This protocol allows investigators in the craniofacial field to quantitatively compare collective movement attributes in control and mutant cells as a means to understand mesenchymal remodeling during palatal shelf elevation. The use of primary mouse embryonic palatal mesenchymal cells and time-lapse imaging provide an accessible proxy method for assessing palatal shelf elevation dynamics. To harvest palatal shells from a mouse embryo, use a sterilized perforated spoon to place an embryonic day 13.5 embryo into a sterile 10 centimeter dish filled with sterile MEPM culture medium under a stereo microscope.
After decapitation, insert one tip of a sterilized fine No.5 forceps into the mouth just inside the cheek and push the tip of the forceps until it exits the back of the skull. Orient the forceps so that the other arm hovers just over the ear canal before pinching the forceps shut to cut the tissue. Repeat the incision on the other side of the head, continuing the pinch cut procedure until the lower jaw, tongue, and inferior portion of the skull have been removed, exposing the palatal shelves.
With the head placed on its side, position the tips of a pair of small, sterile, stainless steel scissors in front of and behind the skull just about the eye level of the embryo and cut just above the eyes to remove the cranium of the skull, creating a flat surface. Next, position the head upside down with the superior aspect resting flat on the bottom of the dish and locate the palatal shelves, which should be visible as two raised bridges on either side of the central groove in the anterior half of the head. To secure the head to the dish, insert one tip of a fine forceps through the tissue near the nasal region of the head anterior to the palatal shelves, and the other through the base of the skull posterior to the palatal shelves.
Insert both points of a second pair of very sharp, fine forceps into the tissue at the base of the lateral surface of the shelf and slowly and carefully pinch to cut the tissue. Repeat the incision along the base of the medial surface of the shelf and at both the anterior and posterior ends of the shelf to detach the shelf from its attachment to the head, then gently lift the shelf, making additional pinches as necessary to completely free the shell from the surrounding tissue. When the second palatal shelf has been removed in the same manner, use a sterile plastic bulb transfer pipette to transfer the shelves into a sterile 1.5 milliliter microcentrifuge tube in approximately 500 microliters of PBS on ice.
To set up an MEPM cell culture, when all of the shelves have been collected, aspirate the PBS without disturbing the tissues and immediately add 200 microliters of 37 degree Celsius 0.25%trypsin to each tube of palatal shelf tissue. Use a 1, 000 microliter pipette to briefly pipette the tissues a few times and incubate the tissues for 10 minutes at 37 degrees Celsius, pipetting briefly after five minutes. At the end of the incubation, pipette the tissues again before adding 800 microliters of MEPM culture medium to each tube and centrifuging the tubes to collect the cells.
After aspirating the supernatant, resuspend the pellets in one milliliter of fresh MEPM culture medium per tube, and plate the cells into individual wells of a six-well tissue culture-treated plate containing two milliliters per well to a final total of three milliliters per well, then allow the cells to adhere to the plastic surface for 12 hours in a sterile cell culture incubator. To set up a 2D collective migration assay, remove the top of one sterile two-well silicone insert to a height of approximately one millimeter for each sample to be analyzed and use sterile forceps to plate the shortened, sterile two-well inserts into the center of individual wells of a six-well plate. Press down along all of the edges to ensure that the inserts are fully adhered to the well bottoms and seed 300 MEPM cells per square millimeter of the shortened silicone inserts in a total volume of 40 to 50 microliters of MEPM culture medium into each insert for an overnight incubation in the cell culture incubator.
To set up a wound repair assay, use sterile forceps to place one unmodified sterile two-well silicone insert into the center of one well of a six-well plate per sample and firmly press the edges of the insert to attach the insert to the culture plate. Then seed 1, 400 cells per square millimeter in 100 microliters of MEPM culture medium into each insert and incubate the cells for 48 hours in the cell culture medium. When performing a 2D collective migration assay, seeding the cells in two-well silicone inserts in a large culture dish typically provides better cell density for imaging.
Small 3D-printed rings placed in a 35 millimeter dish can also be used for 2D motility analysis. For wound repair assays, the cells are grown in two-well silicone inserts until high confluence. The inserts are then removed and the wound is imaged until closure.
MEPM trajectories are persistent and the direction of the cell motility is maintained for several hours. The mean displacement versus time analysis indicates that the persistence in the form of displacement is proportional to the elapsed time. Flow analysis of the motility data reveals that the co-moving MEPM cell clusters are approximately 300 microns in size.
A profound motility difference between wild type and mutant MEPM cells is also observed. This procedure could be used on various transgenic mouse lines and to treat MEPM cells with various biochemical reagents to study their effects on cell movement. We have focused on primary palatal mesenchyme cells, but the time-lapse imaging and quantitative analyses can also be used to explore the migration attributes of any motile cell type in dynamic developmental processes.
