Name: primary culture and plasmid electroporation of the murine organ of corti.
Uploaded: 2010-02-04T00:00:00
Duration: 12 min 29 s
Description: Watch this Scientific Journal Video about Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. at JoVE.com

The authors would like to thank Dem&ecirc;mes Danielle and Douglas Cotanche and for their efforts in teaching us the methods for the isolation of the organ of Corti. Additionally, we would like to thank Ishmael Stefanov-Wagner for engineering the electroporator electrodes; Sherry Lin for her contributing artwork to the video animation; and Matthew Chana, Jason Meeker, and Kendra Marshall (<a href="http://www.goodfightproductions.com">www.goodfightproductions.com</a>) for producing the video. This work was funded by grants (R03DC010065-Parker; RO1DC007174-Edge; P30DC05209- MEEI Core Support for Hearing Research) from the NIDCD.

Day 1. Sterilization and coating of glass coverslips.
<ol>
	<li>Dry sterilize glass microscope coverslips in an autoclave.</li>
	<li>Place the sterilized coverslips into two wells of a pre-sterilized four-well cell culture dish.</li>
	<li>Coat the coverslips with 1:1 poly-L-ornithine and laminin supplemented with 20% Fetal Bovine Serum (FBS) overnight at 4 &#176;C.
		<ol class="lalpha">
			<li>400 &#956;L poly-L-ornithine (0.01% solution stored at 4 &#176;C)</li>
			<li>400 &#956;L laminin (50 &#956;g/mL stock solution stored in aliquots at -20 &#176;C)</li>
			<li>200 &#956;L FBS (stored in aliquots at -20 &#176;C)</li>
		</ol>
	</li>
	<li>Heat-sterilize the dissection tools overnight in a 150&#176;C incubator.</li>
	<li>Make culture medium containing 10% serum and 10 mg /mL ampicillin.
		<ol class="lalpha">
			<li>90 mL Dulbecco's Modified Eagle Medium </li>
			<li>5 mL FBS (stored in aliquots at -20 &#176;C)</li>
			<li>5 mL horse serum (stored in aliquots at -20 &#176;C)</li>
			<li>10 &#956;L ampicillin (10 mg/mL stock solution stored at 4 &#176;C )</li>
		</ol>
	</li>
</ol>
Day 2. Isolation of the organ of Corti.
<ol>
	<li>Sterilize the positive flow dissection hood.
		<ol class="lalpha">
			<li>turn on UV light for 20 min </li>
			<li>spray all surfaces with 70% ethanol and wait 5 min before use. </li>
		</ol>
	</li>
	<li>Decapitate mouse pup (P4) at the base of the foramen magnum using operating scissors.</li>
	<li>Briefly rinse the head in 10 cm dish containing 70% ethanol. </li>
	<li>Remove the epidermis using a scalpel blade.</li>
	<li>Open the cranium along the sagittal suture using a scalpel blade and then bisect the forebrain. Retain the caudal forebrain for further dissection.</li>
	<li>Remove the forebrain, cerebellum and brainstem using blunt dissection. </li>
	<li>Remove the temporal bones (fig 2A), dip them briefly in 70% ethanol, and transfer them to a 3 mm dish containing sterile HBSS.</li>
	<li>Using forceps, remove the bulla and surrounding tissue from the petrous portion of the temporal bone.</li>
	<li>Locate the conch shaped cochlea (fig 2B) and separate it from the vestibular system using forceps.</li>
	<li>At this stage of development, the bony labyrinth is not completely calcified and is easily dissected using forceps. Remove the bony labyrinth of the cochlea by careful separation starting at the basal end and moving apically using forceps.</li>
	<li>The spiral ligament and attached organ of Corti is coiled along the spiral of the modiolus (fig 2C). Carefully remove the organ of Corti by securing the spiral ligament at the hook region of the base using forceps and unwinding it as you move apically. </li>
	<li>Starting at the base, remove the spiral ligament from the organ of Corti using #55 fine forceps (fig 2D). 
		Micro-isolation of the organ of Corti sensory epithelium (Optional). </li>
	<li>Remove the organ of Corti hook region of the base using two &#189; cc Insulin Syringes with permanently attached U-100 28G&#189; needles as forceps.</li>
	<li>Starting at the apex, remove the spiral limbus from the row of inner hair cells and proceed basally (fig 2E-F). 
		Plating the organ of Corti explant</li>
