This work was supported by National Institutes of Health Grant to A.S.M. (EY021784).

다음 프로토콜은 토마스 제퍼슨 대학 기관 동물 관리 및 사용위원회 지침 및 비전 연구에서 동물의 사용에 대한 ARVO 방침을 준수합니다. 예 생체 상처 문화 렌즈 1. 설치 및 준비 <ol><li> 멸균, 층류 후드 세 100mm 페트리 접시를 놓습니다. 실온에서, 중간 트리스 / 우선 당 버퍼와 배양 접시 두 개를 (140 mM의 염화나트륨, 5 mM의 KCl을, 0.7 mM의 나 2 PO 4, 5 mM의 D-포도당, 8.25 mM 트리스 자료, 염산으로 pH 7.4까지 TD 버퍼)를 입력 세 번째 빈 떠나. 사전 따뜻한 배양 배지를 37 ℃로 O (미디어 (199)는, 1 % L- 글루타민 및 1 % 페니실린 / 스트렙토 마이신이 보충 된) 참고 : 생체 내에서 발생으로 문화 매체를 치유 표준 상처, 무 혈청이다 그러나, 상처 수복 배양 혈청 또는 다른 인자를 포함 한정 배지 조건에서 성공적으로 성장시킬 수있다. </li><li> 비옥 한 배아 D를 제거바깥 인큐베이터에서 15 화이트 레그혼 병아리 달걀 (부드러운 락과 37.7 ° C에서 개최) </li><li> 세척 병에서 층류 후드 70 % 에탄올로 쉘의 깨끗한 외부에서 선택한 달걀을 놓습니다. 멸균 솔루션과 장비를 사용하여 층류 후드에서 무균 조건 아래 모든 절차를 실시한다. </li><li> 빈 100mm 페트리 접시에 계란과 장소 내용을 균열. 표준 집게 및 미세 가위를 사용하여 배아 목을 벨. TD 버퍼를 포함하는 배양 접시에 병아리 배아의 머리를 놓고 제대로 배아의 나머지를 폐기하십시오. 선택적으로 15 분보다 더 짧은 시간에 대한 TD 버퍼 병아리 배아 헤드를 유지하지 않는다. </li><li> 페트리 접시 뚜껑에 병아리 배아 머리를 놓습니다. 고정밀 집게를 사용하여, 다음과 같은 순서로 눈로부터 부착 유리체와 함께 렌즈를 제거한다. 눈 뒤쪽의 작은 개구부를 생성하기 위해 집게 눈 뒤쪽 핀치. </li><li> 그런 다음, W 유리체 유머를 파악i 번째 집게 부드럽게 롤링 운동과 유리체에 예인선이 부착 된 렌즈 유리체는 눈에서 탈락됩니다. TD 버퍼를 포함하는 나머지 배양 접시에서 유리체 장소 렌즈 /. 렌즈는 더 이상 30 분 TD 버퍼에 남아 있도록 허용합니다. </li><li> 해부 현미경 새로운 배양 접시 뚜껑에 렌즈를 이동합니다. 이 시점부터 해부 현미경 아래의 모든 단계를 수행합니다. 고정밀 집게 조심스럽게 렌즈 조직이 손상되지 않도록주의를 복용, 집게의 가장자리를 사용하여 렌즈가 빠질 된 모든 모양체 (색소 세포)를 멀리 브러시. 참고 : 모양체 제거는 렌즈에 내생없는 종류의 세포가 상처 수리 문화에 포함되지 않도록합니다. </li><li> 후방 렌즈 캡슐과의 관계에서 유리체 몸을 곤란하게하여 고정밀 집게로 유리체 유머에서 렌즈를 분리합니다. </li><li> 높은 정밀도를 사용하여 작은 방울로 렌즈를 전송 겸자35mm 조직 배양 접시에 TD 버퍼 (약 200 μL)의. </li></ol> 2. 수행 모의 백내장 수술 <ol><li> 이 위를 향하게하여 렌즈의 전방 측면에 35mm 접시에 TD 버퍼의 드롭 렌즈의 방향을. 참고 : 렌즈의 앞쪽에 쉽게 렌즈 상피의 전방과 적도 지역의 경계를주의 조직의 밀도가 고리의 존재에 의해 식별됩니다. 