The goal of this procedure is to culture and electro operate mouse embryonic, cochlea exvivo. In order to study various aspects of cochlear development, including morphogenesis differentiation and growth events, this is accomplished by first obtaining the head from a day 13 embryonic mouse embryo. Next, expose the inner ears by removing the brain and isolate them from the developing temporal bone by detaching from the base of the skull.
Then remove the overlying cartilage and open the cochlear duct to obtain the sensory epithelium of the cochlear. The final step is to culture the dissected cochlear with or without electroporation for up to one week. Ultimately, the implants can be analyzed for live cell imaging or fixed immuno stain and analyzed by confocal imaging, The culturing of mouse embryonic.
Cochlear explan provides an excellent system to study various aspects of organogenesis, such as proliferation, sulfate specification, and polarity in the developing cochlea and how these events contribute to growth and differentiation. So this technique allows one to perform pharmacological manipulations and monitor gene functions ex vivo. And so this technique coupled with the gene transfer via electroporation, one could alter expression of gene of interest and monitor its function at a single cell level.
To begin this procedure, place an E 13 mouse embryo head in a sterile sogar dish containing cold dissecting solution. Under a dissecting microscope, immobilize the embryo head by placing minutiae pins around the eye region. Then using two pairs of sterile forceps, carefully remove the skin and open up the skull on the dorsal side along the midline.
After that, remove the brain from the cranial cavity. The inner ears located within the temporal bones can be identified from the lining of the blood vessel around them. Next, dissect the inner ears from the temporal bones by placing the forceps underneath the tissue and isolating the inner ear from the base of the skull.
Afterwards, transfer the dissected inner ears to a new dish containing cold dissecting solution. Now orient the inner ear so that the ventral side is facing up. Stabilize the inner ear by gently inserting the sharp end of the minuchin pins through the vestibular portion.
Using ultra fineing forceps, cut open the overlying cartilage by making an incision near the oval window. Make sure that the forceps are not inserted too deeply into the cartilage as overlying cartilage is sometimes fused with the cochlear duct. Then carefully remove the cartilage from the cochlear.
Next, expose the sensory epithelium by placing the forceps at the base or at the apex of the cochlear duct and gently pull the roof of the cochlear out. As a last step, carefully remove the underlying connective tissue from the exposed sensory epithelium so that the base of the cochlear explan is flat. After that, isolate the dissected cochlear from the vestibular portion of the inner ear.
Transfer the dissected cochlear sensory epithelium into the basement membrane matrix Coated culture well using a 1.5 millimeter sterile scooper if further cleaning is needed to remove extra tissue, this can be performed during this stage. Then orient the cochlear explan with the luminal surface of the epithelium facing up. Aspirate the basement membrane matrix DMEM solution to carefully flatten the tissue and gently add 150 microliters of fresh culture medium to the dish.
Make sure that each x explan a adheres well to the coated glass cover slip and is not floating in the culture medium. Next, gently transfer the micro dissected cochlear into a sterile 150 millimeter culture dish and place it into a tissue culture incubator. A 37 degrees Celsius with 5%carbon dioxide for three to six DIV.
After 1D IV.Examine the culture under a dissecting microscope to make sure that the cochlear explan has a adhere well to the culture dish. Then continue to incubate the culture in vivo for the desired time period before processing it for immunohistochemistry to set up for electroporation. First sterilize one 100 millimeter cigar coated glass dish by autoclaving or soaking in 70%ethanol for about 20 minutes.
Then allow it to air dry on a clean bench prior to use. Now prepare DNA from an expression vector of choice using an appropriate maxi or MIDI prep kit. The expression vectors used in this protocol are a H one expression construct and neuro D one expression construct.
And the final concentration of DNA should be at least one microgram per microliter in sterile DNA RNAs free water to electro operate DNA into the enic cochlear explan. Add 10 microliters of plasma DNA solution to a fresh 100 millimeter cigar coated dish. Then transfer a fully dissected cochlear explan into the DNA solution with the luminal surface of the epithelium facing up.
Tilt the cochlear slightly so that it is perpendicular to the plane of the dish. Next place the negative electrode paddle towards the sensory epithelium and the positive electrode Paddle next to the base of the cochlear using the electro ator. Deliver nine to 10 pulses of 24 millivolts 30 millisecond pulse duration.
Subsequently add 100 microliters of warm culture medium to the electro rated cochlear. Repeat the procedures for all the cochlear explan and for the DNA of all genes of interest before transferring the electro rated cochlea to the basement membrane matrix coated dish for plating. Then culture all the electro rated cochlea in a 37 degrees Celsius 5%carbon dioxide humidified incubator for desired time period, which will then be processed for immuno cyto chemistry.
These images show the embryonic day 13 cochlear implants from the wild type CD one mouse pubs, which are electroporated with a TH one, EEG FP or neuro D one EGFP Reporter constructs. They were immuno labeled with hair cell specific marker, anti myo seven A or neuronal marker two to one in red. The electro rated cells, which can be visualized by EGFP expression are seen throughout the greater epithelial ridge, lesser epithelial ridge and sensory epithelium cells.
The neuro D one transfected cochlear epithelial cells after five DIV acquired the neuronal phenotype with dendritic processes, most of which were positive for two one or a T oh H one. Transfected cells were positive for myo seven. A expression Once mastered, this technique can be done in three to four hours for the intel liter of eight to 10 pops.
This will allow ample time for dissection culture, electroporation, and plating of the explan.
