The aim of this procedure is to demonstrate the successful establishment of human fungi form taste Pap pilley cell culture. This is accomplished by first obtaining the fungi form taste per pilley from human volunteers and associating them using an enzymatic cocktail. The second step is to transfer the cells from the human fungi formm taste per pilley into a cell culture dish.
Next, allow the cells to grow and change the media to maintain the culture to human fungi. Formm taste per pilley cells. Ultimately, calcium imaging, confocal microscopy, and light microscopy are used to demonstrate the presence of taste, cell markers and physiological responses.
The main advantage of this technique is that it allows the use of cell lines instead of freshly collected tissue each time. One undertakes an experiment. Though this method can provide inside the human taste mechanisms, it can also be applied to other systems, such other species and other studies of regeneration.
This method can help to answer key questions in the taste field, such as how taste cells regenerate and what factors regulate gene expression during taste, cell proliferation and maturation. To begin this procedure, obtain the human fungi form taste per pill by removing it from the dorsal surface of the anterior portion of the tongue, using a pair of curved to bring micro scissors. The details of this protocol were published in a previous JoVE article.
Then place the sample immediately in the isolation solution to digest the fungi pap pill. Isolate them in the isolation solution with collagenase type one elastase and soybean tripsin inhibitor in a 35 degree Celsius water bath with circulation and gentle oxygenation with 95%oxygen, 5%carbon dioxide for 30 minutes. After incubation, wash the papillae with ringer solution.
Ate it with a fire polished glass pasta pipette 10 times, then centrifuge it for three minutes, a 2, 500 RPM at room temperature. After that, remove the isolation solution. Add one milliliter of taste cell culture media to the pape.
Then transfer the digested fungi, formm papillae to a glass dish. Now dissect the fungi formm papillae gently with a surgical razor. Add 250 microliters of the dissected papillae onto the cover slip coated with rat tail collagen.
Type one in the cloning cylinder. Afterward, add one milliliter of taste cell culture medium to the well. In this procedure, incubate the plate at 36 degrees Celsius in a humidified incubator containing 5%carbon dioxide for two days.
Then change the complete medium taste cells may not be visible in the first three days, but they will eventually bind to the coated cover slip. Next, remove the cloning cylinder from the plate and the medium completely. Add one milliliter of taste cell medium to the well.
After that, replace one third of the medium every six to seven days. In the meantime, check the cells onto the microscope Every other day, most of the new cells grow underneath the cell clusters. Cell clusters usually detach from the surface of the cover slip after two to three weeks in culture leaving the newly generated cells, once 40 to 50%of the cloning cylinder is covered by the expanding taste cells.
Trypsin is the cells using 0.25 weight per volume trips in EDTA for two to three minutes at 36 degrees Celsius. Then transfer the cells from the well into a 15 milliliter tube. Subsequently dilute the taste cell culture medium fourfold with the medium containing fetal bovine serum centrifuge to 3000 rotations per minute for five minutes of room temperature.
After that, remove the supinate and re suspend the cells with one milliliter of taste cell medium. Now transfer the cells to a T 25 plate. Then add four milliliters of taste cell medium.
Maintain the cells at 36 degrees Celsius in a humidified incubator containing 5%carbon dioxide. Next, replace one third of the medium every six to seven days until the taste cells have reached 100%confluence. To enable taste cells for harvesting, wash the cells once with sterile PBS.
Then trypsin eyes them using 0.25%weight per volume trips in EDTA for two to three minutes at 36 degrees. Cell cs, repeat the centrifugation as described previously, then re suspend them in taste cell complete medium afterward, transfer the cells to a fresh T 75 flask and replace one third of the medium every six to seven days. When the cells are close to 100%confluence, split the cells to no more than one to four dilution in a T 75 flask for maintaining adequate growth of the cells over time.
To freeze the stock of primary taste cells. After trypsin, add three milliliters of complete taste cell medium, transfer the cells to a 15 milliliter conical centrifuge tube, then centrifuge at 2, 500 RPM for five minutes of room temperature. Carefully remove the supinate and gently resuspend the cells with an appropriate volume of freezing medium containing 95%fetal bovine serum and 5%DMSO.
Next, transfer the cells to a labeled sterile cryo vial. Cap the vial tightly, then place it in a freezing container containing isopropanol and place the container in the minus 80 degrees Celsius freezer for at least one day before transferring to the liquid nitrogen tura vial of frozen primary human fungi formm taste per pill cells. Place it in the 37 degrees Celsius water bath in a lamina flow cell culture hood until just thawed.
After that, transfer the cells and freezing medium to a sterile 50 milliliter conical centrifuge tube. Add five milliliters of taste cell culture medium to it. Next, centrifuge the cells at 2, 500 RPM for five minutes.
At room temperature, carefully discard the snat and gently resuspend the cells with complete taste. Cell culture medium. Then transfer it to a sterile T 25 tissue culture flask.
Cells within these cultures display many molecular and physiological features characteristic of mature taste cells. Gus din and phospholipase C beta two mRNA were detected by reverse transcriptase polymerase chain reaction, and the products were confirmed by sequencing. The expression of Gus Din and phospholipase C beta two was detected Immuno chemically indicating the presence of type two like cells.
Cultured cells also exhibited robust increases in intracellular calcium in response to appropriate concentrations of several taste stimuli. In these studies, sweet and bitter stimuli elicit an increase in intracellular calcium that may correspond to a depolarization. Once master, this technique can be done in one to two hours if it is performed properly Following this procedure.
Other methods such as calcium imaging and immunochemistry can be performed in order to answer additional questions like what taste receptors are present or what is the rate of proliferation in response to certain growth factors. After watching this video, you should have a good understanding of how to dissociate and grow human taste cells.
