We thank Dr. A. Jacobo, Dr. J. Salvi, and A. Petelski for their contributions to the original research on which this protocol is based. We also thank J. Llamas and W. Makmura for technical assistance and animal husbandry. We acknowledge NIDCD Training grant T32 DC009975, NIDCD grant R01DC015530, Robertson Therapeutic Development Fund, and the Caruso Family Foundation for funding. Finally, we acknowledge support from Howard Hughes Medical Institute, of which Dr. Hudspeth is an Investigator.

All methods described here have been approved by the Animal Care and Use Committees of Rockefeller University and of the University of Southern California.
1. (Optional) Preparation of Collagen I Solution from Mouse-tail Tendons
Note: Collagen I solutions are available commercially. Follow the manufacturer&#39;s instructions for gel preparation.
<ol>
	<li>Euthanize 5 - 10 young adult (3 - 5 weeks old) mice of any wild type strain with carbon dioxi...

<ol>
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The authors have nothing to disclose.

The molecular signals that mediate growth and differentiation in the inner ear during development have been studied extensively5,6,7,8,9,10. However, evidence obtained from the utricular model system suggests that mechanical cues, sensed through cell junctions and the activation of Hippo signaling, also play an important role...

The study of most aspects of mammalian organ development has been facilitated by in vitro systems. Two principal methods are now used for the culture of vestibular sensory organs: free-floating1 and adherent2 preparations. Both methods permit the investigation of hair cell vulnerabilities3 and regeneration1,4&#160;in vitro. In addition, the developmental roles of the Notch5,6, Wnt7,8, and epidermal growth factor receptor (EGFR)9,10 signaling cascades in the inner ear have been established, in part, through the use of in vitro cultures of sensory epithelia. However, cell growth and differentiation are controlled, not only through signaling by morphogens, but also through physical and mechanical cues such as intercellular contacts, the stiffness of extracellular matrix, and mechanical stretching or constriction. The role of such mechanical stimuli is challenging to investigate in the developing inner ear in vivo. Moreover, existing free-floating and adherent culture methods are not suitable for such studies in vitro. Here we describe a method for three-dimensional organotypic culture in collagen I gels of varying stiffness. This method largely preserves the in vivo architecture of the vestibular and cochlear sensory organs and allows investigation of the effects of mechanical force on growth and differentiation11.
Because mechanical stimuli are known to activate downstream molecular events, such as the Hippo signaling pathway12,13,14,15, it is important to be able to combine mechanical stimulation with biochemical and genetic manipulations. The culture method described here permits virus-mediated gene delivery and can therefore be used to study both mechanical and molecular signaling during inner ear development11.

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		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Transferrin</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">T8158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan blue&#160;</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">T6146</td>
			<td></td>
		</tr>
	</tbody>
</table>

three-dimensional organotypic cultures of vestibular and auditory sensory organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Vestibular and auditory sensory organs from embryonic ears, cultured in 40-Pa collagen I gels mimicking low stiffness embryonic conditions11, retain relatively normal three-dimensional structures (Figure 1) and maintain hair cells and supporting cells (Figure 2 and Figure 3). Although supporting cell density decreases by over 30% (Student&#39;s t-test: n = 4, p</e...

Watch this Scientific Journal Video about Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs at JoVE.com