대조적으로, 렌즈 섬유 세포가 부착되는 렌즈 캡슐의 후부에 마킹 부재가있다. </li><li> 각 손에 하나 집게로 조직을 파지하여 두 고정밀 집게는 전방 렌즈 캡슐 렌즈 조직을 둘러싸는 굵은 기저막 및 관련 전방 렌즈 상피의 중앙에 작은 절개 (약 850μm)하게 사용 부드럽게 반대 방향으로 잡아 당 겼. </li><li> 렌즈 용 티슈, dislod의 대부분을 구성하는 섬유의 균체를 분리렌즈 상피 세포에 첨부 파일에서 그것을 카 및 (그림 1A에서 모델링 고전적인 백내장 수술에 사용되는 방식) 수력 용출에 의해 수정체 낭 주변. </li><li> TD 버퍼 300 μL와 27.5 G 바늘 끝 1 ML의 주사기를 채우십시오. 앞쪽에 렌즈 캡슐에서 만든 절개로 바늘 끝을 삽입하고 대한 중간 렌즈로. </li><li> 부드럽게 렌즈 섬유 세포 덩어리로 TD 버퍼를 주입 주사기 우울. 50 ~ 200 μL의 TD, 결코 300 개 이상의 μl를 주입한다. 상피 및 렌즈 캡슐에서 자신을 풀어 섬유 세포 질량을 관찰한다. </li><li> 고정밀 집게를 사용하여, 전방 절개 부위를 통해 렌즈로부터 풀어 섬유 세포 덩어리를 제거한다. 참고 :이 절차는이 사이트에 바로 인접한 섬유 세포가 부착 된 세포의 무 결함했다되는 후방 렌즈 기저막 캡슐 및 부상 렌즈 상피 세포를 떠난다. </li></ol> 3. Preparin전의 VIVO 문화 G 부상 렌즈 <ol><li> 낭의 전방 측면에서 다섯 삭감하여, 배양 접시에 위에서 설명한 백내장 수술, 셀 사이드의 결과 렌즈 낭을 평평하게. </li><li> 렌즈의 균분 원을 통해 절개 부위에 수직으로 잘라. 아래로 배양 접시 캡슐 측에 부착 된 상피 세포 쪽이 위로 렌즈 캡슐의 결과 다섯 &quot;날개&quot;를 평평하게. 스타 또는 flowerlike 모양 (그림 1B 참조)해야 지금 생체 부상 렌즈를합니다. </li><li> 별의 각 지점에서 부드럽게 아래로 집게, 접시를 눌러 캡슐을 확보합니다. 이 이식편의 다섯 가장 바깥 끝에서 작은 들여 쓰기를하고 접시에 지속적인 첨부 파일에서 발생합니다. 주 :이 절차 중에 캡슐 손상 가능하며 그래서 FLA의 선단에 가깝게 접시에 캡슐을 고정하는 것이 중요가능한 PS뿐만 아니라 가능한 확보 지점 최소량을 만들기 위해 (일반적으로 두 플랩 당 최대 세). </li><li> 35mm 접시에서 TD 버퍼를 제거하고 미리 예열 미디어 1.5 ㎖로 교체합니다. 인큐베이터에서 뚜껑과 장소 35mm 접시 커버 (37 ° C를, 5 % CO 2). </li></ol> 후방 캡슐의 부상 영역의 재 상피화가 정량 분석​​을위한 렌즈 상피 세포의 원래 첨부 지대 (OAZ)에서 발생 중앙 마이그레이션 지대 (CMZ), 4. 분리. 참고 : 세포 손상에 대한 응답으로 즉시 CMZ 영역으로 이동하기 시작한다. 문화의 날 하나 충분한 세포는 미세 해부 (23)에 의해 CMZ와 OAZ의 분리 다음, 분자, 생화학 적 분석을 위해 CMZ를 통해 마이그레이션. 이 프로토콜은 캡슐의 상처 영역으로부터 한 번에 하나의 플랩 (OAZ)의 제거를 수반한다. <ol><li> demar을 준수양이온과 OAZ CMZ 사이, 해부 현미경 명확하게 볼 (도 2a 참조). 두 고정밀 집게를 사용하여, 라인의 양측에, OAZ / CMZ 라인에 바로 인접한 다른 하나의 에지에서 두 집게로 잡고 (도 2A, B, 화살표 참조). </li><li> 한 손으로 / CMZ 측의 한 집게는 다른 손 / 집게로 부드럽게 OAZ / CMZ 라인을 따라 OAZ을 끌어 동안, 부상자 문화에 개최하는 것을 계속한다. CMZ 쉽게이 선을 따라 OAZ에서 분리합니다. 두 지역이 완전히 분리 될 때까지 전체 문화 주위에이 라인을 따라 계속합니다. </li><li> 이러한 웨스턴 블롯 또는 공동 면역 (23, 25)와 같은 RNA-SEQ (24) 또는 생화학 적 분석과 같은 분자 분석을위한 분리 OAZ 및 CMZ 분수를 공부한다. </li></ol>

