Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

The overall goal of this procedure is to establish explan cultures of zebrafish keratocytes for use in collective cell migration and epithelial wound healing studies. This is accomplished by first removing scales with attached epithelial tissue from the anesthetized adult zebrafish. The scales with attached tissue are placed into a culture dish to allow the cells to adhere.

Then the culture medium is added along with any treatments indicated by the experimental design. Finally, the cells are incubated and observed by video microscopy for the desired timeframe. Ultimately, a variety of assays can be used to demonstrate changes in migration or cellular morphology, as well as expression of proteins or other markers over time.

The main advantage of this technique over existing method, like the transform cell culture method, is that explant culture are primary, which closely reassemble to the properties of cell in vivo. This method can help answer key questions in the epithelial wound healing field, such as the role of collective migration and epithelial to mesenchymal transition. Generally, individuals new to this method will struggle because scales may not attach to the growth surface or may fail to grow in any given culture dish.

Demonstrating this procedure will be Jose Rap PanIN medical student, Chana TI and Agnes Pasquale, both senior research associates in my laboratory. To prepare for this experiment, de chlorinate approximately 1.5 liters of tap water using either a commercial dechlorination agent or by letting the water stand for 24 hours, then use 500 milliliters to fill each of three one liter beakers. Label one beaker for holding fish before any procedure, and a second one for recovery to the third beaker.

Add 100 micrograms per milliliter of trica. For anesthetizing fish fill a shallow tray with ice. Warm the culture medium to room temperature and gather clean jewelers tweezers.

Clean the tweezers by immersing the tips in 70%ethanol and passing them through a bunsen burner, allowing the ethanol to burn off. Catch the desired number of fish and place in a holding beaker. Transfer a single fish to the anesthesia beaker.

After anesthetizing the fish according to the text protocol, transfer it to ice and place it horizontally with the tail facing the hand holding the tweezers. To remove the scales position the tweezers parallel to the fish. With the tip at the edge of a scale, depress the flat side of the tweezers slightly into the side of the fish to cause the scale to elevate from the body of the fish.

Use the tweezers to grasp the scale, then pluck it and without inverting it, place it into the tissue culture dish. Repeat the procedure, removing a maximum of 12 scales from a large adult, avoiding the gill region, lateral line, and fins. Place the fish in the recovery beaker and monitor to ensure it recovers from the effects of anesthesia.

Then transfer the fish into an individual tank and allow the fish to recover for 21 days to allow complete scale regeneration. Depending on the experiment, place up to 20 scales per plastic. 35 millimeter tissue culture treated dish, or four to six scales per glass.

Bottom dish, allow the scales to adhere to the dish before gently adding 1.2 milliliters of complete medium to each culture dish. Incubate the X explan cultures at 28 degrees Celsius for the desired period of time with or without treatment to carry out microscopy. After removing a dish containing cell sheets from the incubator, position it into the microscope stage.

Use a four x objective to initially focus for facilitating image taking over time. Mark the location of each cell sheet and or the orientation of the dish on the stage. Photograph the cell sheets at various time points according to the experimental protocol, and if only still images are needed for the experiment placed back into the incubator.

To fix samples for immunofluorescence microscopy, prepare and prewarm solutions at room temperature. To view OSE sites under fluorescence, use glass bottom 35 millimeter dish culture dishes to grow eggplants as described earlier in this video, without removing the culture medium, add 0.8 milliliters of 4%paraldehyde in 1.1 XPBS to each dish. Incubate at room temperature for five minutes.

Then aspirate the solution. Add an additional 0.8 milliliters of fixative to each dish, and after a second five minute incubation, aspirate the solution. Unless using external ligands or epitopes, perme the cells by adding 0.5 milliliters of 0.2%TRITTON X 100 in one XPBS incubate for five minutes.

Before removing the solution, add 0.5 milliliters of 1%BSA in one XPBS, and incubate at room temperature for one hour or at four degrees Celsius overnight after the incubation, aspirate the solution and proceed with antibody staining. Alternatively, add one to two milliliters of 1%BSA in one XPBS and store at four degrees Celsius for three to four weeks. Carry out immunofluorescence.

According to the text protocol as illustrated here, sheets of keratocytes can be observed migrating from beneath the scale within hours of establishing the EXPLAN culture. Occasionally, an individually migrating keratinocyte, which has broken away from the collectively migrating sheet can be observed. The initial rate of sheet advancement is extremely rapid, but this rate quickly slows as cells spread during the first 48 hours of culture.

The rapid increase is not associated with cell proliferation since after labeling for 24 hours with the fluorescent EDU, approximately 10%of cells show evidence of cell division a rate far lower than seen in transformed cell lines. When treatment is added at the time X explan are established, some compounds alter collective cell migration. For example, as shown here, some MMP inhibitors decrease cell sheet area after 24 hours.

In culture, when soluble RGD containing peptide is added to the culture medium to disrupt adhesion formation, the lamely at the leading edge of the sheet rapidly shrink and subsequently the leading edge of the sheet detaches and retracts and less than two minutes to assess localization of proteins within the sheet or within liter and follower cells, the entire sheet can be fixed and stained as shown in this figure. Once mastered, this technique can be done in 10 to 15 minutes if it is performed properly. While attempting this procedure, it's important to remember to add prewarm culture media only after allowing time for the cytes to adhere to the culture dish Following this procedure.

Other method like QPCR can be performed to answer any additional question, such as changes in gene expression in relation to time and or treatment condition. After watching this procedure, you should have a much better idea of how to establish IDE explan cultures for use in a variety of procedures.