Name: in vitro time-lapse live-cell imaging to explore cell migration toward the organ of corti
Uploaded: 2020-12-04T14:45:00
Description: Watch this Scientific Journal Video about In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti at JoVE.com

이 작품은 한국국립연구재단(NRF)과 한림대학교 연구기금(NRF)의 연구보조금(NRF-2018-R1D1A1B07050175, HURF-2017-66)에 의해 지원되었다.

ICR 마우스와 관련된 모든 연구 프로토콜은 원주의 의과대학의 연세대학교 기관동물관리및이용위원회(IACUC)의 승인을 받았습니다. 실험은 세계 의학 협회의 윤리 강령에 따라 수행되었다. 이 프로토콜에서 임신 한 ICR 마우스는 음식과 물에 무료로 액세스 할 수있는 12/12 h 빛 / 어두운 주기에 보관되었습니다.
1. 달팽이관 해부
<ol><li>30분 동안 자외선을 켜서 라미나르 유동 조?...

<ol>
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손상된 세포의 재생을 촉진하기 위해 손상된 부위로 MSC를 이식하는 것은 광범위하게 연구되었으며 치료 효과가 분명합니다. MSC의 이식 및 후속 분화는 3-니트로프로피온산(13)에 의해 유도된난청을 가진 쥐의 청력을 회복하는 것으로 보고되었다. Lee et al.은 인간에게 MSC를 적용했지만,14번 청각에서 상당한 개선을 이루지 못했습니다. 최근까지, MSC 이식에 의한 ?...

청력 손실은 선천적으로 발생할 수 있으며 노화, 약물 및 소음을 포함한 여러 가지 요인에 의해 점진적으로 발생할 수 있습니다. 청력을 담당하는 모발 세포가 손상되면 손상된 기능을 복원하기가 매우 어렵기 때문에 청력 손실은 종종 치료하기가어렵습니다. 세계보건기구(WHO)에 따르면 전 세계 4억 6,100만 명이 청력 손실을 입은 것으로 추정되며, 이는 전 세계 인구의 6.1%를 차지합니다. 청력 상실을 가진 사람들 중 93%는 성인이고 7%는 어린이입니다.
청력 손실을 치료하기 위해 여러 가지 접근 법이 시도되었습니다. 특히, MSC를 이용한 재생 접근법은 유망한 치료법으로 부상했습니다. 조직이 손상되면, MSC는 자연적으로 순환 계통으로 방출되고 다양한 분자를 분비하는 상해 부위로 이동하여 재생을 촉진하는 미세 환경을 형성한다2. 따라서, 손상된 세포 기능3, 4, 5의 복원을 향상시키기 위해 강력한 면역 조절, 혈관신생 및 항 세포멸을 유발하는 분자의 장기 및 후속 분비를 표적으로 하는 외부 이식된 MSC의 이동을 통해 손상된 조직을 치료하는 방법을 개발하는 것이 중요하다3,4,5.
MSC가 손상된 조직으로 마이그레이션하는 호밍 프로세스가 극복해야 할 가장 중요한 장애물이 될 수 있습니다. MSC는 테더링 /롤링, 활성화, 체포, 트랜스 마이그레이션 / diapedesis 및 마이그레이션6,7,8의순차적 단계를 갖는 전신 호밍메커니즘을가지고 있습니다. 현재 이러한 단계를 개선할 방법을 파악하기 위한 노력이 진행 중입니다. 유전자 변형, 세포 표면 공학, 체외 프라이밍 및 자기 지도를 포함한 다양한 전략이6,7로테스트되었습니다. 또한 손상된 달팽이관 부위에 MSC를 호밍하여 청각 모발 세포의 보호 및 재생을 촉진하기 위한 여러 시도가 있었습니다. 그러나 생체 내에서 MSC를 추적하는 것은 시간이 많이 걸리고 노동 집약적이며 고도로 전문화된 기술9이필요합니다.
이러한 문제점을 해결하기 위해, 몇 시간 동안 세포의 이주를 촬영한 시간 경과 공초점 현미경검사를 통해 달팽이관내의 MSC의 호밍을 관찰하는 방법이 개발되었다(도1). 그것은 20세기 초에 개발 되었으며 최근 특정 세포의 마이그레이션을 공부 하기 위한 강력한 도구가 되고있다.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61947/61947fig01.jpg" style="font-size: 14px;"> 도 1: 그래픽 추상화. (A)코르티의 해부 기관이 집게를 사용하여 플라스틱 커버슬립에 부착된 후, 커버슬립은 35mm 유리 바닥의 공초점 미세한 접시에 배치되고(B)유리 실린더가 배치된다. (C)유리 실린더 내부를 중간으로 채운 후,(D)GFP 라벨이 부착된 MSC를 매체로 기르기 바깥에 신중하게 첨가한다. (E)하룻밤 동안 인큐베이션 후유리실린더가 제거되고, 이미지는 공초점 현미경으로 촬영된다. 약어: GFP = 녹색 형광 단백질; MSC = 중간 엽 줄기 세포. <a href="https://www.jove.com/files/ftp_upload/61947/61947fig01large.jpg" style="font-size: 14px;" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10X PBS Buffer</td>
			<td>GenDEPOT</td>
			<td>P2100-104</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Formalin</td>
			<td>T&#38;I</td>
			<td>BPP-9004</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>sigma&#160;</td>
			<td>A5354-10ml</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>sigma&#160;</td>
			<td>A4503-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>confocal dish</td>
			<td>SPL</td>
			<td>200350</td>
			<td></td>
		</tr>
		<tr>
			<td>confocal microscope&#160;</td>
			<td>ZEISS</td>
			<td>LSM800</td>
			<td></td>
		</tr>
		<tr>
			<td>coverslip</td>
			<td>SPL</td>
			<td>20009</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>10565-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher scientific</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorsheild with DAPI</td>
			<td>sigma&#160;</td>
			<td>F6057</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>Dumont</td>
			<td>0508-L5-P0</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Thermo Fisher scientific</td>
			<td>14065056</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin-iFluor 647 Reagent</td>
			<td>abcam</td>
			<td>ab176759</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage Top Incubator</td>
			<td>TOKAI HIT</td>
			<td>WELSX</td>
			<td></td>
		</tr>
		<tr>
			<td>Strain C57BL/6 mouse messenchymal stem cells with GFP</td>
			<td>cyagen</td>
			<td>MUBMX-01101</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>sigma&#160;</td>
			<td>T8787</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro time-lapse live-cell imaging to explore cell migration toward the organ of corti