	<li>Remove the poly-L-ornithine/laminin/FBS solution from the culture wells and add 130 &#956;l of culture medium.</li>
	<li>Transfer the dissected organ of Corti to the coated glass coverslip in the culture well and orient the explant so that the cilia of the hair cells are pointing up.</li>
	<li>Remove the medium in the culture well using a 200 &#956;L pipette. Make sure that the basilar membrane makes solid contact with the coated glass coverslip. </li>
	<li>Carefully add 130 &#956;L of culture medium to the organ of Corti using a 200 mL pipette. Apply two drops onto the surface of the organ of Corti and then slowly add the remaining volume to the side of the coverslip. Make sure that the organ of Corti does not float in the culture media, but remains affixed to the coverslip. </li>
	<li>Incubate overnight at 37 &#176;C in the presence of 5% CO2.</li>
</ol>
Day 3. Electroporation of the reporter gene into cultured organ of Corti.
<ol>
	<li>Remove the culture medium from the organ of Corti culture.</li>
	<li>Add 130 &#956;L H2O for 1 min and then remove using a 200 &#956;L pipette.</li>
	<li>Add 30 &#956;L of DsRed reporter plasmid (2mg /mL H2O stored at -20 &#176;C).</li>
	<li>Advance the electrodes of the electroporator using a micromanipulator so that the anode and cathode are on either side of the culture.</li>
	<li>Generate a pulse (27V, 30 ms duration, 10 pulse trains) to electroporate the reporter gene into the organ of Corti explant culture.
		<ol class="lalpha">
			<li>Optional: reverse the polarity of the pulse to ensure electroporation of the transgene on both the modiolar and spiral ligament sides of the explant.</li>
		</ol>
	</li>
	<li>Wait 5 min. </li>
	<li>Add 130 &#956;L of the Fugene 6: DNA solution (3 parts Fugene to 2 parts DNA). This solution should be prepared prior to the start of the electroporation procedure (step 20) using a 3 mL round bottom polystyrene test tube in a laminar flow hood. To make this solution:
		<ol class="lalpha">
			<li>Add 2.4 &#956;L of Opti-MEM (stored at 4 &#176;C) to the test tube.</li>
			<li>Add 0.6 &#956;L of Fugene 6 reagent (stored at 4 &#176;C) to the test tube. Make sure to add the Fugene directly to the Opti-MEM and avoid direct contact with the sides of the test tube.</li>
			<li>Vortex for 1 second.</li>
			<li>Incubate 5 min at room temperature.</li>
			<li>Add 2.0 &#956;L DsRed reporter plasmid double stranded DNA (stored at -20 &#176;C at 100 &#956;g DNA/mL H2O aliquots).</li>
			<li>Vortex for 1 second.</li>
			<li>Incubate 15 min at room temperature.</li>
			<li>Add 200 &#956;L of culture medium.</li>
			<li>Vortex for 1 second.</li>
		</ol>
	</li>
	<li>Incubate overnight at 37 &#176;C in 5% CO2.</li>
	<li>Add 2 mL culture medium to the culture well and incubate for 37 &#176;C for up to 10 days.</li>
</ol>
Representative Results
We present a method for the isolation of the organ of Corti from a perinatal mouse. The procedure can be used for mice as young as embryonic day 16 up to about postnatal day 6, at which point the bony labyrinth becomes sufficiently calcified to render the dissection cumbersome. Once the organ of Corti is dissected, it may be plated and cultured either in its entirety (fig 3) or as micro-isolated sensory epithelium (fig 4). We have further presented a technique to express exogenous genes in the cultured organ of Corti. The organotypic culture is useful for many other types of study, such as the analysis of organ of Corti gene expression using RT-PCR or in situ hybridization, organ of Corti co-culture with spiral ganglion cells or exogenous stem cells using the micro-isolate3, or in vitro analysis of hair cell death and regeneration.
<img src="/files/ftp_upload/1685/1685fig1tmb.jpg" alt="Figure 1" border="0" /> 
