Name: a protocol for decellularizing mouse cochleae for inner ear tissue engineering
Uploaded: 2018-01-01T14:00:00
Description: Watch this Scientific Journal Video about A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering at JoVE.com

Det aktuella projektet finansierades av University of Kansas bevis på konceptet fonden. Vi vill tacka vårdpersonalen på KUMC (Kansas City, KS) för att hjälpa oss att få mänskliga navelsträngar, och David Jorgensen bistå med cochleae kulturer.

Alla förfaranden, inklusive djur dödshjälp, genomfördes enligt godkända institutionella djur vård och användning kommittén (IACUC) protokoll (misstänkt #2014-2234) vid University of Kansas Medical Center (KUMC).
Obs: HWJCs isolerades från mänskliga navelsträngar som skänktes av patienter som gett informerat samtycke och prover användes i enlighet med de protokoll som godkänts av University of Kansas mänskliga försökspersoner kommittén (KU-IRB #15402).
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<ol>
	<li>Raphael, Y., Altschuler, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+innervation+of+the+cochlea.">Structure and innervation of the cochlea.</a> Brain Res Bull. 60 (5-6), 397-422 (2003).</li><li>LeMasurier, M., Gillespie, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair-Cell+Mechanotransduction+and+Cochlear+Amplification.">Hair-Cell Mechanotransduction and Cochlear Amplification.</a> Neuron. 48 (3), 403-415 (2005).</li><li>Neal, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+cell+counts+in+a+rat+model+of+sound+damage:+Effects+of+tissue+preparation+&amp;+identification+of+regions+of+hair+cell+loss.">Hair cell counts in a rat model of sound damage: Effects of tissue preparation &amp; identification of regions of hair cell loss.</a> Hear Res. 328, 120-132 (2015).</li><li>Ivory, R., Kane, R., Diaz, R. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospects+for+replacement+of+auditory+neurons+by+stem+cells.">Prospects for replacement of auditory neurons by stem cells.</a> Hear Res. 297, 106-112 (2013).</li><li>Mellott, A. J., Shinogle, H. E., Nelson-Brantley, J. G., Detamore, M. S., Staecker, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+decellularized+cochleae+as+scaffolds+for+inner+ear+tissue+engineering.">Exploiting decellularized cochleae as scaffolds for inner ear tissue engineering.</a> Stem Cell Res Ther. 8, (2017).</li><li>Santi, P. A., Johnson, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+ear+tissues+as+scaffolds+for+stem+cell+differentiation.">Decellularized ear tissues as scaffolds for stem cell differentiation.</a> J Assoc Res Otolaryngol. 14 (1), 3-15 (2013).</li><li>Davies, D., Holley, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+alpha+3+and+alpha+6+integrins+in+the+developing+mouse+inner+ear.">Differential expression of alpha 3 and alpha 6 integrins in the developing mouse inner ear.</a> J Comp Neurol. 445 (2), 122-132 (2002).</li><li>Gerchman, E., Hilfer, S. R., Brown, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+extracellular+matrix+in+the+formation+of+the+inner+ear.">Involvement of extracellular matrix in the formation of the inner ear.</a> Dev Dyn. 202 (4), 421-432 (1995).</li><li>Goodyear, R. J., Richardson, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrices+associated+with+the+apical+surfaces+of+sensory+epithelia+in+the+inner+ear:+molecular+and+structural+diversity.">Extracellular matrices associated with the apical surfaces of sensory epithelia in the inner ear: molecular and structural diversity.</a> J Neurobiol. 53 (2), 212-227 (2002).</li><li>Mellott, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+Viability+and+Transfection+Efficiency+with+Human+Umbilical+Cord+Wharton's+Jelly+Cells+Through+Use+of+a+ROCK+Inhibitor.">Improving Viability and Transfection Efficiency with Human Umbilical Cord Wharton's Jelly Cells Through Use of a ROCK Inhibitor.</a> Cell Reprogram. , (2014).</li><li>Mellott, A. J., Shinogle, H. E., Moore, D. S., Detamore, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Photo-conversion:+A+second+chance+to+label+unique+cells.">Fluorescent Photo-conversion: A second chance to label unique cells.</a> Cell Mol Bioeng. 8 (1), 187-196 (2015).</li><li>Mitchell, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+cells+from+Wharton's+jelly+form+neurons+and+glia.">Matrix cells from Wharton's jelly form neurons and glia.</a> Stem Cells. 21 (1), 50-60 (2003).</li><li>Mellott, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonviral+Reprogramming+of+Human+Wharton's+Jelly+Cells+Reveals+Differences+Between+ATOH1+Homologues.">Nonviral Reprogramming of Human Wharton's Jelly Cells Reveals Differences Between ATOH1 Homologues.</a> Tissue Eng Part A. 21 (11-12), 1795-1809 (2015).</li><li>Buytaert, J. A., Johnson, S. B., Dierick, M., Salih, W. H., Santi, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+versus+sTSLIM+3D+imaging+of+the+mouse+cochlea.">MicroCT versus sTSLIM 3D imaging of the mouse cochlea.</a> J Histochem Cytochem. 61 (5), 382-395 (2013).</li><li>Nonoyama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+ototoxicity+of+gadoteridol+(ProHance)+and+gadodiamide+(Omniscan)+in+mice.">Investigation of the ototoxicity of gadoteridol (ProHance) and gadodiamide (Omniscan) in mice.</a> Acta Otolaryngol. 136 (11), 1091-1096 (2016).</li><li>Dai, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhesus+Cochlear+and+Vestibular+Functions+Are+Preserved+After+Inner+Ear+Injection+of+Saline+Volume+Sufficient+for+Gene+Therapy+Delivery.">Rhesus Cochlear and Vestibular Functions Are Preserved After Inner Ear Injection of Saline Volume Sufficient for Gene Therapy Delivery.</a> J Assoc Res Otolaryngol. , (2017).</li><li>Sutherland, A. 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Vi har framgångsrikt visat att infödda cochlear celler kan tas bort från snäckan via en decellularization process, vilket möjliggör användning av snäckan som en intrikat, tredimensionella vävnad byggnadsställning. Santi et al. 15 utvecklade den första metoden för decellularizing cochleae, och har korrekt uppskattade volymerna för många cochlear strukturer genom med hjälp av lätta blad mikroskopi23. Sådana tidiga arbete fungerat som en stark grund f...