세포 재생 및 치료에 대한 중간엽 줄기 세포 (MSC)의 효과를 연구하기 위해이 방법은 달팽이관 상피와 공동 배양 후 MSC 마이그레이션 및 형태 학적 변화를 추적합니다. 코르티의 장기는 해부 중에 생성된 Reissner 막의 일부를 눌러 플라스틱 커버슬립에 고정되었습니다. 실린더를 제거할 때 유리 실린더에 의해 수감된 MSC가 달팽이관 상피로 이동했습니다. 그들의 지배적인 국소화는 코르티의 기관의 modiolus에서 관찰되었다, 신경 섬유의 것과 유사한 방향으로 정렬. 그러나 일부 MsMC는 림푸스 지역에 국한되어 수평으로 길쭉한 모양을 보였습니다. 또한, 모발 세포 영역으로의 이동이 증가되었고, MSC의 형태는 가나마이신 치료 후 다양한 형태로 변경되었다. 결론적으로, 본 연구의 결과는 달팽이관 상피와 MSC의 공존이 세포 이식을 통해 치료의 발달과 다양한 조건과 요인을 검사 할 수있는 세포 재생연구에 유용 할 것임을 나타냅니다.

3차원 모드에서 MSC의 체외 이동은 트랜스웰 시스템 또는 전통적인 상처 치유 방법에 의해 2차원(2D)모드(11)에서마이그레이션을 관찰하도록 평가되었다. 코르티의 기관은 보처 세포, 클라우디우스 세포, 디터 세포, 기둥 세포, 헨센의 세포, 외부 헤어 셀, 내부 모발 세포, 신경 섬유, 바실라 막 및 망상 라미나12와같은 다양한 세포로 구성된...

Watch this Scientific Journal Video about In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti at JoVE.com

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

본 연구에서는, 우리는 코르티의 기관을 포함하는 달팽이관 상피와 전 생체 배양에 의해 손상된 조직을 향해 이동하는 세포를 관찰하는 공초점 현미경을 사용하여 실시간 이미징 방법을 제시한다.

코르티 의 장기를 향해 세포 마이그레이션을 탐구하는 체외 시간 경과 라이브 세포 이미징

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after ...

