The overall goal of the following experiment is to follow tooth development over several days. In vitro explan culture, this is achieved by dissecting the developing mandible of fetal mice into live slices to expose the developing tooth germs as a second step. The slices are selected, placed on filters over a culture medium and incubated to allow development.
Next slices are labeled with a lineage tracer in order to track cellular movement during the culture period, results are obtained that show the dynamic nature of dental tissues as they develop during explan culture. The main advantage of this technique over conventional explant culture is that internally developing organs such as tooth gems can be observed throughout their development within the context of the surrounding tissue. Visual demonstration of this method is critical as the chopping and selection steps can be difficult if the setup of the machine or the orientation of the tissue is inaccurate.
First, prepare the dissection and culture medium for the embryonic organs by adding 1%glutamate and 1%penicillin streptomycin to 500 milliliters of advanced ECCOs modified eagle medium F 12. Then as described in the video article by Takahashi Sumi dissect mice embryos between stages E 11.5 and E 15.5 of development and place them into a Petri dish containing fresh medium. Next, decapitate the embryos using a freshly sharpened sterile tungsten needle and transfer the heads into a fresh dish of pre chilled medium on ice.
Then isolate the mandibles from the heads by placing a needle into the oral cavity and cutting towards the back of the head temporarily. Store the isolated mandibles in pre chilled medium on ice prior to being sectioned. Prior to slicing the mandible tissue, clean the mounting disc and blade of the tissue chopper with 70%ethanol, and make sure the blade lies flush with the mounting disc.
When it falls, the blade will need to be replaced after slicing approximately 200 mandibles, set the cutting distance to between 200 and 400 micrometers according to the age of the specimen, and depending on whether the whole tooth germ is required for an E 14.5 embryo where isolation of the tooth germ is required, a cutting distance of 250 micrometers is used. Next, transfer a mandible to the plastic mounting disc and arrange it so that the developing tongue is in a frontal transverse orientation. When dissecting molars to isolate incisors, take sagittal slices through the mandible.
Once correctly oriented, remove the excess medium from around the tissue. Using filter paper, this will slightly drive a mandible fixing it into place on the mounting disc. Immediately after drying with the filter paper, place the disc on the circular stainless steel table of the chopper.
Then set the blade force on maximum, turn the machine on and press start. The table traverses automatically from left to right while at the same time the chopping arm carrying the blade is raised and dropped at up to 200 strokes per minute. Once the tissue has been completely sliced, turn off the machine.
Make sure the blade arm is in the raised position, and then remove the disc. Be sure not to place fingers under the blade arm or leave the machine unattended. When chopping tissue, take the mounting disc and immediately add medium to the slices on the disc in order to prevent excessive drying.
Then carefully dislodge the slice tissue from the bottom of the disc using a tungsten needle or pipette and pipette them into a small Petri dish of medium. Next, separate the individual slices using a needle and then select the tissue slices containing the area of interest under a stereo microscope. Place selected slices in culture dishes with fresh, medium, prepare metal grids for the culture system by cutting strips, measuring four centimeters by 1.5 centimeters from sheets of stainless steel plain weave.
Then punch a hole in the center using a standard stationary hole punch and bend the sides so that the grid fits flat across the central well and lies parallel to the bottom of the dish. Once the grids are shaped correctly, sterilize them in an autoclave before use. Next, prepare the central well organ culture dishes for culture by adding two milliliters of autoclave water to the outer well to prevent dehydration.
Then position a sterile metal grid across the center of the dish. Cut a piece of transparent polyethylene terephthalate membrane with a pore size of 0.4 micrometers so that it is large enough to cover the hole in the grid. Then place the cut membrane into the dish with a selected mandible slice if lineage tracing is desired.
First prepare a glass capillary needle by drawing the glass with a needle puller. Then break the tip of the needle and place it tip down into a lipophilic dye solution such as dai eye or dai o, and draw up some of the dye solution by capillary action. After a few minutes, place the filled needle into a holder and attach it to a mouth Aspirator.
Position the tip of the needle in the culture medium so that it touches the area to be labeled, such as the dental follicle shown here. Then displace a small amount of dye onto the tissue. Next, transfer the slice onto a fluorescent microscope stage and check that the slice is successfully labeled.
Once successfully labeled carefully lift the slice on top of the membrane and then transfer the membrane out of the medium with the slice flattened in the center. Then place the filter over the circular hole in the center of the grid at approximately one milliliter of culture medium to the well until the level of medium is at the level of the membrane. At this point, add any proteins or small molecule inhibitors into the medium.
Also, image the slice for a record of the morphology at day zero using a dissecting microscope for the best photography, view the slices with a light source from underneath. Maintain the dishes in a humidified atmosphere of 5%carbon dioxide at 37 degrees Celsius, and change the media every two to three days at regular intervals. Continue to image the cultures in order to track progress.
Shown here is a series of images documenting the development of the first molar dental follicle, which is labeled with dye eye fluorescent stain. The slice is a 250 micron thick frontal slice dissected from the mandible of a mouse at E 14.5. The tooth which is outlined with black dots begins in the cap stage at day zero.
By day four, the tooth germ has reached the bell stage and the dental follicle cells are seen spreading out in an arc around the developing outer enamel. Epithelium Once mastered, this technique can be done in three hours from embryo to final culture. After watching this video, you should have a good understanding of how to successfully visualize the development of organs in culture.