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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=Distinct+roles+for+N-Cadherin+linked+c-Src+and+fyn+kinases+in+lens+development.">Distinct roles for N-Cadherin linked c-Src and fyn kinases in lens development.</a> Developmental dynamics : an official publication of the American Association of Anatomists. 242, 469-484 (2013).</li><li>Sieg, D. 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=FAK+integrates+growth-factor+and+integrin+signals+to+promote+cell+migration.">FAK integrates growth-factor and integrin signals to promote cell migration.</a> Nature cell biology. 2, 249-256 (2000).</li><li>Sieg, D. J., Hauck, C. R., Schlaepfer, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Required+role+of+focal+adhesion+kinase+(FAK)+for+integrin-stimulated+cell+migration.">Required role of focal adhesion kinase (FAK) for integrin-stimulated cell migration.</a> Journal of cell science. 112 (Pt 16), 2677-2691 (1999).</li><li>Hauck, C. R., Hsia, D. A., Schlaepfer, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+focal+adhesion+kinase--a+regulator+of+cell+migration+and+invasion).">The focal adhesion kinase--a regulator of cell migration and invasion).</a> IUBMB life. 53, 115-119 (2002).</li><li>Zhao, X., Guan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+adhesion+kinase+and+its+signaling+pathways+in+cell+migration+and+angiogenesis.">Focal adhesion kinase and its signaling pathways in cell migration and angiogenesis.</a> Advanced drug delivery reviews. 63, 610-615 (2011).</li><li>Menko, A. S., Bleaken, B. M., Walker, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional-specific+alterations+in+cell-cell+junctions,+cytoskeletal+networks+and+myosin-mediated+mechanical+cues+coordinate+collectivity+of+movement+of+epithelial+cells+in+response+to+injury.">Regional-specific alterations in cell-cell junctions, cytoskeletal networks and myosin-mediated mechanical cues coordinate collectivity of movement of epithelial cells in response to injury.</a> Experimental cell research. 322, 133-148 (2014).</li><li>Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing--aiming+for+perfect+skin+regeneration.">Wound healing--aiming for perfect skin regeneration.</a> Science. 276, 75-81 (1997).</li><li>Ferguson, M. W., O'Kane, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scar-free+healing:+from+embryonic+mechanisms+to+adult+therapeutic+intervention.">Scar-free healing: from embryonic mechanisms to adult therapeutic intervention.</a> Philosophical transactions of the Royal Society of London. Series B, Biological. 359, 839-850 (2004).</li><li>Redd, M. J., Cooper, L., Wood, W., Stramer, B., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+and+inflammation:+embryos+reveal+the+way+to+perfect+repair.">Wound healing and inflammation: embryos reveal the way to perfect repair.</a> Philosophical transactions of the Royal Society of London. Series B, Biological. 359, 777-784 (2004).</li><li>Nodder, S., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+in+embryos:+a+review.">Wound healing in embryos: a review.</a> Anatomy and embryology. 195, 215-228 (1997).</li><li>Gurtner, G. C., Werner, S., Barrandon, Y., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair+and+regeneration.">Wound repair and regeneration.</a> Nature. 453, 314-321 (2008).</li></ol>

The authors declare that they have no competing financial interests.

Here is described a technique for preparing a culture model of wound repair that involves performing an ex vivo cataract surgery on chick embryo lenses after their removal from the eye. The lens epithelium responds to this clinically relevant wounding with a repair process that closely mimics that which occurs in vivo, and shares features with wound repair in other epithelial tissues2,4. While the protocol is straightforward and simple to follow, performing mock cataract surgery with embryoni...

