Introduction:

       Wound healing involves cell migration and invasion which are processes that offer rich targets for intervention in key physiologic and pathologic pathways. With the advent of high-throughput and high content imaging systems, there has been a movement towards the use of physiologically relevant, phenotypic cell-based assays earlier in the testing paradigm. This allows more effective identification of lead compounds and recognition of undesirable effects sooner in the drug discovery screening process. At V&P Scientific, we have created tools for mechanically scratching the cell substrate with a 384 pin array.  Scientists can create characteristically sized wounds in all wells of a 384 well plate.

 

Rationale:

       Many of our customers have used our pin tools to "wound" cell culture monolayers and then study the effects of different treatments on wound healing. Initially, our standard 96 wound healing pin tools and a special wounding Library Copier (VP 381NW, VP 381NW4.5 or VP 381NW5) were used to make the wounds. For most applications, these wounding tools give good results. However, some customers who were using 384 well plates needed the wounds to be more precisely localized. For these customers, we have developed specialized pin tools that have very tight hole tolerances in the floating fixture so the wounds are consistently located in the same position in each well.

       A recent addition to our line of wounding pin tools are pins with a 0.05 mm layer of Parylene coating, deposited using a vapor deposition process to produce a soft lining on the pin tips. Each pin now acts as “eraser” to remove the cell monolayer without scratching the plastic well surface under the monolayer. Another new innovation and problem solved only by V&P Scientific.

       Another factor that can lead to wound variation is a loose fit between the microplate and the registration device (e.g., our VP 381NW Library Copier).  This is due to variation in the molds used to make the microplates. One way of dealing with the problem is to use our adjustable Library Copiers (VP 381NWGV4.5 or VP 381NWGH4.5).

 

Results:

       We recently developed a new Library Copier for creating wounds in 384 well microplates in the horizontal direction (VP 381NWGH4.5). We added a second set of slots to this library copier which allows the user to use a 384 pin tool containing only 192, or 96 pins which significantly reduces the cost of the wound creating pin tool, and still allows for the production of 384 wounds by simply changing the pin tool and library copier orientation.

 

These pictures show the V&P fixture, AFIX384FPWP, filled with 192 or 96 FP-WP pins:

 

The following pictures show the V&P fixture, AFIX384FPWP, filled with 192 or 96 FP-WP pins, being used to produce wounds in 384 plates half-a-plate at a time, or in quadrants by using the library copier: VP 381NWGH4.5.  Standard carbon paper was used to trace the scratch produced by the wound healing tool.

 

 

Top Half of 384 Well Plate

 

Bottom Half of 384 Well Plate

Result, a Complete 384 Well Plate

 

1st Quadrant(Upper Left) of a 384 Well Plate

 

2nd Quadrant(Lower Left) of a 384 Well Plate

 

Rotate Plate 180 Degrees, Repeat Scratches, Result a complete 384 Well Plate:

 

CHO-K1 (ATCC® CCL-61™) Cells Before and After Scratching:

 Before                                                                                                                                                                                                  After

 

Summary:

 

       V&P Scientific's pin tools are an effective way to perform wound healing experiments. The use of 384 well plates allows for high-throughput wound creation allowing many compounds to be screened for effects on cell migration or invasion. The use of V&P Scientific's Library Copiers helps to create wounds uniform in length and width. Parylene coated pins produce a soft lining on the pin tips. Each pin now acts as an “eraser” to remove the cell monolayer without scratching the plastic well surface under the monolayer.