The overall goal of this procedure is to image an embryonic cochlea taken from a fluorescent reporter mouse at 30 minute intervals over five days. This is accomplished by first harvesting embryonic day 13 cochlea from fluorescent reporter mice. Next, the cochlea are cultured in vitro.
Then the cochlear explan cultures are placed in an incubator microscope. Finally, an automated imaging routine is set up and the cochlea E are imaged. Ultimately, long-term time lapse imaging of cochlear explan cultures can be used to show morphogenetic movements of the cochlear epithelium as a whole, as well as changes in reporter gene expression over time, and the effects of slow acting pharmacological agents.
The main advantage of this technique over existing methods, like manually photographing X fund cultures over a time course is that this system is entirely automated. It allows more time points to be collected, and it ensures that the same region of the cochlear or set of cells is imaged at the same orientation at every time point, all while growing in irregular tissue culture incubator. After supplementing KO's modified eagle medium or DMEM and Hank's balance salt solution or HBSS according to the accompanying text protocol.
Under a laminar flow hood, use five milliliters of unsu supplemented DMEM to resuspend, 200 microliters of basement membrane substrate pipette 150 microliters into the center of each 10 millimeter well of a 35 millimeter diameter glass bottomed culture dish. Incubate for 40 minutes in a CO2 incubator at 35 degrees Celsius. To prepare the workstation and tools, turn on the laminar flow clean bench, and use 70%ethanol to spray it down and to soak forceps, spoons, and a black silicone coated dish.
In the clean workstation, harvest embryos of the appropriate gestation in ice. Cold HBSS supplemented with 1%heaps. Using a fluorescent stereo microscope, identify embryos that exhibit reporter activity externally or within a visible organ and transfer them to fresh ice cold HBSS with 1%heaps for collecting temporal bones, using a cool light source and fine forceps.
Work quickly to clip at the cervical vertebrae and below the jaw to collect the heads. After collecting the embryo heads according to the text protocol, carefully open the skull, then remove the brain trim off the front of the head and transfer the posterior skull to a dish with fresh ice.Cold. HBSS heaps, carefully dissect out the peanut shaped temporal bones, taking care to keep the vestibular system intact.
To dissect the cochlea, transfer the temporal bones to a black silicone elastomer coated dish in ice cold HBSS heaps. Pin the vestibular region of the bone by inserting pins at an oblique angle in order to stabilize the temporal bone and create room for the forceps. Next carefully remove the cartilage surrounding the cochlea by inserting one tie of the forceps into the cartilage at the outer edge of the base of the cochlea, and clipping a hole in the cartilage.
Clip horizontally across the top and diagonally and carefully lift off the front section of the capsule. Insert a prong of the forceps in between the remaining cartilage and the duct, and gently clip off the remaining cartilage. The apical surface of the cochlea is now exposed.
Starting at the base, catch the area where the roof of the duct meets the cochlear epithelium and open the cochlear duct. Gently peel off the roof trimming when necessary until it is completely removed. Trim off any portions of membrane left on the medial side of the duct.
Then clean the duct of excess mesenchymal tissue and detach the duct from the vestibular system to culture implants. Place one luminal surface up in the center of a substrate coated glass bottom culture dish. Carefully draw off the liquid and leave for two minutes.
Then dropwise. Add 150 microliters of supplemented DMEM to the X explan, taking care not to disturb it so that it can settle to the substrate. Place the glass bottom dishes in a deep 12 centimeter diameter Petri dish with a small dish of sterile water to maintain humidity.
Put the cultures into a 35 degrees Celsius incubator overnight in order for the explan to attach to the substrate and flatten to perform live imaging. After selecting samples, use a fluorescent stereo microscope to evaluate the condition of each explanted cochlea, and select only explants where the duct is intact and attach to the glass from the base to apex. Set the incubator at 35 degrees Celsius with 5%CO2 to culture.
Cochlear tissue gently aspirate the supplemented DMEM from the cultures and replace it with at least 500 microliters a fresh medium. In cases where explan are loosely attached, pipette a ring of medium around the edge of the dish. This extra liquid will make a miniature humidified chamber without disturbing the explan while it continues to attach to the matrix.
The microscope in this example has a rotating platform that holds 8 35 millimeter sample dishes under the laminar flow hood. Replace the plastic lids with glass lids and insert the dishes into the sample dish holder. If it is necessary to change the media during the course of the experiment.
To avoid disturbing the explan, use hinged dish covers that will allow opening of the lids while the dish stays in its fitting in the microscope, place the sample dish holder inside the incubator microscope, taking care not to dislodge the explan from the bottom of the dish or to disturb the medium. To set up the imaging routine, switch on the microscope UV lamp and camera, and open the imaging software. For each sample located, pick an imaging area plane of focus and exposure, keeping in mind not only the view of the explan at the time of starting, but also how the tissue will move during the course of the run.
In the fluorescence channel, set a Z stack centering around the selected focal plane. Then set the frequency and length of the sampling for the experiment. This will be limited by the time it takes to collect images to generate a movie at the end of a time lapse period.
Use a software such as METAMORPH to open the image files for a sample frame by frame. Pick the best focal plane for showing the cell population of interest. Finally, convert the images to a dot a VI file, or export them as a montage to generate a set of images that can be opened and analyzed in image processing software or converted to other formats.
Shown here is a movie demonstrating how an organotypic cochlear explan will grow if plated. On E 13.5 a SOX two reporter mouse was used to visualize the pros sensory region as seen in green as the cells of the nons sensory regions divide and move. The non-proliferative sensory region in green undergoes tissue rearrangements and convergent extension movements.
A second time lapse experiment centering on the mid base of a different cochlear explan demonstrates that as it extends the pros. Sensory region narrows when first in culture. The tissue thickness makes it possible to identify regions of expression but not individual cells.
However, after three days in culture, the tissue has flattened considerably, making it possible to visualize individual fluorescent cells Following this procedure. Other methods like immunofluorescent staining or protein and RNA extraction can be performed in order to evaluate changes in gene expression at the molecular level. After watching this video, you should have a good understanding of how to dissect and culture cochlea and how to use an incubator microscope to perform live imaging on whole explan over a period of days.