Snäckan är en intrikat spiralstrukturen Funna i tinningbenet. Den består av en yttre benig labyrint och en koncentrisk, inre membranös labyrint1. Membranös labyrinten består av tre flytande utrymmen: Scala vestibuli, Scala media och Scala tympani1. Scala media hus sensoriska epitelet, som består av en mängd celltyper, men sensoriska hårcellerna (HC), som transduce mekanisk energi i ljudvågor till nervimpulser2, är av särskilt intresse. Exponering för akustisk trauma3,4,5, medicinering6, sjukdom7,8och åldrande9 kan alla leda till nedsatt hörsel funktion via HC död. Hår cellförlust hos däggdjur är permanent, till skillnad från aviär HCs, som kan regenerera efter skada10.
En mängd samtida forskningsansträngningar har försökt att återställa förlorade HCs, även om de specifika experimentella metoderna varierar. Manipulation av genuttryck i sensoriska epitelet och implantation av stamcellerna differentierade utanför kroppen är dominerande ansatser inom, men induktion metoder som syftar till att differentiera stamceller till cochlear organoids har varit försökt11,12,13. Alla dessa metoder är antingen direkt beroende av stamceller eller utvecklingsmässiga ledtrådar används av stamceller; dock en andra delade, och potentiellt kritiska, elementet är ECM av snäckan själv14,15.
ECM ger inte bara fysiska stöd för celler och vävnad, som omfattar en yta för celladhesion, spridning, överlevnad och migration, men också spelar avgörande roller i utvecklingen av HCs och spiral ganglion15,16 ,17. Naturligt ger förekommande ECM induktiva signaler som kan vägleda cell fenotyp bestämning eller cell adhesion, spridning och överlevnad18. Därför, användningen av cell-lösa snäckan i kombination med odlade hWJCs erbjuder en unik möjlighet att utforska rollen av ECM och HC regeneration. HWJCs är en lättillgänglig, icke-kontroversiella celltyp som isolerats från mänskliga navelsträngar som beter sig som mesenkymala stamceller19. HWJCs har visat förmågan att skilja ned sensorineural cell härstamningar20,21. Således, det nuvarande protokollet uppgifter om isolering, decellularization och genomblödning av cochleae från C57BL mus slaktkroppar med hWJCs för innerörat vävnadsteknik.

