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

현재 프로젝트 개념 펀드의 캔자스의 대학 증거에 의해 투자 되었다. 우리는 cochleae 문화 지원에 대 한 인간의 탯, 그리고 데이비드 조 젠 슨은 취득에 우리 위한 KUMC (캔사스 시, KS)에서 간호 직원을 감사 하 고 싶습니다.

동물 안락사를 포함 하 여 모든 절차는 승인된 기관 동물 관리 및 사용 위원회 (IACUC) 프로토콜 (ACUP #2014-2234) 캔자스의 대학 의료 센터 (KUMC)에 따라 실시 했다.
참고: HWJCs 동의 제공 하는 환자에 의해 기증 된 인간 탯 으로부터 격리 되었고 표본 캔자스의 대학 인간 주제 위원회 (구 IRB #15402)에 의해 승인 하는 프로토콜에 따라 사용 했다.
1. 측 뼈 수확과 달팽?...

<ol>
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우리는 성공적으로 복잡 한 3 차원 조직 발판으로 달팽이 관의 사용을 허용 하는 decellularization 과정을 통해 달팽이 관에서 네이티브 인공 세포를 제거할 수 있습니다 시연 했다. 산 티 외. 15 decellularizing cochleae에 대 한 초기 방법을 개발 하 고 정확 하 게 빛 시트 현미경23의 도움으로 많은 인공 구조를 통해 볼륨 견적 했다. 이러한 초기 작업 조직 엔지니어?...

달팽이 관은 측 뼈에서 발견 하는 복잡 한 나선형 구조. 그것은 외부 뼈 미로 동심, 내부 membranous 미로1로 구성. Membranous 미로 3 유체 공간 구성: 스칼라 vestibuli, 스칼라 언론과 Scala tympani1. 스칼라 미디어 하우스 수많은 종류의 세포로 구성 되어, 감각 상피, 하지만 감각 머리 세포 (HC), 신경 충 동2음파 기계적 에너지 transduce는 특정 관심의. 음향 외상3,,45, 약6, 질병7,8, 및 노화9 노출 모두 HC 죽음 통해 장애인된 청각 기능 발생할 수 있습니다. 포유류에서 머리 세포 손실 부상10후 재생성 수 조류 HCs 달리 영구적 이다.
다양 한 현대 연구 노력 하고자 했다 손실된 HCs 복원 특정 실험적인 접근 다르지만. 감각 상피에 유전자 발현의 조작 및 몸 밖에 서 분화 하는 줄기 세포의 주입은 필드에서 지배적인 접근 인공 organoids로 줄기 세포를 분화 하는 유도 방법 있다 지만 시도11,,1213. 이 방법의 각각은 줄기 세포 나 줄기 세포; 사용 발달 큐에 직접 의존 그러나, 두 번째 공유 하 고 잠재적으로 중요 한 요소는 달팽이 관 자체의 ECM14,15.
ECM 뿐만 아니라 세포와 조직, 세포 접착, 확산, 생존, 및 마이그레이션에 대 한 서피스를 포함 하지만 또한 HCs와 나선형 ganglion15,16의 개발에 중요 한 역할에 대 한 물리적 지원 제공 ,17. 자연스럽 게 발생 ECM는 세포 표현 형 결정 및 세포 접착, 확산, 그리고 생존18가이드 수 유도 신호를 제공 합니다. 따라서, 교양된 hWJCs와 함께에서 decellularized 달팽이 관의 사용 ECM HC 재생의 역할을 탐구 하는 독특한 기회를 제공 합니다. HWJCs는 중간 엽 줄기 세포19처럼 인간의 탯에서 고립 된 쉽게 사용할 수, 비 논란이 셀 유형입니다. HWJCs는 neurosensory 세포 계보20,21아래로 차별 하는 능력을 보여주었다. 따라서, 현재 프로토콜 절연, decellularization, 및 cochleae 내가 조직 공학에 대 한 hWJCs와 C57BL 마우스 시체에서의 관류 자세히 설명합니다.

<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

포유류에서 청각 부족을 재생성 하는 기능을 촉진 하는 mechanosensory 세포는 청력 손실에 대 한 치료를 제한 했다. 현재 재생 의학 전략 이식 줄기 세포 또는 주변 청각 상실을 해결 하기 위해 손상 된 줄기 세포의 교체를 장려 하는 내가 지원 세포의 유전자 조작에 집중 했다. 그러나, 기질 (ECM) 유도 하 고 세포의 기능 유지에 중요 한 역할을 재생할 수 있습니다 그리고 잘 조사 하지. 인공 와우를 사용 하 여 성체 줄기 세포를 성장 하는 발판으로 ECM 어떻게 구성 및 extracellular 환경 아키텍처 셀에 에이즈 청력 기능을 유지 하는 독특한 통찰력을 제공할 수 있습니다. 여기 우리는 분리 하 고 건설 기계 성인 줄기 세포 끼얹는다 수락으로 사용 하는 마우스에서 cochleae를 decellularizing 하는 방법을 제시. 현재 프로토콜에서 cochleae 안락사 쥐, decellularized, 그리고 석 으로부터 격리 됩니다. 그 후, 탯 줄에서 고립 된 인간의 튼 젤리 셀 (hWJCs) 각 달팽이 관에 끼얹는다 신중 하 게 했다. cochleae 생물 반응 기, 사용 하 고 셀 분석에 대 한 처리를 진행 하기 전에 30 일 동안 배양 되었다. Decellularized cochleae 식별 extracellular 구조를 유지 하지만 DNA의 현저한 조각 또는 셀의 존재를 공개 하지 않았다. 셀 달팽이 관에 끼얹는다 인테리어와 달팽이 관의 외관의 대부분을 침공 하 고 30 일의 기간 동안 사고 없이 성장 했다. 따라서, 현재 방법 어떻게 인공 ECM에 영향을 미치는 세포 발달 및 행동 연구를 사용할 수 있습니다.

여기에 제시 하는 방법을 사용 하 여, cochleae의 성공적인 decellularization 4'를 통해 DNA의 유무를 검사 하 여 평가 했다 6-diamidino-2-phenylindole (DAPI) 얼룩. Cochleae는 DNA 이었다 decellularized 달팽이 관 내에서 확인 되지 않는 경우 완전히 decellularized로 간주 됐다. Decellularization 또는 decalcification을 받을 하지 않았다 이전 실험에서 네이티브 달팽이 관 구조와 전통적으로 C57BL 마우스 달팽...

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

내가 조직 공학에 대 한 마우스 Cochleae Decellularizing에 대 한 프로토콜

이 프로토콜의 목표 decellularize 알려줍니다 마우스 cochleae 조직 공학 응용 프로그램에 대 한 공중 발판으로 이용 하는 효과적인 방법 설명 하는 것입니다.

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.

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