isolating vestibular ganglion neurons from a mouse inner ear

Isolating Vestibular Ganglion Neurons From a Mouse Inner Ear

Scoop out the brain of the euthanized mouse using closed surgical scissors. Sever and remove the cranial nerve to completely detach the brain from the skull. Starting at the back of the head, pull the clear membranous material off with forceps, and then cut the top of the skull out using surgical scissors.
Remove the excess tissue from the back of the head and neck, to make the dissection area and otic capsule, cleaner and easier to access. Transfer the tissue to a second Petri dish with fresh L-15 solution. Then, locate the otic capsule and the auditory, superior vestibular, and inferior vestibular nerves. Separate the superior and inferior ganglia using small spring scissors, and cut away the auditory nerve.
Using a scalpel, gently shave off the bony ridge to weaken the bony area under which the nerve dives into the bony capsule. Carefully remove the debris with a scalpel, exposing the entire swollen portion of the ganglion.
Use the fine spring scissors to cut and separate the ganglion from the peripheral nerve branch that is diving toward the utricle. Remove the superior ganglion using fine forceps, and transfer it to a 35-millimeter Petri dish with fresh L-15 solution.
After preheating the enzyme solution, pour the enzyme mixture into a 35-millimeter Petri dish, and place it in a 37 degrees Celsius incubator for 10 to 15 minutes. Clean the ganglion using fine forceps and small spring scissors by removing bone, excess tissue, nerve fibers, and any other superfluous structures. Take care to minimize the removal of any ganglionic tissue.
Transfer the cleaned ganglia into the preheated enzyme solution, and place it back in the incubator for 10 to 40 minutes. Then, transfer the ganglia to the 35-millimeter Petri dish with fresh L-15 solution.
After two to three minutes, transfer the ganglia to another 35-millimeter Petri dish filled with filtered culture media. Transfer approximately 150 microliters of filtered culture media onto a coated glass bottom dish. Then, transfer the desired number of ganglia to the coverslip.
Draw a small amount of culture media from the culture dish, and rinse the trituration pipette with medium to prevent the tissue from sticking to the sides of the glass pipette. Triturate by gently and repeatedly passing the tissue through the pipette until the ganglia are sufficiently dissociated.
After five minutes, check under a light microscope to see if the cells have settled on the coverslip. Carefully place a culture dish into a 37 degrees Celsius incubator for 12 to 24 hours.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparations
NOTE: The solutions and tools described in this section can be made well ahead of time and used on the day of dissection and recording.
<ol>
	<li>Prepare Liebovitz media supplemented with 10 mM HEPES (L-15 solution). The following steps describe making 1 L of L-15 solution.
	<ol>
		<li>Pour 990 mL of deionized H2O into one beaker. Measure and add 2.386 g (10 mM) of HEPES. Add one bottle of 1 L of L-15 solution powder. 
		&#8203;NOTE: Powdered L-15 has a longer shelf life and takes up less storage space. Alternatively, purchase L-15 solution and supplement with HEPES. The latter option also has the option of using a phenol-free solution, which is useful for fluorescence imaging.</li>
		<li>Stir the solution on a stir plate. Using a pH meter, slowly add 1 N sodium hydroxide (NaOH) to obtain a pH between 7.34 and 7.36.</li>
		<li>Add deionized H2O until the solution reaches a volume of 1,000 mL. Filter the solution using a 0.22 &#181;m pore size membrane. 
		NOTE: L-15 solution can be stored at 4 &#176;C for 1-2 weeks. If using L-15 made with phenol-red, the solution will turn pink if too basic (pH &#62; 7.4) or orange if too acidic (pH &#60; 7.34).</li>
	</ol>
	</li>
	<li>Prepare the culture medium for vestibular ganglion neurons (VGNs). Follow the steps below to make 50 mL of culture medium:
	<ol>
		<li>Add 47.5 mL of GluMAX-I minimum essential medium to a clean glass beaker.</li>
		<li>Measure and add 0.1194 g (10 mM) of HEPES. This additional buffer resists pH changes while the cells settle before being moved to the incubator.</li>
		<li>Add 2.5 mL of fetal bovine serum (FBS). This makes 5% by volume FBS in a total volume of 50 mL of medium.</li>
		<li>Stir the solution on a stir plate. Using a pH meter, slowly add 1 N NaOH to obtain a pH between 7.38 and 7.4.</li>
		<li>Add 0.5 mL of penicillin-streptomycin. Filter the solution through a 0.22 &#181;m sterile filter. Store the solution at 4 &#176;C.</li>
		<li>At least 30 min before use, transfer the culture media to a tissue culture flask with a vented cap. Place the flask with culture media in an incubator with 5% CO2&#160;concentration at 37 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Fabricating trituration pipettes