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

The overall goal of this procedure is to establish three-dimensional organotypic cultures of the murine vestibular and auditory sensory organs. This method can help answer the key questions in the inner ear research field, such as, what are the roles of mechanical force and tissue stiffness during development of the inner-ear sensory organs. The main advantage of this technique is that it largely preserves innate architecture of vestibular cochlea and allows us to study both molecular and mechanical signaling during inner ear development.Dr.Hudspeth and I first had the idea of this method when we realized that three-dimensional cultures can provide morphysiologic conditions to study vestibular organ growth in-vitro. First, use 70%ethanol to clean and disinfect the working area and all the necessary dissection instruments, including number five forceps and a hair knife. Then, use a sterile scalpel blade to make a longitudinal cut, split the head of the mouse into two halves, and extract the temporal bones.Transfer in 15 milliliters of HBSS buffer in a 60 millimeter Petri dish on ice. Next, use two pairs of fine number five forceps to isolate the inner ears from the temporal bones. Then, transfer three to four ears at a time in a 60 millimeter Petri dish containing ice-cold HBSS buffer.Next, orient the ears with the medial side up to locate the utricle. Use the two pairs of number five forceps to remove the cartilage surrounding the vestibular organs. Next, cut the vestibular nerve, the connection between the utricle and saccule, and the semi-circular canals to free the utricle and the attached ampullae from the ears.Trim excess semi-circular canals if needed. Alternatively, use the two pairs of number five forceps to remove the cartilaginous tissue surrounding the hearing organ. Then, isolate the cochlea.Next, use a 200-microliter pipette fitted with a wide, non-stick tip to transfer the cochlea and the utricles to a 30-millimeter Petri dish filled with the DMEM/F12-based growth medium. Then, transfer the cochlear and the utricle preparations to a tissue culture incubator, gassed with 5%carbon dioxide at 37 degrees Celsius. This is to achieve organ cultures with enclosed lumens.Prepare 100 microliters of the collagen one polymerization solution in a sterile 1.5-milliliter tube. Then, leave the tube on ice. Next, remove the cochlear and utricle preparations from the tissue culture incubator.Prepare the neutralized collagen one solution. To do so, add 400 microliters of collagen one to a chilled 1.5-milliliter tube with 100 microliters of polymerization solution by pipetting several times on ice. Transfer 500 microliters of the neutralized collagen one solution to a chilled 30-millimeter Petri dish with a 10-millimeter glass bottom insert.Immediately transfer the cochlea or the utricles to the neutralized collagen one solution using a 200-microliter pipette fitted with a wide, non-stick tip. Use a sterile hair knife, or a pair of fine forceps to adjust the organs to their desired positions under a binocular dissecting microscope. In order to ensure complete polymerization, incubate the Petri dish at 37 degrees Celsius for 20 minutes.After 20 minutes, add three milliliters of growth medium, supplemented with 0.5%fetal bovine serum in each Petri dish. After adding the growth medium, incubate the culture at 37 degrees Celsius. First, defrost the virus on ice.Then, mix the viral suspension with Trypan blue solution in a 0.5-milliliter conical tube to adjust the final dye concentration of 0.05%After mixing the Trypan blue solution, leave the viral particles on ice. Then, use a binocular dissecting microscope to observe the breaking of the tip of the glass needle, prepared on a micro-pipette puller with clean, fine forceps. First, remove the sensory organ culture from the incubator.Attach the needle to the micro-injector. Fill the needle with two to three microliters of dye and viral mixture. Look through the binocular dissecting microscope while setting the needle into the sensory organ.Keep forwarding the needle tip through the mesenchymal and epithelial layers of the roof of a three-dimensional utricular culture. Keep injecting the viral mixture until the cavities of the utricle and ampullae fill with the blue dye. Then, incubate the organ culture in a 37 degree Celsius tissue culture incubator gassed with 5%carbon dioxide.These are the light microscopic images of the utricle and cochlear culture embedded in the three-dimensional collagen one gel. The utricle and the cochlea are obtained from 17.5 and 14.5 days old embryos, respectively, and cultured for 48 hours. These confocal microscopic images show 18.5 days old embryonic utricle just before and after explantation in the 40PA collagen one gel.After three days in the culture, almost 80%of the Myo7A positive hair cells remain intact. Supporting cell density decreases by over 30%and hair cell density decreases by 60%after three days in utricular cultures. These confocal microscopic images show an embryonic 14.5 days old cochlea just before and after explantation in the 40PA collagen one gel.After three days in culture, Myo7A and Sox2 positive inner and outer hair cells are morphologically distinguishable and are organized in about four to five rows. Here, the images show successful viral infection of the supporting cells at the base of the cochlear and throughout the utricular three-dimensional cultures obtained from 15.5 and 17.5 days of embryos, respectively. After watching this video, you should have a good understanding of how to dissect intact inner-ear sensory organs establish three-dimensional cultures of utricle and cochlea and collagen type one gel, and perform viral transfections in those three-dimensional cultures.Remember that working with viral particles, depending on gene cargo, can be hazardous. Precautions, such as wearing personal protective equipment and proper viral disposal, should always be practiced.

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Developmental Biology

8.1K vistas.  Keck School of Medicine of the University of Southern California. The overall goal of this procedure is to establish three-dimensional organotypic cultures of the murine vestibular and auditory sensory organs. This method can help answer the key questions in the inner ear research field, such as, what are the roles of mechanical force and tissue stiffness during development of the inner-ear sensory organs. The main advantage of this technique is that it largely preserves innate architecture of vestibular cochlea and allows us to study both molecular and mec...