The overall goal of this procedure is to obtain cells for culture from the mouse incisor, labial cervical loop in a reliable and consistent manner. This is accomplished by first dissecting the lower Hemi mandible from the adult mouse and removing the bone covering the cervical loop region. The second step is to isolate the cervical loop region and separate the meen kind from the epithelium.
Next, the cervical loop is micro dissected. The final step is to dissociate then culture, the isolated epithelial cells. Ultimately, this video is used to show the correct procedure for successful isolation of cells from the labial cervical loop.
Demonstrating the procedure will be myself, postdocs Kirsten, SEL, and Jimmy Hu and ing Lee, a visiting faculty member here in the Klein laboratory. After euthanizing the animal, remove the skin from the lower lip to the neckline. Then use a scalpel to make an incision between the two lower incisors, which are loosely connected.
Upon applying pressure, the mandible will be split into two halves. Next, WeCh the scalpel between the condyle of the jaw and the temporal mandibular joint, and cut alongside the jaw muscle to detach the jaw after word. Remove all the muscles, tendons, and ligaments until only the bone remains.
Now carefully shave off the bone at a 45 degree angle from the proximal end to the distal end of the membranes region. Use the tip of the scalpel to remove any remaining bone, including the bone fragments at the edge. Then slowly start just below the third molar to remove the lingual cortical plate of the mandible.
Continue to clean. Working carefully around the area containing the cervical loop. Make a clean cut to first remove the bone and tissues proximal to the cl.
Then make a second cut distally with the scalpel roughly below the second molar. After that, remove the inferior alveolar nerve bundle. The proximal incisor region is then dissected out by inserting the scalpel between the bone and the medial surface of the incisor.
This must be done carefully so as not to tear the tissue. Then place the dissected tissue in 2%collagenase in one XPBS for three to four hours at four degrees Celsius in a low adherence plate for one to 10 incisors. Use 100 microliters per well.
In a 12 well plate, and for greater than 10 incisors, use 500 microliters per well in a six well low adherence plate. In this procedure, remove the CL region from the collagenase and place in cold D-M-E-M-F 12 media. Next, pull gently at the lower end of the CL using either forceps or an insulin syringe.
The mesenchyme will fall apart while the epithelium remains intact. Then the CL and the adjacent epithelium that extend laterally as a wing leg structure will be visible. Remove the apical bud from the adjacent epithelium by making a V-shaped incision on either side and place it in cold DMMF 12 on ice.
Immediately repeat for all CLS and collect them in a 1.5 milliliter micro fuge tube. Next, spin down the micro tube with the specimens. Remove the media and replace it with 100 microliters of cell detachment solution.
Then incubate the tissues in cell detachment solution for 30 minutes at 37 degrees Celsius. After word dissociate the tissues to produce a single cell suspension by gently pipetting up and down. Using a 1000 microliter low adhesion pipette tip count cells with a hemo cytometer subsequently plate the cells on tissue culture, plastic, or other desired material.
Now plate the cells in a six well plate at one to six times 10 of the fourth cells per milliliter. In DESC media Grow the cells plated at 37 degrees Celsius in 5%carbon dioxide. Do not disturb the initial cultures for five days after plating to allow for cell adhesion and colony formation for the first media change.
Replace 500 microliters of the old media with 500 microliters of new media. Check the colonies under a microscope when changing the media after the first media change change with one to two milliliters of media every two days and check the colonies under a microscope When ready to passage cells, aspirate the media and wash the cells three times with prewarm PBS. Then add the PREWARM 0.25%tripsin EDTA in saline solution and incubate at 37 degrees Celsius for 10 to 15 minutes for the DSCs to detach.
Next, add DESC media to neutralize the trypsin. Gently pipette along the wall of the tissue culture plate to collect the cells and place them in a 15 milliliter conical tube After word spin down the cells at 800 Gs for two minutes. Shown here is the visualization of the cervical loop.
Using a fluorescent reporter mouse, the bone is relatively autofluorescent. Removal of the bone exposes the cervical loop and the rest of the epithelium, which can then be easily excised. All structures of the cervical loop can be easily visualized with the reporter gene.
This figure shows the representative results of the successful colony formation in vitro. This image of DSCs is taken under the phase contrast microscope after plating and culture for seven days. Tight colony formation indicates successful epithelial isolation with no mesenchymal contamination.
After 10 days of incubation, colonies are larger in size, and the cells have a typical epithelial cobblestone morphology. After watching this video, you should have a good understanding of how to isolate and microdisect the cervical loop and culture dental epithelial stem cells from the adult mouse incisor.