NOTE: Trituration pipettes can be reused for multiple experiments. Flush the pipettes with ethanol, water, and L-15 solution before each use and clean them with water and ethanol after each use. Carefully store between uses.
<ol>
	<li>To prepare trituration pipettes for cell dissociation, hold a glass Pasteur pipette to the flame of a Bunsen burner. Once the glass tip begins to melt, stretch the glass out to the desired tip diameter. Pull one end of the pipette away from the flame to create a small bend.</li>
	<li>Bring the bent pipette near to a microscope. Using a scoring tile, score and break the glass at the desired diameter.</li>
	<li>Pass the tip of the pipette over the top of the Bunsen burner flame. This will quickly polish the rough edges near the tip. Check to ensure that the tip is not sealed by the flame.</li>
	<li>Repeat the process until four or five pipettes have been prepared with varying sizes.</li>
</ol>
3. Extraction of vestibular ganglion and plating of vestibular neurons
<ol>
	<li>Preparation for dissection
	<ol>
		<li>Prepare an enzyme mixture of L-15 solution with 0.05% collagenase and 0.25% trypsin. For example, add 0.001 g of collagenase and 0.005 g of trypsin to 2 mL of L-15 solution into a beaker, add a small magnetic stirrer, and stir until fully mixed. Set aside at room temperature.</li>
		<li>Prepare a commercially obtained guillotine by placing paper towels to the side and underneath the guillotine to minimize exposure of the benchtop and other surfaces to blood and other biological materials.</li>
		<li>Lay out large stainless-steel scissors, large forceps, and large spring scissors. Keep a large spray bottle with 70% ethanol handy for head cleaning.</li>
		<li>Fill a 100 mL beaker with L-15 solution and place it on ice. Oxygenate the L-15 solution.</li>
		<li>Lay out the spring scissors, surgical scissors, forceps, scalpel, and transfer pipette (see&#160;Table of Materials).</li>
		<li>Prepare a 60 mm Petri dish (for the gross dissection) and two 35 mm Petri dishes (for the cleaning/fine dissection of the ganglia). Fill the 60 mm&#160;dish with oxygenated L-15 solution using a 30 mL syringe with a 0.22 &#181;m filter tip.</li>
	</ol>
	</li>
	<li>Euthanasia, head-cleaning, and hemisection
	<ol>
		<li>Weigh the pups to compute the appropriate dosage of fatal-plus dilution to administer intraperitoneally (~0.01 mL per 10 g).</li>
		<li>Once a deep plane of anesthesia is reached, as assessed by a lack of response to the toe pinch, decapitate using the guillotine.</li>
		<li>Rinse the head thoroughly with 70% ethanol and then thoroughly with L-15 solution.</li>
		<li>Completely remove the skin from the skull. Bisect the head using large spring scissors by starting at the entry point of the spinal cord to the brain and making two cuts, the first cut through the top of the skull and the second through the bottom of the jaw. Finish bisecting the head using surgical scissors.</li>
		<li>Place both halves of the head brain side down in a 60 mm Petri dish filled with L-15 solution. Place the fresh tissue not currently being dissected on ice.</li>
	</ol>
	</li>
	<li>Extracting the superior vestibular ganglion
	<ol>
		<li>Scoop out the brain using closed surgical scissors. Sever and remove the cranial nerve to completely detach the brain from the skull. 
		NOTE: At this point, the otic capsule (shaped like the number 8) should be visible at the back of the head.</li>
		<li>Using forceps and starting at the back of the head, pull clear membranous material off.</li>
		<li>Cut the top of the skull out using surgical scissors. Remove excess tissue from the back of the head and neck to make the dissection area and otic capsule cleaner and easier to access. 
		NOTE: Avoid making the dish too cloudy by discarding excess tissue throughout the procedure.</li>
		<li>Transfer the tissue to a second Petri dish with fresh L-15 solution. Locate the otic capsule and the auditory, superior vestibular, and inferior vestibular nerves. Cut away the auditory nerve and separate the superior and inferior ganglia using small spring scissors. 
		NOTE: Although the somata of the vestibular nerve are found in both the inferior (thinner) and superior (thicker) ganglia, cells extracted from the superior ganglion have better survival.</li>
		<li>Using a scalpel, gently shave off the bony ridge to weaken the bony area under which the nerve dives into the bony capsule. Carefully remove debris with fine forceps, exposing the entire swollen portion of the ganglion.</li>
		<li>Use the fine spring scissors to cut and separate the ganglion from the peripheral nerve branch that is diving toward the utricle.</li>
		<li>Remove the superior ganglion using fine forceps, making sure not to pinch too close to the ganglionic tissue. Transfer to a 35 mm Petri dish with fresh L-15 solution.</li>
		<li>Before proceeding, pre-heat the enzyme solution: pour the enzyme mixture into a 35 mm Petri dish and place in a 37 &#176;C incubator for 10-15 min.</li>
