内耳組織工学用マウス蝸牛を Decellularizing するためのプロトコル

The overall goal of this procedure is to demonstrate an effective method to decellularize and decalcify mouse cochleae to use a scaffold for inner ear tissue engineering applications. This method can help answer key questions in the auditory and tissue engineering fields such as, how do 3D cochlea and various components of the extracellular matrix influence stem cell differentiation? The main advantage of this technique is that it enables the researcher to study how cells directly interact with the extracellular matrix of the cochlea to form sensory epithelium.

Visual demonstration of this method is critical as the temporal bone isolation and cell profusion apparatus can be difficult to visualize correctly based solely on text descriptions. To begin this procedure bisect the skull in the midsagittal plane using a sharp pair of surgical scissors. Next, remove the brain tissue from the skull.

Then identify the temporal bone through the presence of the auditory and vestibular nerve roots on the interior of the skull and the ear canal from the exterior. Carefully cut through the skull to isolate the temporal bone from the remainder of the skull. Afterward, insert the fine forceps into the opening of the ear canal approximately five millimeters so the tips do not risk puncturing the cochlea.

Gently break open the bone of the bola so it fractures. Using fine forceps, pry away the remainder of the bola from the temporal bone exposing the bony labyrinth of the cochlea. Subsequently, submerge the temporal bone in PBS with the oval and round windows of the cochlea facing up.

Using an ultra-fine pair of forceps remove the stapedial artery which passes through the stapes. Then insert one tip of the ultra-fine forceps through the arch of the stapes where the artery pass through and delicately lift the stapes upward. Following that, puncture the oval and round windows.

Afterward, position the tubing which is connected to a PBS-filled 28.5 gauge syringe over the oval window. Profuse two millimeters of 10%Antibiotic-Atimycotic and 10%Penicillin-Streptomycin in PBS through the cochlea over five minutes to remove the perilymph. Then remove and discard any remaining muscle tissue and bone fragments.

For decellularization, using the transfer pipet gently draw the cochlea up into the pipet so it just passes the cut off edge by a few millimeters. Next, expel the cochlea into the 1%SDS solution-filled scintillation vial. Circulate two milliliters of 1%SDS in de-ionized water through the cochlea in the scintillation vial using a transfer pipet.

Place the scintillation vial into a rotator and allow it to rotate at 10RPM for 72 hours at room temperature. Replace with fresh, 1%SDS solution every 24 hours over a 72-hour period and be careful not to expose the cochlea to air during the solution replacement. After that, wash the cochlea three times in the same scintillation vial for 30 minutes each in DI water.

At this stage, the cochlea should be decellularized. For decalcification, remove the remaining DI water from the scintillation vial leaving just enough to keep the cochlea submerged. To begin decalcification, add five milliliters of 10%EDTA and DI water to the scintillation vial containing the cochlea.

Circulate two milliliters of 10%EDTA in de-ionized water through the cochlea in the scintillation vial using a transfer pipet. Place the scintillation vial back in the rotator and allow the scintillation vial to rotate at 10RPM for 72 hours at room temperature. Change the 10%EDTA solution every 24 hours over a 72-hour period.

Take care when changing solutions so that the cochlea is not exposed to the air. Then rinse the cochlea three times for two hours each with PBS. At this stage, the cochlea should be decalcified.

Before infusing cells, begin by incubating decellurized and decalcified cochlea in a 5%Carbon Dioxide, 37 degrees Celsius cell culture incubator for a minimum of one hour. To profuse the cochleae, next add a drop of MSCGM to each cochlea to prevent any cochlea from drying out. Transfer them to a new 24-well plate using a transfer pipet.

Delicately orient the cochlea using ultra-fine forceps so that the oval and round windows are facing up. Then using a sterile 28.5 gauge Insulin syringe with connected tubing, draw up 0.2 milliliters of re-suspended Human Wharton's jelly cells. Subsequently, position tubing over the oval window using sterilized fine forceps.

Delicately and slowly profuse the cochlea with 0.2 milliliters of re-suspended Human Wharton's jelly cells over five minutes. This profusion step is important. Successful profusion of fluids through the cochlea greatly impacts the outcome of this protocol.

After profusion, add 0.8 milliliters of 37 degree Celsius pre-warmed MSCGM to the well containing cochlea to bring the total volume to one milliliter. Repeat the procedure for each cochlea. Using a fresh syringe and tubing each time.

Place the profused cochlea in a 37 degree Celsius cell culture incubator and change the media three times per week. Shown here is the successful decellularization and decalcification of a mouse cochlea without any injected cells. Overall, cochlea anatomy is preserved with voids left with no dappy staining.

Here are the representative results of successfully cultured Human Wharton's jelly cells on a cochlea scaffold. Abundant dappy staining is observed throughout the cochlea and on the outer bony surface. Closer examination of individual turns reveals that fine cochlea structures are preserved following decellularization and decalcification and that injected cells are attached to and or interacting with the native cochlea extra-cellular matrix.

Decellularization in subsequent cell culture were also investigated using H&E staining. The decellularized cochlea contained none of the cells of the native cochlea but retains most of the anatomy of the organ of Corti because the extra-cellular matrix remains. Injected in cultured cells can be observed through the organ of Corti, interacting with the decellularized extra-cellular matrix.

Once mastered, the profusion of cells into a decellularized cochlea can take approximately 10 to 15 minutes if the cochlea is already prepared and the required tools are laid out appropriately. While attempting this procedure it is important to handle the cochlea with care as it is fragile. It is also important to avoid the introduction of air into the cochlea as bubbles will block the flow of fluids during profusions.

This general procedure can be applied to cochleae of other species and a wide array of cell types can be injected as well. Specific decellularization and decalcification times will likely vary but the underlying concepts will remain the same. Dr.Peter Santi at the University of Minnesota initially developed the decellularization technique that we modified for this protocol.

We hope to make our technique and cell culture on cochlea scaffold available to other tissue engineers. After watching this video, you should have a good understanding of how to successfully isolate, decellularize and decalcify temporal bones prior to injecting cells for the use in culture experiments.