that the scratch area is in the central position of each well. Also, the height of the guiding bars can be adjusted so that the wounder is applicable for various brands of culture plates. Finally, the adjustable wounding pin design enables an even contact of the pipette tips with the culture plate surface to achieve simultaneous and reproducible wounding^7,8.

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Protokół

1. Introduction to the Parts of the Wounder (Figure 1)

1. Prepare the pin holder by fixing the wounding pins at equal distances. Hold the pipette tips and adjust the tip height by moving the adjustable wounding pins up and down.
2. Fit the adjustable guiding-bar on different brands of 96-well culture plates and ensure that the wounding area is in a fixed position.
3. Fix the adjustable wounding pin by tightening the hex screw. Fix the guiding-bar in position by tightening the hex socket head caps on both ends.

2. Setting the Width of the Pin Holder (Figure 2)

1. Loosen the hex socket head caps with the M5 hex wrench.
2. Insert a suitable number of adjusting rings to both sides of the pin holder so that the guiding bars fit perfectly with the width of the 96-well culture plate.
   NOTE: Corning and Iwaki plates requires 1 pair of large rings and 1 pair of small rings. Falcon and Nunc plates need 1 pair of large rings.
3. Tighten the hex socket head caps to fix the guiding bar.

3. Adjusting the Guiding Bars (Figure 3)

1. Fit the wounding pins with sterile 10-µL plastic pipette tips.
2. Loosen the hex socket head caps with the M5 hex wrench.
3. Hold the wounder and dip the wounding pins into one column of a 96-well culture plate.
4. Adjust the height of the guiding bars until all of the tips barely touch the bottom of the wells.
5. Tighten the hex socket head caps. Ensure that the wounder now perfectly fits on the culture plate and that the pins are well-positioned in the middle of the wells.

4. Calibration of the Pins

1. Hold the wounder perpendicular to a flat sterile surface (i.e. Petri dish) and loosen all the hex screws with the M3 hex wrench.
2. Tap the wounder until all of the tips evenly touch the flat sterile surface.
3. Tighten the hex screws again to lock the pins in position.
4. Check the evenness of each tip by tapping the wounder gently on the flat sterile surface.

5. Scratching Cell Monolayer (Figure 4)

1. Seed human umbilical vascular endothelial cells on a 0.1% gelatin-coated 96-well cell culture plate. Culture cells in Medium 199 supplemented with 20% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin and 0.09 g/L heparin. Maintain cells in a humidified incubator with 5% carbon dioxide at 37 ºC overnight before being used. Cells should reach 100% confluency before wounding.
2. Place the wounding tips at the leftmost (or the rightmost) side of each well within the same column of the culture plate. Slide the wounder across to the other side of the well; make sure the wounding tips are touching the bottom of the well.
3. Repeat the scratching (Step 5.2) for all columns.
4. After wounding, discard the medium in each well and replace with fresh medium containing testing compounds.

6. Data Acquisition and Image Analysis

1. Image the whole well with the mechanical wound on the cell monolayer using a low-power microscope with digital camera (e.g. 10X objective) immediately after wounding (t=0 h).
2. Add test compounds if desired and incubate for the desired length of time. Image the whole well with the wound area again after the desired incubation time (t=Δh).
3. Using image analysis software such as ImageJ (https://imagej.nih.gov/ij/), measure the wounded area on the image using the freehand tool.
4. Calculate the percentage of wound closure:
   

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Wyniki

This 8-channel mechanical wounder is constructed to scratch a cell monolayer in order to perform the cell migration assay. It is a user-friendly device that can carry out cell scratching assays in 96-well plates in less than a minute without special training. This wounder can introduce wound areas on cell monolayers with a uniform width of about 600 µm and with sharp edges (Figures 5 and 6). The width of the wounds depends on the brand of pipette tip...

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Dyskusje

Our cell wounder has several unique features in solving the problems of traditional cell migration assays. The 8-channel mechanical cell wounder is made of high grade stainless steel (Steel 304) with a long lifespan that can be sterilized by autoclaving. Almost all commercial brands of 96-well culture plates available on the market can be used with this mechanical cell wounder because of the adjustable guiding bar. The design of the adjustable guiding bar also ensures that the scratching area is at the center of each wel...

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Materiały

Name	Company	Catalog Number	Comments
96-well cell culture plate	Nunc	167008	Other brands of 96-well cell culture plate can also be used
P10 pipette tips	Axygen	301-03-051	Short P10 pipette tip is more easy to create a clear wound
Wounder	R&P Technology Limited
Medium 199	Sigma	M2520-1L	For cell culture of human umbilical vein endothelial cells. Use appropirate culture medium and condition for other type of cells.
Fetal bovine serum	Gibco	26140079	For cell culture of human umbilical vein endothelial cells. Use appropirate culture medium and condition for other type of cells.
Penicillin/Streptomycin	Gibco	15140122	For cell culture of human umbilical vein endothelial cells. Use appropirate culture medium and condition for other type of cells.
Heparin sodium salt from porcine intestinal mucosa	Sigma	H3393	For cell culture of human umbilical vein endothelial cells. Use appropirate culture medium and condition for other type of cells.
Gelatin from bovine skin	Sigma	G9391	For cell culture of human umbilical vein endothelial cells. Use appropirate culture medium and condition for other type of cells.