		<li>Clean the ganglion using fine forceps and small spring scissors by removing bone (which appears white and crystallized in structure), excess tissue, nerve fibers, and any other superfluous structures. Take care to minimize the removal of any ganglionic tissue.</li>
	</ol>
	</li>
	<li>Tissue dissociation and plating
	<ol>
		<li>Transfer cleaned ganglia into the pre-heated enzyme solution and place it back in the incubator for 10 to 40 min. Enzyme treatment is complete once the tissue starts to break apart but remains in one piece. Overtreating the tissue with enzymes will result in complete dissolution of the ganglia before trituration. 
		&#8203;NOTE: The amount of time that the tissue undergoes enzymatic treatment depends on the age of the animal. For example, ganglia from P9 rats are treated with enzyme for 25 min, and ganglia from P15 rats are treated with enzyme for 35 min.</li>
		<li>Transfer the ganglia to the 35 mm Petri dish with fresh L-15 solution and incubate for 2-3 min.</li>
		<li>Transfer the ganglia to another 35 mm Petri dish filled with filtered culture media.</li>
		<li>Using a 200 &#181;L micropipette, pipette a ~150 &#181;L drop of filtered culture media onto a coated glass-bottom dish. Do not add too much solution, as it is important to maintain surface tension to form a bubble of solution that remains over the glass coverslip.</li>
		<li>Transfer the desired number of ganglia (one to four) to the coverslip.</li>
		<li>Draw a small amount of culture medium from the culture dish to rinse the trituration pipette with medium to prevent the tissue from sticking to the sides of the glass pipette. Triturate by gently and repeatedly passing the tissue through the pipette until the ganglia are sufficiently dissociated. 
		NOTE: Do not overwork the tissue to attempt single-cell suspensions or allow the cells to crash to the bottom of the dish using excessive positive pressure. Also, avoid forming air bubbles, as this will reduce cell survival. Gentle trituration is the key to achieving successful cell survival.</li>
		<li>Let the cells rest for 5 min. Check under a light microscope to see if the cells have settled on the coverslip.</li>
		<li>Carefully place a culture dish into a 37 &#176;C incubator for 12-24 h. Again, make sure to keep the bubble of solution intact. 
		NOTE: When culturing for longer than 24 h, refresh the culture media daily. This can be a possible pause point.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>B27 Supplement (50x), serum free</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td>additive to culture medium, for SGN</td>
		</tr>
		<tr>
			<td>Beakers (1000, 100, 10) milliliter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bench-top centrifuge</td>
			<td>USA Scientific</td>
			<td>2641-0016</td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma-Aldrich</td>
			<td>C5318</td>
			<td>one out of three enzyme to digest tissue</td>
		</tr>
		<tr>
			<td>Coverglass, rectangular, #1 thickness, 22x40&#160;</td>
			<td>Warner Instruments</td>
			<td>64-0707</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Biotium</td>
			<td>90082</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I,from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>11284932001</td>
			<td>one out of three enzyme to digest tissue</td>
		</tr>
		<tr>
			<td>Dumont #3 Forceps (Blunt)</td>
			<td>Fine Science Tools</td>
			<td>11231-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps (Fine)</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #55 Forceps (Fine)</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>2716</td>
			<td>for cleaning head and around dissection bench</td>
		</tr>
		<tr>
			<td>Filamented Borosilicate Capillaries for electrodes</td>
			<td>Sutter Instruments</td>
			<td>BF140-117-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-edged dissection blade</td>
			<td>Fine Science Tools</td>
			<td>10010-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur Pipettes</td>
			<td>VWR</td>
			<td>14673-010</td>
			<td>to pull trituration pipettes</td>
		</tr>
		<tr>
			<td>Heat-inactivated Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>16140063</td>
			<td>additive to culture medium</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-100G</td>
			<td>for pH buffering all solutions in protocol</td>
		</tr>
		<tr>
			<td>Hot plate / magnetic stirrers&#160;</td>
			<td>VWR</td>
			<td>76549-914</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulated bucket filled with ice</td>
			<td></td>
			<td></td>
			<td>to keep all samples and solutions cool</td>
		</tr>
		<tr>
			<td>Large Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>14133-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz medium&#160;</td>
			<td>Sigma Aldrich</td>
			<td>L4386</td>
			<td>dissection and bath solutions&#160;</td>
		</tr>
		<tr>
			<td>Low-profile-bath recording chamber for culture dishes</td>
			<td>Warner Instruments</td>
			<td>64-0236</td>
			<td></td>
		</tr>
		<tr>
			<td>luer-lok syringes, 30 ml</td>
			<td>BD</td>