<table>
	<tbody>
		<tr>
			<td>Allegra X-14R Centrifuge</td>
			<td>Beckman-Coulter</td>
			<td>B08861</td>
			<td></td>
		</tr>
		<tr>
			<td>Intramedic Semi-Rigid Tubing</td>
			<td>Becton Dickinson</td>
			<td>427401</td>
			<td></td>
		</tr>
		<tr>
			<td>New Brunswick Innova 2000 Orbital Shaler</td>
			<td>Eppendorf</td>
			<td>M1190-0002</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors</td>
			<td>Fine Science Tools</td>
			<td>14060-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11370-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>18155-13</td>
			<td></td>
		</tr>
		<tr>
			<td>50-mL Conical Tubes</td>
			<td>Fisher Scientific</td>
			<td>12565271</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Fisher Scientific</td>
			<td>FB087579B</td>
			<td></td>
		</tr>
		<tr>
			<td>U-100 Insulin Syringe</td>
			<td>Fisher Scientific</td>
			<td>14-829-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Scintillation Vial</td>
			<td>Fisher Scientific</td>
			<td>03-341-73</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td>Fisher Scientific</td>
			<td>88-861-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Pipette</td>
			<td>Fisher Scientific</td>
			<td>22-170-404</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor Blade</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Fisher Scientific</td>
			<td>15-240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Fisher Scientific</td>
			<td>15-140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Plate</td>
			<td>Fisher Scientific</td>
			<td>07-200-84</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost PLUS Glass Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Pipette</td>
			<td>Fisher Scientific</td>
			<td>22-170-404</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI</td>
			<td>Fisher Scientific</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear-Rite 3</td>
			<td>Fisher Scientific</td>
			<td>22-046341</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Forma Series II 3110 Water-Jacekted CO2 Incubator</td>
			<td>Fisher Scientific</td>
			<td>13-998-078</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesenchymal Stem Cell Growth Medium</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Lonza</td>
			<td>CC-3232</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP T-75 Culture Flask</td>
			<td>MidSci</td>
			<td>TP90076</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP T-150 Culture Flask</td>
			<td>MidSci</td>
			<td>TP90151</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP T-300 Culture Flask</td>
			<td>MidSci</td>
			<td>TP90301</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Microscope</td>
			<td>Nikon Instruments</td>
			<td>SMZ800</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ts2R-FL Inverted Microscope</td>
			<td>Nikon Instruments</td>
			<td>MFA51010</td>
			<td></td>
		</tr>
		<tr>
			<td>NuAire Class II, Type A2 Biosafety Cabinet</td>
			<td>NuAire</td>
			<td>NU-425-600</td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>Sigma-Aldrich</td>
			<td>P5368-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>1% SDS Solution</td>
			<td>Sigma-Aldrich</td>
			<td>436143-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>10% EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884-100G</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol for decellularizing mouse cochleae for inner ear tissue engineering

Hos däggdjur, mechanosensory hårcellerna som underlättar förhandlingen saknar förmågan att regenerera, som har begränsad behandlingar för hörselnedsättning. Nuvarande strategier för regenerativ medicin har fokuserat på transplantation av stamceller eller genetisk manipulation av omgivande stödjeceller i innerörat att uppmuntra utbyte av skadade stamceller att korrigera hörselnedsättning. Men den extracellulär matrixen (ECM) kan spela en viktig roll i inducerande och upprätthålla funktion av hårceller, och har inte undersökts väl. Med hjälp av cochlear kan ECM som en byggnadsställning växa adulta stamceller ge unika insikter i hur sammansättning och arkitekturen av den extracellulära miljön hjälper celler upprätthålla förhandlingen funktion. Här presenterar vi en metod för att isolera och decellularizing cochleae från möss att använda som ställningar att acceptera perfunderade adulta stamceller. I det nuvarande protokollet isoleras cochleae från euthanized möss, cell-lösa och urkalkade permanenta. Efteråt, var mänskliga Wharton's jelly celler (hWJCs) som var isolerade från navelsträngen noggrant perfusion in varje cochlean. Cochleae användes som bioreaktorer och celler odlades i 30 dagar innan de genomgick behandling för analys. Cell-lösa cochleae behöll identifierbar extracellulära strukturer, men avslöja inte förekomsten av celler eller märkbar fragment av DNA. Celler perfusion in i cochlean invaderade de flesta av interiör och exteriör av cochlean och växte utan incidenter under en löptid på 30 dagar. Dagskursmetoden kan således användas för att studera hur cochlear ECM påverkar cell utveckling och beteende.

Med hjälp av metoder som presenteras här, lyckad decellularization av cochleae bedömdes genom att undersöka närvaron eller frånvaron av DNA genom 4', 6-diamidin-2-fenylindol (DAPI) färgning. Cochleae ansågs fullt cell-lösa om DNA inte identifierades inom cell-lösa snäckan. En infödd snäckan från ett tidigare experiment som inte genomgår decellularization eller urkalkning användes som en positiv kontroll för att illustrera strukturer och celler som traditionellt finns i en...

Watch this Scientific Journal Video about A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering at JoVE.com

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

Ett protokoll för Decellularizing mus Cochleae för innerörat Tissue Engineering

Målet med detta protokoll är att demonstrera en effektiv metod för att decellularize och om avkalkning mus cochleae för utnyttjande som ställningar för tissue engineering program.

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

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.

Research

JoVE Journal

Bioengineering

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