	Figure 1. Cross section of the P4 murine organ of Corti. (A) A cross section from the basal turn of a cryosectioned cochlea obtained from a P4 mouse illustrates the general structures of the murine cochlea described in this protocol. The scala media is bordered by the spiral ligament and stria vascularis laterally, the Reissner s membrane superiorly, the spiral limbus medially, and the basilar membrane inferiorly. The box indicated the region expanded in B. (B) The organ of Corti is located on the superior side of the basilar membrane and includes one row of inner hair cells, three rows of outer hair cells, and their respective supporting cells. Dashed line 1 indicates the location along the basilar membrane that is removed during this organ of Corti dissection. Dashed line 2 indicates the location along the basilar membrane that is removed during the micro-isolation procedure. Green indicates immunohistochemical labeling of calbindin, which labels interdental cells of the spiral limbus, cochlear hair cells, spiral ganglion neurons, as well as cells of the spiral ligament and stria vascularis 7. DAPI labeling of nuclei is in blue. To see a larger version of this image please <a href="https://www.jove.com/files/ftp_upload/1685/1685fig1.jpg">click here</a>.
<img src="/files/ftp_upload/1685/1685fig2tmb.jpg" alt="Figure 2" border="0" /> 
	Figure 2. Organ of Corti dissection. Images from the accompanying video of the organ of Corti dissection highlight A) the cochlea and vestibular system located within the isolated temporal bone (red), B) the bony labyrinth of the cochlea, C) the spiral ligament and attached organ of Corti after removal of the bony labyrinth, D) removal of the spiral ligament and stria vascularis (red) from the organ of Corti, E) micro-isolation of the sensory epithelium from the spiral limbus (red), and F) the isolated spiral limbus (left) and sensory epithelium (right). To see a larger version of this image please <a href="https://www.jove.com/files/ftp_upload/1685/1685fig2.jpg">click here</a>.
<img src="/files/ftp_upload/1685/1685fig3tmb.jpg" alt="Figure 3" border="0" /> 
	Figure 3. Cultured organ of Corti explant. DIC image showing the organ of Corti of a P4 Atoh1-nGFP mouse that was isolated, plated, and cultured for five days as described. This mouse has been genetically engineered so that cells that express the pro-hair cell gene Atonal homolog 1 (Atoh1 aka Math1) exhibit a green fluorescent protein that is localized to the nucleus8. The organs of Corti from these mice exhibit a nuclear GFP label in all hair cell nuclei and therefore allow for easy visualization of the sensory epithelium using an epifluorescent microscope. The relatively large spiral limbus can be seen lateral to the sensory epithelium. Mesenchymal cells that have originated from the organ of Corti have migrated away from the explant. Blue nuclear label is DAPI. To see a larger version of this image please <a href="https://www.jove.com/files/ftp_upload/1685/1685fig3.jpg">click here</a>.
<img src="/files/ftp_upload/1685/1685fig4tmb.jpg" alt="Figure 4" border="0" /> 
	Figure 4. Micro-isolated sensory epithelium. Epifluorescent image obtained from the sensory epithelium that has been isolated from a P4 murine organ of Corti as described and cultured overnight. The micro-isolate was then fixed in 4% paraformaldehyde and processed for immunolabeling of Myosin 7a which labels cochlear hair cells. Note the absence of the comparatively larger spiral limbus from the figure 3. Blue nuclear label is DAPI. To see a larger version of this image please <a href="https://www.jove.com/files/ftp_upload/1685/1685fig4.jpg">click here</a>.
<img src="/files/ftp_upload/1685/1685fig5tmb.jpg" alt="Figure 5" border="0" /> 
	Figure 5. Electroporation of DsRed reporter gene into the cultured organ of Corti. Whole organs of Corti from P4 Atoh1-nGFP mouse pups were isolated, plated, and then electroporated with the DsRed reporter gene as described and then viewed under an epifluorescent microscope. Cells of the organ of Corti explant that expressed the transgenic DsRed reporter protein exhibit a red fluorescence and endogenous cells of the sensory epithelium exhibit a green nuclear fluorescence. Transgenic cells can be seen throughout the spiral limbus and sensory epithelium. To see a larger version of this image please <a href="https://www.jove.com/files/ftp_upload/1685/1685fig5.jpg">click here</a>.