			<td>302832</td>
			<td>for drawing L-15/HEPES/HEPES solution.</td>
		</tr>
		<tr>
			<td>MEM + Glutamax Supplement</td>
			<td>Fisher Scientific</td>
			<td>41-090-101</td>
			<td>base of the culture medium</td>
		</tr>
		<tr>
			<td>microFil needle for filling micropipettes - 34 gauge&#160;</td>
			<td>World Precision Instruments</td>
			<td>MF34G</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Supplement (100x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502-048</td>
			<td>additiive to culture medium, for SGN</td>
		</tr>
		<tr>
			<td>NaOH (1 M)</td>
			<td>Thomas Scientific</td>
			<td>319511-500ML</td>
			<td>for titration pH</td>
		</tr>
		<tr>
			<td>Oxygen, Medical grade, with adequate regulator and tubing</td>
			<td>USC Material Management</td>
			<td>MEDOX200 (Identifier: 00015)</td>
			<td>for dissolving into dissection and bath solutions</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM992</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette bulb (3 ml)</td>
			<td>Fisher Scientific</td>
			<td>03-448-25</td>
			<td>bulb for trituration pipettes</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>additive to prevent contamination of culture medium</td>
		</tr>
		<tr>
			<td>Pentobarbital based euthanasia solution (e.g., Fatal Plus. 50 &#8211; 60 mg/kg dosing)&#160;</td>
			<td>MWI Animal Health</td>
			<td>15199</td>
			<td>for euthanasia</td>
		</tr>
		<tr>
			<td>PES membrane filters ,&#160; 0.2 micrometer&#160;</td>
			<td>Nalgene</td>
			<td>566-0020</td>
			<td>for filtering solutions</td>
		</tr>
		<tr>
			<td>PES membrane sterile syringe filters, 0.22 um, 30 mm&#160;</td>
			<td>CELLTREAT</td>
			<td>229747</td>
			<td>for filtering solutions drawn into syringes</td>
		</tr>
		<tr>
			<td>Petri dishes, 35 x 10 mm</td>
			<td>Genessee Scientific</td>
			<td>32-103</td>
			<td>for micro dissection and to hold Tip dip solution in perforated-patch configuration</td>
		</tr>
		<tr>
			<td>Petri Dishes, 60 x 15 mm</td>
			<td>Midland Scientific</td>
			<td>P7455</td>
			<td>for gross dissection</td>
		</tr>
		<tr>
			<td>pH Meter</td>
			<td>Mettler Toledo</td>
			<td>Model S20</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettors (1000, 200, 10) microliter</td>
			<td>USA Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-d-lysine coated glass bottomed culture dish</td>
			<td>Mattek</td>
			<td>P35GC-0-10-C</td>
			<td>to plate neurons for culture</td>
		</tr>
		<tr>
			<td>Quick change platform, heated base, for 35 mm culture dishes</td>
			<td>Warner Instruments</td>
			<td>64-0375</td>
			<td></td>
		</tr>
		<tr>
			<td>Reference Cell</td>
			<td>World Precision Instruments</td>
			<td>RC1T</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Miltex</td>
			<td>4-315</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Scientific Scale</td>
			<td>Mettler Toledo</td>
			<td>XS64</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes (10, 25) milliliter</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone Grease Kit (for sealing coverglass and chamber)</td>
			<td>Warner Instruments</td>
			<td>64-0378</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Animal Guillotine</td>
			<td>Kent Scientific</td>
			<td>DCAP</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal guillotine</td>
			<td>Kent Scientific</td>
			<td>DCAP</td>
			<td>for decapitation if dissecting rats older than P15.</td>
		</tr>
		<tr>
			<td>Stereo Dissection Microscope&#160;</td>
			<td>Zeiss</td>
			<td>Stemi 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (3, 10, 30) milliliter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma Aldrich</td>
			<td>T1426</td>
			<td>one out of three enzyme to digest tissue</td>
		</tr>
		<tr>
			<td>Tuberculin syringe&#160;</td>
			<td>Covidien</td>
			<td>8881500105</td>
			<td>for delivering euthanasia solution by intraperitoneal injection</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissor, 2.5 mm Cutting Edge</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Volumetric flask, 1000 milliliter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>VWR</td>
			<td>945300</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, sterile u ltrapure, R&#62;18.18 megaOhms cm (e.g., filtered by a Millipore-Sigma water purification system)</td>
			<td>Millipore-Sigma</td>
			<td>CDUFBI001</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Iyer, M. R. et al., <a href="https://app.jove.com/v/64908">Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings.</a>&#160;J. Vis. Exp. (2023).
This video showcases a method for isolating vestibular neurons from a mouse pup&#39;s inner ear. It meticulously outlines the steps involved in dissecting the inner ear, harvesting the vestibular ganglion, and subsequently dissociating the ganglion into single cells. These cells are then cultured on glass-bottom dishes to establish a vestibular neuron culture.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparations
NOTE: The solutions ...

