Manual Replicators

  1. Wound Healing Using 384 Well Microplates

    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.

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  2. Cleaning V&P Pins with Caustic Soda

         For daily and between-transfer cleaning, V&P Scientific recommends the VP 110 Pin Cleaning Solution. This proprietary liquid uses a combination of acid and detergent to remove soiling caused by cell debris, salts, proteins and nucleic acids. However, it may become necessary to use a more aggressive cleaning agent; caked on protein, organic soils, or burnt-on carbon deposits caused by flaming handheld replicators can affect transfer volume and consistency, and can be difficult to remove.

         For these cases, a solution of Sodium Hydroxide, or caustic soda, is recommended for deep cleaning. V&P Scientific pins are made from stainless steel and are not damaged by high pH, though, we recommend against exceeding solutions of more than 3% sodium hydroxide to avoid pitting of the stainless steel. Start with a 1% solution and increase for subsequent cleanings if needed. For best results, use the NaOH solution hot- between 60 and 80 degrees C. You can adjust the concentration, temperature, and residence time of the pins in the solution to accommodate the level of soiling.

         A sonicator may also be used to aid in cleaning, and can help maintain the temperature of the cleaning solution. Be sure to cover the pin tool during sonication to avoid splashing and wear all appropriate PPE as hot caustic solutions are extremely dangerous. An example cleaning procedure is described below:

    1.      Prepare a solution of 2% NaOH in distilled or deionized water.

    2.      Heat the solution to 60C.

    3.      Immerse the Pin Tool in the hot solution, making sure that the float plate and frames do not come    into contact with the solution. Only the pins should be submerged.

    4.      Sonicate with heat on 60C for 30 minutes.

    5.      Remove pins from cleaning solution and blot using V&P Scientific's VP 540DB blotting station.

    6.      Rinse pins well with distilled water. If any cleaning solution has splashed onto float plates, rinse the float plates. Blot.

    7.      Rinse pins well with 70% ethanol. If the float plates are wet, rinse the float plates with ethanol. Blot.

    8.      Air Dry Pin Tool

    9.      If pins are fully clean, perform the standard cleaning procedure with VP 110 Pin Cleaning solution and return pin tool to service.

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