<ol>
	<li>Sobkowicz, H. M., Bereman, B., Rose, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+Development+of+the+Organ+of+Corti+in+Culture.">Organotypic Development of the Organ of Corti in Culture.</a> Journal of Neurocytology. 119 (4), 543-543 (1975).</li><li>Zheng, J. L., Gao, W. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Math1+induces+robust+production+of+extra+hair+cells+in+postnatal+rat+inner+ears.">Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears.</a> Nature Neuroscience. 3 (6), 580-580 (2000).</li><li>Martinez-Monedero, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=THE+POTENTIAL+ROLE+OF+ENDOGENOUS+STEM+CELLS+IN+REGENERATION+OF+THE+INNER+EAR.">THE POTENTIAL ROLE OF ENDOGENOUS STEM CELLS IN REGENERATION OF THE INNER EAR.</a> J Neurobiol. 66 (4), 319-319 (2006).</li><li>Saffer, L. D., Gu, R., Corwin, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RT-PCR+analysis+of+mRNA+for+growth+factor+receptors+in+damaged+and+control+sensory+epithelia+of+rat+utricles.">An RT-PCR analysis of mRNA for growth factor receptors in damaged and control sensory epithelia of rat utricles.</a> Hearing Research. 94 (1,2), 14-14 (1996).</li><li>Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+growth+and+differentiation+of+hair+cell+progenitors+from+the+newborn+rat+cochlear+greater+epithelial+ridge.">Isolation, growth and differentiation of hair cell progenitors from the newborn rat cochlear greater epithelial ridge.</a> Journal of Neuroscience Methods. 164 (2), 271-271 (2007).</li><li>Jones, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+Differentiation+and+DNA+Binding+(Ids)+Regulate+Math1+and+Hair+Cell+Formation+during+the+Development+of+the+Organ+of+Corti.">Inhibitors of Differentiation and DNA Binding (Ids) Regulate Math1 and Hair Cell Formation during the Development of the Organ of Corti.</a> J. Neurosci. 26 (2), 550-550 (2006).</li><li>Daniela, B., Josef, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calbindin+and+S100+protein+expression+in+the+developing+inner+ear+in+mice.">Calbindin and S100 protein expression in the developing inner ear in mice.</a> The Journal of Comparative Neurology. 513 (5), 469-469 (2009).</li><li>Helms, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoregulation+and+multiple+enhancers+control+Math1+expression+in+the+developing+nervous+system.">Autoregulation and multiple enhancers control Math1 expression in the developing nervous system.</a> Development. 127 (6), 1185-1185 (2000).</li><li>Zheng, J. L., Wei-Qiang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Damage+to+Auditory+Neurons+and+Hair+Cells+by+Ototoxins+and+Neuroprotection+by+Specific+Neurotrophins+in+Rat+Cochlear+Organotypic+Cultures.">Differential Damage to Auditory Neurons and Hair Cells by Ototoxins and Neuroprotection by Specific Neurotrophins in Rat Cochlear Organotypic Cultures.</a> European Journal of Neuroscience. 8 (9), 1897-1897 (1996).</li></ol>