Take one-half of a mouse pup&#39;s head and remove the brain.
Cut the top of the skull and excise the surrounding tissue to visualize the otic capsule, a bony outer layer protecting the inner ear structures.
Transfer the tissue to a culture plate containing a buffered medium.
Separate the superior and inferior vestibular ganglia, which contain the vestibular nerve cell bodies.
Shave off the bony ridge and harvest the underlying superior ganglion.
Transfer the superior ganglion to a fresh plate containing the buffered medium.
Remove adherent tissues from the surface to clean the isolated ganglion.
Place the ganglion in a solution containing proteolytic enzymes.
The enzymes degrade the tissue&#39;s extracellular matrix, loosening the cells.
Transfer the ganglion to a polymer-coated glass-bottom dish containing culture medium and mechanically dissociate the tissue to release the cells.
Incubate, allowing the cells to adhere and establish a vestibular ganglion neuron culture.

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Video: Aislamiento de neuronas ganglionares vestibulares del oído interno de un ratón - Experimento

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

Un protocolo para Decellularizing ratón cócleas para ingeniería de tejidos del oído interno

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

Investigación

JoVE Journal

Bioingeniería

Isolation and Culture of Chick Ciliary Ganglion Neurons

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the eye. This ganglion is constituted by a homogenous population of ciliary and choroidal neurons that innervate striated and smooth muscle fibers, respectively. Each of these neuronal types regulate specific eye structures and functions. Over the years, neuronal cultures of the chick ciliary ganglia were shown to be effective cell models in the study of muscle-nervous system interactions, which communicate through cholinergic synapses. Ciliary ganglion neurons are, in its majority, cholinergic. This cell model has been shown to be useful comparatively to previously used heterogeneous cell models that comprise several neuronal types, besides cholinergic. Anatomically, the ciliary ganglion is localized between the optic nerve (ON) and the choroid fissure (CF). Here, we describe a detailed procedure for the dissection, dissociation and in vitro culture of ciliary ganglia neurons from chick embryos. We provide a step-by-step protocol in order to obtain highly pure and stable cellular cultures of CG neurons, highlighting key steps of the process. These cultures can be maintained in vitro for 15 days and, hereby, we show the normal development of CG cultures. The results also show that these neurons can interact with muscle fibers through neuro-muscular cholinergic synapses.

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system. Neuronal cultures of chick CG neurons were shown to be effective cell models in the study of nerve muscle interactions. We describe a detailed protocol for the dissection, dissociation and in vitro culture of CG neurons from chick embryos.

Aislamiento y cultura de chick Ciliary Ganglion Neurons

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the ...

Neurociencias

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Cocultura de neuronas ganglionas retinal de rata axotomizada con glia olfativa, como modelo in vitro de regeneración axonal adulta

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

Isolating Vestibular Ganglion Neurons From a Mouse Inner Ear

Transcripción

Isolating Vestibular Ganglion Neurons From a Mouse Inner Ear