임상 적으로, 모의 백내장 수술은, 여기에서 설명한 생체 상피 상처 치유 모델 부상에 응답하여 상피 조직의 복구를 조절 메카니즘을 조사하기위한 도구를 제공하기 위해 개발되었다. 이 모델을 창출을 목적으로 한 주요 기능은 밀접하게 복구 프로세스를 문화 설정, 2), 수리의 규제 요소를 변조의 완화에 부상에 대한 생체 반응을 복제하고 이미지 3) 기능 1) 제공하는 조건을 포함 실시간 전체에서. 과제는, 따라서, 세포의 미세 환경에서 원시 상피 상처 복구를 연구하는 것이 가능시킨 배양 모델을 생성하고 조작하는 것이었다. 이 상처 수리 모델의 유용성은 복구 프로세스를 조절하는 매트릭스 단백질, 사이토 카인 및 케모카인에서 내인성 시그널링 신호를 식별하기위한 새로운 가능성을 연다. 또한, 모델을 조사하기위한 이상적인 방법N 상피 창상 면적 2,3-을 다시 epithelialize하는 단체 시트 같이 부상 상피 4 집단 이주를 연출 기능 감긴 가장자리 간엽 리더 셀의 혈통을 결정하기위한 이동시킬 수있다. 이 모델은 또한 효과적인 상처 치유를 촉진하고 비정상적인 상처 수리 (5)을 방지 할 수 치료제를 식별하는 데 사용할 수있는 플랫폼을 제공한다. 가능한 상처 수리 모델의 수는 문화와 오늘 상처 복구 프로세스에 대해 알려진 대부분을 제공 한 생체, 모두, 이미있다. 이러한 각막 6-12 및 13-17 등 피부 손상 동물 모델에서의 기여를 포함하는 과정에 관여 할 수있는 모든 수리 매개체의 맥락에서 부상으로 조직의 반응을 연구 할 수있는 기회가있다 혈관 및 신경계. 그러나, experi 조작에 한계가있다생체 내에서의 정신적 상태 및 연속적으로 시간이 지남에 따라, 생체 내에서 수리 응답의 영상 검사를 실시 아직 가능하지 않다. 대조적으로, 이러한 스크래치 상처와 같은 대부분의 시험 관내 상처 수복 배양 모델들은 쉽게 조작 될 수 있고, 시간이 지남에 따라하지만 생체 조직에 상처 치유 공부의 환경 문맥이 부족하다. 생체 외 모델이 프로세스의 모든 시점에서 수리 분자 조절기를 조절하는 능력과 결합 된 세포의 미세 환경의 콘텍스트에서 시간에 걸쳐 연속적으로 부상 복구 프로세스를 연구하는 이점을 제공하지만, 이들에 맞 몇몇 모델이 있습니다 매개 변수를 설정합니다. 여기에 생리 상처 상피 조직의 반응을 재현 배양 치유 재현성 높은 생체 상피의 상처를 생성하는 절차를 설명한다. 조직 공급원, 생체 MOC으로 병아리 배아 렌즈 사용K 백내장 수술이 행해진 다. 렌즈는 무 혈관, 신경 지배, 및 관련 기질 (18,19)의 자유되지는 자체 포함 두꺼운 기저막 캡슐 내에 있기 때문에이 연구에 사용하는 이상적인 조직이다. 인간의 질환, 백내장 수술로 인해 렌즈의 혼탁으로 시력 상실을 해결하고, 렌즈의 대부분을 포함하는 렌즈 섬유 세포 덩어리의 제거를 포함한다. 다음은 백내장 수술의 비전은 인공 수정체의 삽입을 통해 복원됩니다. 백내장 수술 절차, 섬유 세포의 제거를 통해, 섬유 세포에 의해 점유되었던 렌즈 캡슐의 후방 영역의 재 상피화함으로써 응답 인접한 렌즈 상피의 상처 반응을 유도한다. 백내장 수술에서 가장 상처 복구 응답 같이 때때로 렌즈 후방 Capsu로 알려져 근육 섬유 모세포의 출현과 관련된 창상 치유 반응을 비정상적 섬유화 결과,이 발생제작 혼탁 20-22. 백내장 수술 상처 치유 모델을 생성하기 위해, 백내장 수술 절차는 생리 부상을 생성 병아리 배아로부터 제거 눈 렌즈에 모방한다. 렌즈 상피 세포에 의해 둘러싸인 매우 일관된 원형 상처 영역에서 렌즈 섬유 세포 결과 미세 수술 제거. 이 세포 집단 단단히 렌즈 기저막에 부착 된 캡슐 유지되며 외과 적 절차에 의해 부상한다. 상피 세포는 리더 셀 1로 복구 프로세스에 공지 멘틴 풍부한 간엽 세포 집단 이끄는 상처를 치유하는 내인성 기저막의 무 결함 영역으로 이동한다. 이 모델 부상 상피의 응답 쉽게 시각화 할 수 있고, 세포의 미세 환경의 콘텍스트에서 시간에 따라. 세포 발현 또는 상처 복구하는 역할을 할 것으로 기대 분자의 활성화의 변형에 용이하게 접근 할 수있다. 일의 강력한 기능모델 분리 및 상처 치유의 틀에서 마이그레이션 별 변화를 연구 할 수있는 기능입니다. 연구를 위해 세 유사한 생체 상처 치유 배양 다수를 준비하는 능력은이 모델의 다른 이점이다. 따라서,이 모델 시스템은 상처 치유 과정에 미치는 영향에 대한 상처 수리의 메커니즘과 테스트 치료제 떨어져 애타게 할 수있는 독특한 기회를 제공합니다. 생체 모의 백내장 수술 모델은 손상 복구 메커니즘의 연구를위한 중요한 자원을 제공하는, 다양한 적용 가능성을 가질 것으로 기대된다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Use at 140mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td>Use at 5mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Sodium Phosphate (Na2HPO4)</td>
			<td>Sigma</td>
			<td>S0876</td>
			<td>Use at .7mM in TD Buffer</td>
		</tr>
		<tr>
			<td>D-glucose (Dextrose)</td>
			<td>Fisher Scientific</td>
			<td>D16-500</td>
			<td>Use at 0.5mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td>Use at 8.25mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144-500</td>
			<td>Use to pH TD buffer to 7.4</td>
		</tr>
		<tr>
			<td>Media 199</td>
			<td>GIBCO</td>
			<td>11150-059</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Corning/CellGro</td>
			<td>25-005-CI</td>
			<td>Use at 1% in Media199</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Corning/CellGro</td>
			<td>30-002-CI</td>
			<td>Use at 1% in Media199</td>
		</tr>
		<tr>
			<td>100mm petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875711Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Filter Unit</td>
			<td>Millipore</td>
			<td>SCGPU01RE</td>
			<td>Use to filter sterilize Media</td>
		</tr>
		<tr>
			<td>Dumont #5 forceps (need 2)</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm Cell Culture Dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>27 Gauge 1mL SlipTip with precision glide needle</td>
			<td>BD</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Forceps</td>
			<td>Fine Science Tools</td>
			<td>91100-12</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Other Items Needed: General dissection instruments,&#160; fertile white leghorn chicken eggs,&#160;</td>
		</tr>
		<tr>
			<td colspan="4">check egg incubator (humidified, 37.7&#176;C), laminar flow hood, binocular stereovision dissecting&#160;</td>
		</tr>
		<tr>
			<td>microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of a clinically relevant ex vivo mock cataract surgery model for investigating epithelial wound repair in a native microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