There are several details that are critical for the success of this procedure. The shorter the time from temporal bone isolation to organ of Corti incubation, the greater the chance that the organs will attach to the coverslip and result in viable organ cultures. Therefore, it is important to limit the amount of time between dissection and placing the organs in the incubator. The choice of antibiotic is also crucial, since many aminoglycoside antibiotics are ototoxic and will result in hair cell death. Although it is preferable to forgo the use of antibiotics altogether, this leaves open the possibility for contamination. Therefore, we suggest the use of 10 &mu;g /mL ampicillin as a general rule to overcome potential contamination problems.
The most troublesome aspect of this, and other procedures for the primary culture of the organ of Corti, is tendency of the organs to float off the plates during incubation. Although floating organ cultures can remain viable for 5-7 days, there are drawbacks of culturing floating organs. For instance, floating organ cultures often fold onto themselves after 4-5 days rendering microscopy problematic. Subsequently, the structural integrity of the organ can become compromised when compared to organs that have been affixed to the coverslip. 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 coverslip. First, coat the glass coverslips in 1:1 polyornithine/laminin supplemented with 20% FBS as described. The overnight incubation called for in this protocol is the minimum time that the plate should be coated. In our laboratory, we often coat all of the plates that we need for one week and keep them at 4 &deg;C until the day prior to use when we move them to the incubator for an overnight incubation. Second, after the organs are transported to the coated coverslips, orient the explant so that the cilia of the hair cells face up. This orientation will facilitate the adherence of the basilar membrane to the culture dish. Third, remove the media within the well to affix the explant to the coated dish. This will ensure contact between the culture dish and the basilar membrane and enhance the ability of the explants to adhere to the glass. This will also assist in maintaining the structural integrity of the rows of hair cells. Lastly, carefully drip 2 drops of culture medium onto the surface of the organ of Corti using a 200 &mu;L capacity pipette and then slowly fill the well by dripping the remaining volume (of the total 130 &mu;L) on the side of the coverslip. It is important to work quickly to assure the attachment of the organ of Corti to the coated coverslip. From the initiation of the dissection to the incubation of the explants, it typically takes 10 minutes for a practiced operator to complete this organ of Corti isolation procedure.
In this protocol, we also present a method for the micro-isolation of the sensory epithelium from the spiral limbus of the organ of Corti. In this procedure, the spiral limbus is dissected away from the sensory epithelium using 28G&frac12; insulin needles as dissection tools. The resulting micro-isolate consists of the rows of hair cells and their corresponding supporting cells (fig 3). The isolated sensory epithelium can then be cultured as described in this protocol. This micro-isolation procedure should be compared to the enzymatic separation of the sensory epithelia from surrounding tissue4. In mammals, as well as non-mammalian species such as chickens, thermolysin digestion of isolated vestibular organs results in the isolation of sensory epithelia from the basement mesenchymal cells4. In the rat cochlea, thermolysin digestion results in the separation of the greater epithelial ridge, lesser epithelial ridge and accompanying sensory epithelia from the basement membrane5. However, it is unclear whether the cochlear sensory epithelium can attach to the coated plates without the accompanying mesenchymal cells. While both the mechanical micro-isolation method and enzymatic digestion method result in the separation of the sensory epithelia from the spiral limbus, advantages of the micro-isolate dissection over the thermolysin digestion include a relatively shorter protocol, cheaper reagents, and potentially less stress to the explant due to enzymatic effects of the digestion. Additionally, the basement membrane is left intact in this approach, which may enhance attachment of the sensory epithelium to the culture plate. Disadvantages of this method include the need to develop the skills for this delicate dissection and potential mechanical damage to the sensory epithelium resulting from the micro dissection.
As an example of the utility of this procedure, we also present one example of the use of the organ of Corti cultures; the electroporation of exogenous genes into the explant culture. The electroporation procedure described above is based on previous methods of organ of Corti electroporation. Notably, Zheng and Gao (2000) describe the electroporation of isolated rat organs of Corti where the explants are held in place for electroporation by a molded groove of agarose and then plated on collagen coated 8-well LabTek slide in serum-free medium2. An advantage of their approach is that the organs are oriented so that the top surfaces of the explants face the cathode, which should theoretically result in an even distribution of electroporated cells across the explant. In our hands however, the method that we describe improved on this procedure because the organ of Corti remained affixed to the coverslip throughout the procedure thereby reducing the manipulation of the explant after electroporation. Additionally, a higher percentage of organs of Corti remained attached to the coverslips using this presented method. Our method is taken from Jones, et al. (2006) which uses the addition of the Fugene 6 reagent to increase the efficiency of gene expression after the electroporation. In the Jones et al. (2006) protocol, the organs are electroporated, incubated for 5 minutes with 100 &mu;L of Fugene 6 transfection reagent, and plated 6. Our method differs in the use of a 3:2 ratio of Fugene 6 reagent to plasmid DNA, which we found empirically to provide optimal transgenic expression with minimum organ toxicity. We do not use undiluted Fugene 6 reagent, which can result in toxicity to the cultures. The electrode configuration in our protocol, as well as Jones et al. (2006), results in primary gene expression in either the spiral limbus or sensory epithelium depending upon the position of the cathode. Although there are DsRed positive cells on the side of the culture distant to the cathode, there is a higher concentration of transgenic cells closer to the cathode. To ensure a complete expression of the transgene in both sides of the organ of Corti explant, the current can be reversed for a second pulse train by simply inverting the leads. The presented protocol results in robust expression of the transgene throughout the organ of Corti (fig 5).