Existing in vitro method to investigate migration of MSCs include Transwell or wound healing protocols. The method described here can be used to track migration of MSCs to explant in real time. In terms of culture of explant, the advantage of this method compared to others is that it achieves a reduction in time and cost, as well as a simplification of experimental steps.This technique can be used to observe stem cell homing to target in real time leading to development of models with optimized homing efficiency. Removal of tectorial membrane is difficult for unexperienced researchers. Depending on the development stage, understanding the right time to remove the tectorial membrane is important for the success of this protocol.Demonstrating the procedure will be Dr.Jeong-Eun Park and Dr.Su Hoon Lee, researcher professors from my lab. Begin by sterilizing the laminar flow tissue culture hood by turning on the ultraviolet light for approximately 30 minutes. Spray all surfaces with 70%ethanol prior to use and allow them to dry.Place dissection instruments in 70%ethanol for 10 minutes, then dry them before using. After decapitating a three to four-day-old mouse, soak the ahead in 70%ethanol and place it under a serial microscope in the laminar flow hood. Quickly soak the tissue in tissue dissection solution to remove the ethanol.Cut the center line of the skull with the surgical blade, then expose the skull by pulling down the skin anteriorly and cutting the external auditory canal of the ear. Cut from the anterior to the posterior part of the cranium across the eye line, then open the skull and remove the forebrain, cerebellum, and brainstem with blunt forceps. Using microforceps, separate the cochlea from the temporal bone and transfer it to a Petri dish containing tissue dissection solution.Carefully dissect all of the cochlear otic capsule leaving only the internal cochlear soft tissue. Hold the modiolus of the cochlea with forceps and the cochlear duct with another pair of forceps, then slowly separate the two tissues. Remove the stria vascularis and tectorial membrane by gently peeling them away.Place a sterilized plastic coverslip in new tissue dissection solution, then place the organ of Corti on the coverslip making sure that the basilar membrane faces downward. Immobilize the tissue by pressing the Reissner's membrane and the remaining modiolus tissue onto the coverslip with forceps. Transfer the coverslip with the embedded tissue to the center of a 35 millimeter confocal dish.Place the glass cloning cylinder on the dish with the cochlear explant positioned in the center of the dish and add 100 microliters of explant culture medium into the cylinder. Place 5, 000 bone marrow derived GFP-tagged mesenchymal stem cells or MSCs in two milliliters of culture medium outside the glass cylinder and put the dish in the incubator. After culturing the cells as described in the text manuscript, aspirate all medium inside and outside the cylinder and remove the glass cylinder from the confocal dish.Add two milliliters of fresh culture medium to the dish. Place it in a humidified incubator until ready for analysis. Use a confocal microscopy system with a stage top incubator.Turn on the confocal microscope, fluorescent light and computer. Set the conditions of the stage top incubator to 37 degrees Celsius and 5%carbon dioxide atmosphere. Place the sample dish on the dish fixing vessel, cover it with the dish fixing lid and close the chamber with the top heater lid.Adjust the zoom and focus to localize the organ of Corti and MSCs in the field of view. Open the image processing software. Under the locate option, select a 20X plan-apochromat objective or numerical aperture 0.8 and a 0.5X crop area.Under acquisition, click on smart setup and select EGFP. Open the channel tab under acquisition and set the laser power to 0.2%the pinhole to 44 micrometers, the master gain to 750 volts, and the digital gain to 1.0. Click on ESID under imaging setup and set the ESID gain to four and the digital gain to 7.5.Click on tiles and stake to produce 210 tiles. Open focus strategy and select the focus mode. Under time series, set the duration to 24 hours and the interval to 10 minutes.Under acquisition, set the frame size to 512 by 512 pixels, scan speed to eight, the direction to bidirectional, the averaging to 4X, and the bits per pixel to 16. Click on start experiment to begin the experiment. The two-dimensional path of mesenchymal stem cells or MSCs was tracked in a wound healing method by using a glass cylinder as a barrier between the MSCs and the organ of Corti.When the organ of Corti was cultured, fibroblasts grew very quickly outward from the outer hair cells in the outermost layer. Most GFP-labeled MSCs were pushed out by fast-growing fibroblasts. Nonetheless, some penetrated the layer of fibroblasts and successfully migrated to the organ of Corti.The results from 72 hours of incubation showed that MSCs migrated to the organ of Corti and were mainly localized in the modiolus and the area with the cochlear nerve fibers separated from the modiolus. The morphology of the MSCs was altered to a radial shape similar to that of nerve fibers. The cells were transformed into a linear shape along the limbus line, rather than the hair cell area.The MSCs were able to migrate and grow in the nerve fiber collection area by penetrating the various cell layers and physical barriers. When damage to hair cells was induced by a 16-hour treatment with kanamycin and cells were cultured for a further 72 hours after the removal of kanamycin, MSCs were localized not only in the modiolus, but also in the outer hair cells. Immobilizing the tissue is an important step in this protocol was the organ of Corti is attached.It does not fall off easily. This is a trivial, but helpful tip for successful explant culture. This method may be adapted to study the efficacy of exosomes secreted from MSCs.

Research

JoVE Journal

Medicine

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

Compartmentalizing 마이크로 유체 장치와 라이브 세포 이미징을 사용하여 암 줄기 세포 마이그레이션 평가

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

연구

의학

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

종양의 4D 라이브 세포 이미징을위한 MAME 모델 : Microenvironment 상호 작용 영향을 미치는 악성 진행

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

사전 임상 연구에서 Pharmacological - 도전 fMRI의 사용 : 5-HT 시스템 신청

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

기본 Preneoplastic 유방 상피 세포의 시간 경과 영상은 유방암의 유전자 변형 마우스 모델에서 파생

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

개인 신장 세포 암에서 단백질 표현 변화를 탐험하기 위해 역 위상 단백질 배열 (RPPA)의 사용

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

치과 복합 재료의 Cytocompatibility 평가하기위한 효율적인 방법으로 공 촛점 시간 경과 영상

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Bowditch 효과 조사 : 압력 볼륨 관계를 측정하기 위해 마우스의 심장 도관

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Lepr<sup&gt; DB</sup&gt; 2 형 당뇨병의 마우스 모델 : 본래 독도 췌장 섬 세포의 분리 및 라이브 셀 2 광자 이미징

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

유리를 통해 보면 : 시간 ​​경과 현미경 및 단일 세포의 종 추적 항암 치료를 공부하기

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

종양 미세 환경에서 실시간 다세포 역학의 확장 시간 경과 생체 내에 영상

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...