Hello, I'm Mark Parker. Welcome to the Eaton Peabody Laboratory at the Massachusetts Eye and Ear Infirmary in Boston, Massachusetts. Today we'll present a protocol for the isolation and primary culture of the marine organ of kti.
This protocol is fundamental in our work because it allows for the in vitro analysis of the organ essential for audition. To highlight the utility of this approach, we'll also demonstrate one of the many uses of the organs in our laboratory, the electroporation of exogenous genes into the cultured explants. Let's get to work.
The outline of this procedure is as follows, neonatal mice will be decapitated and their temporal bones removed using blunt dissection. Next, the cochlea will be isolated from the temporal bone using blunt dissection. The bony labyrinth of the cochlea will then be opened using forceps and the spiral ligament will be removed from the inner modis of the cochlea.
Finally, the spiral ligament will be separated from the organ of corde using forceps, translucent roses of hair cells can be seen running along the length of the organ of kti. A portion of the RISE's membrane, which has been ruptured during the removal of the spiral ligament, can be seen running the length of the modular side. As an example of the utility of this procedure, organ corde explants will be cultured for 24 hours and then be electroporated with an exogenous fluorescent reporter gene.
The initial steps of this procedure involve the preparation of the culture plates. Place a sterilized glass cover slip that has been stored in 70%ethanol into two wells of a pre sterilized four ring cell culture dish. Using only two of the four wells will limit the amount of time the cultures are out of the incubator tote.
The cover slips with the solution consisting of one-to-one poly l ornithine and laminin. Supplemented with 20%fetal bovine serum, placed the culture dish inside a larger 10 centimeter dish in order to facilitate its transfer and then incubate overnight at 37 degrees Celsius. Finally, heat sterilize that is section tools in a 150 degrees Celsius incubator overnight.
Sterilize the positive flow dissection hood by turning on the UV light for 20 minutes prior to the start of the dissection. Sterilize the working space within the dissection hood by spraying all the surfaces with 70%ethanol. Place the head of a decapitated mouse pup in a Petri dish containing 70%ethanol, and then remove the epidermis using a scalpel blade.
Open the cranium along the sagittal suture using a scalpel blade and then bisect the for brain. Retain the coddle forebrain for further dissection. Remove the forebrain, cerebellum and brainstem using blunt dissection.
Note the location of the parametal shaped temporal bone on the inferior lateral side of the cranium. Transfer the TAL bones to a three millimeter dish containing sterile hanks ball salt solution. The remaining dissection is to be conducted under a dissection microscope.
Remove any surrounding tissue from the temporal bone. Locate the conch shaped cochlea and remove it from the vestibular system using forceps. At this stage of development, the bony labyrinth is not completely calcified and is easily dissected.
Remove the bony labyrinth of the cochlea by careful separation starting at the basal end and moving apically. Using forceps carefully remove the spiral ligament and attached sensory epithelium from the modis by securing it at the hook region of the base using forceps and unwinding it. As you move aply Starting at the base, remove the spiral ligament from the sensory epithelium using number 55 fine forceps.
In some of our experiments, the removal of the spiral limbus from the sensory epithelium is desired. In this micro ol isolate procedure, the organ of cordi is isolated from the spiral limbus using two one half cc insulin syringes as forceps. First, the hook region of the organ corde is removed using the insulin syringes.
Next, the spiral limbus is separated from the row of inner hair cells starting at the apex and proceeding basally either the whole organ of Corte with attached spiral limbus. The micro isolated sensory epithelium or the micro isolated spiral limbus can be cultured as follows, remove the coating solution from the culture wells and replace it with 130 microliters of culture media containing 10%serum transfer, the dissected organ of corde to the coated glass cover slip in the culture. Well, we have found that the following techniques help to ensure that the organ of corti does not float in the culture media but remains affixed to the cover slip.
First coat, the glass cover slips as described. Second, remove the media to affix the explan to the coated dish. This will ensure contact between the culture dish and the X explan.
Third, orient the explan so that the cilia of the hair cells face up. This orientation will facilitate the adherence of the explan to the cultured dish. Lastly, add 130 microliters of serum culture media using a 200 microliter pipette by carefully dripping two drops onto the surface of the organ accord, and then slowly adding the remaining volume to the side of the well carefully move the explan to an incubator and then incubate a 37 degrees Celsius in the presence of 5%carbon dioxide.
These cultures can be maintained for seven to 10 days. There are numerous applications for the isolated organ of core T.Some examples include the analysis of gene expression using R-T-P-C-R or NC two hybridization, organ of cortico culture with spiral ganglion cells or exogenous stem cells or the in vitro analysis of hair cell death. To demonstrate the utility of this procedure, we will show you one technique commonly used in our laboratory to express exogenous genes in the explan cultures.
For this demonstration, the isolated organ of corti will be electro parade with an expression vector that constitutively expresses the DS red reporter gene. After electroporation, the cultures will be incubated with a few gene six transfection reagent for 24 hours to enhance the transfection procedure. Using this system, the transfected cells will exhibit a cytoplasmic red fluorescence within 24 hours.
First, prepare a three to two ratio of FU gene six transfection reagent to target DNA in a three milliliter round bottom polystyrene test tube. In a laminar flow hood, remove the culture media from the organ of horticulture. Add 130 microliters of water for one minute and then remove using a 200 microliter capacity pipette.
Next, add 30 microliters of DS red reporter plasmid, which is stored in a stock solution of two milligrams of plasmid DNA per milliliter of water. For electroporation, we use a BioRad gene Pulser Excel to generate the electrical pulse. The electrodes had been customized by our machine shop to meet our purposes, the electrodes consist of bare wire that are bent at a 90 degree angle and have been crimped into a paddle shape.
Advance the electrodes of the electro using a micro manipulator so that the anode and cathode are on either side of the culture. Generate 10 pulse trains consisting of 27 volts and 30 millisecond duration to electro purate the reporter gene into the organ accord explan culture. Then wait five minutes.
Next, add 130 microliters of the FU gene six DNA solution to the explan, and then incubate at 37 degrees Celsius in the presence of 5%carbon dioxide overnight. The next day add two milliliters of serum to culture, media to each plate. The explan cultures can be maintained for six or more days after 24 hours.
Transfected cells can be viewed using a SI three filter on an epi fluorescent microscope. We've just shown you a method for the isolation and primary culture of the organ Corte. This method works with mice as young as embryonic day 16 up to postnatal day, at which point the cochlea becomes sufficiently calcified to render the dissection cumbersome.
In addition, we've demonstrated a method for the expression of exogenous genes into the cultured organ of corde. This is an example of one type of experiment that can be conducted on the cultured explan. No doubt there are a multitude of uses for this technique.
So that's it. Thanks for watching and good luck with your experiments.