<table border="1">
	<thead>
		<tr>
			<th>Material Name</th>
			<th>Type</th>
			<th>Company</th>
			<th>Catalogue Number</th>
			<th>Comment</th>
		</tr>
	</thead>
	<tbody>
		<tr>
			<td>Glass microscope coverslips</td>
			<td>&nbsp;</td>
			<td>DYNALAB Corporation (Rochester, NY)</td>
			<td>2010</td>
			<td>10mm diameter, circle #1, 1mm thickness, 1 ounce</td>
		</tr>
		<tr>
			<td>4 ringed cell culture dish</td>
			<td>&nbsp;</td>
			<td>Greiner Bio-One (Frickenhausen, Germany)</td>
			<td>627170</td>
			<td>Sterilized 35 X 10 mm cell culture dish with 4 inner rings</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine</td>
			<td>&nbsp;</td>
			<td>Sigma-Aldrich Company (St. Louis, MO)</td>
			<td>P4957</td>
			<td>0.01% Solution</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>&nbsp;</td>
			<td>BD Biosciences (Franklin Lakes, NJ) </td>
			<td>354232</td>
			<td>made in mouse</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>&nbsp;</td>
			<td>Invitrogen (Carlsbad, CA)</td>
			<td>26140-095</td>
			<td>Qualified</td>
		</tr>
		<tr>
			<td>Operating scissors</td>
			<td>&nbsp;</td>
			<td>Roboz Surgical Instrument Co. (Gaithersburg, MD)</td>
			<td>RS-6806</td>
			<td>Straight, sharp-blunt length 5"</td>
		</tr>
		<tr>
			<td>#11 Scalpel Blade</td>
			<td>&nbsp;</td>
			<td>Becton Dickenson (Franklin Lakes, NJ)</td>
			<td>372611</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>#4 Dumoxel forceps</td>
			<td>&nbsp;</td>
			<td>Fine Science Tools (Foster City, CA)</td>
			<td>11241-30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>#55 Dumostar fine forceps</td>
			<td>&nbsp;</td>
			<td>Fine Science Tools (Foster City, CA)</td>
			<td>11295-51</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle Medium </td>
			<td>&nbsp;</td>
			<td>Invitrogen (Carlsbad, CA)</td>
			<td>10564-011</td>
			<td>High Glucose</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>&nbsp;</td>
			<td>Invitrogen (Carlsbad, CA)</td>
			<td>2605088</td>
			<td>Heat Inactivated</td>
		</tr>
		<tr>
			<td>Ampicillin Sodium Salt </td>
			<td>&nbsp;</td>
			<td>Invitrogen (Carlsbad, CA)</td>
			<td>11593-027</td>
			<td>Irradiated</td>
		</tr>
		<tr>
			<td>&frac12; cc Lo-Dose Insulin Syringe </td>
			<td>&nbsp;</td>
			<td>Becton Dickenson (Franklin Lakes, NJ)</td>
			<td>329465</td>
			<td>U-100 28G&frac12;</td>
		</tr>
		<tr>
			<td>Fugene 6 Transfection Reagent</td>
			<td>&nbsp;</td>
			<td>Roche (Mannheim, Germany)</td>
			<td>11-815-091-001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polystyrene test tube</td>
			<td>&nbsp;</td>
			<td>Fisher Scientific (Pittsburgh, PA)</td>
			<td>14-956-5A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>&nbsp;</td>
			<td>The Baker Company (Stanford, ME)</td>
			<td>Model SG603a</td>
			<td>SterileGARD III 
				Advanced Class II 
				Biological Safety Cabinet</td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced-Serum Medium</td>
			<td>&nbsp;</td>
			<td>Invitrogen (Carlsbad, CA)</td>
			<td>31985</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Reporter plasmid</td>
			<td>&nbsp;</td>
			<td>Clontech (Mountain View, CA)</td>
			<td>632539</td>
			<td>pCMV DsRed-Express 2</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>&nbsp;</td>
			<td>BioRad (Hercules, CA)</td>
			<td>165&#x2013;2662</td>
			<td>BioRad Gene Pulser Xcell</td>
		</tr>
	</tbody>
</table>

primary culture and plasmid electroporation of the murine organ of corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Watch this Scientific Journal Video about Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. at JoVE.com

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

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Research

JoVE Journal

Biology

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological ...

Unveiling Inflammatory Arthritis&amp;#58; Exploring Functions of Synovial Cells

Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central roles in the pathogenesis of rheumatoid arthritis. It is important to understand the functions of both cell populations to reveal the mechanisms underlying pathological progression and remission in inflammatory arthritis. In general, in vitro experimental conditions should mimic the in vivo environment as much as possible. Primary tissue-derived cells have been used in experiments characterizing synovial fibroblasts in arthritis. In contrast, in experiments investigating the biological functions of macrophages in inflammatory arthritis, cell lines, bone marrow-derived macrophages, and blood monocyte-derived macrophages have been used. However, it is unclear whether such macrophages actually reflect the functions of tissue-resident macrophages. To obtain resident macrophages, previous protocols were modified to isolate and expand both primary macrophages and fibroblasts from synovial tissue in an inflammatory arthritis mouse model. These primary synovial cells may be useful for in vitro analysis of inflammatory arthritis.

Author Spotlight: Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue  

The present study provides a modified protocol to isolate synovial macrophages and fibroblasts from murine inflammatory arthritis tissue.

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central ...