예 생체 내 모델은 세포의 기본 미세 환경에서 상처 치유 과정을 연구하기 위해 만들어 세포의 기본 미세 환경 내에서 상피의 상처 치유 조절에 관여하는 메커니즘을 조사하기 위해, 임상 적으로 생체 모의 백내장 수술 모델을 만들었습니다. 이 모델은 그의 고유 특성 때문에 많은 이점을 제공 렌즈 조직으로부터 생성된다 : 1) 렌즈 수정체 낭 불리는 두꺼운 기?...

Watch this Scientific Journal Video about Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment at JoVE.com

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

임상 적으로 구축<em&gt; 전의 VIVO</em기본 미세 환경에서 상피 상처 복구 조사를위한&gt; 모의 백내장 수술 모델

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

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Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

6.5K 조회수.  Thomas Jefferson University. Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment. 

비디오: Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

시간 경과 브라이트 실체 현미경으로 Zebrafish의 애벌레에서 조직 복구를 캡처

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

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Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

심 외막 파생물 문화 분석 및<em&gt; 전의 VIVO</em&gt; 심 외막 유래 세포 마이그레이션 평가

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

당뇨병 각막에 대한 아데노 바이러스 유전자 치료 : 인간 장기 배양 각막 및 윤부 상피 세포에 상처 치유에 효과 및 세포 마커의 발현 줄기

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

장기를위한 잠수 필터 종이 샌드위치<em&gt; 예 비켜</em초기 병아리 태아의&gt; 시간 경과 영상

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

약물 치료와<em&gt; 생체</em&gt;은 메다카 물고기 골다공증 모델에서 조골 파골 세포 상호 작용의 영상

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

BMPR-IB +의 신속한 격리 두개골 결함 치유 모델에서 사용을위한 기질 세포를 지방이는 유래

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

96 웰 플레이트에 세포 마이그레이션 / 상처 치유를 수행하기위한 장치

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

조사 Trypanosoma cruzi 운동 비보 를 동물 모델로 서 애벌레 Zebrafish의 설립

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

Microtubules 및 전 비보 장 조직 및 장 Organoids 3 차원 생체 외에서 Centrosomal 단백질의 면역 형광 라벨

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch&#039;s Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

세 인간 Bruch 막의 Ex Vivo 모델에서 망막 안료 상피 세포의 배양

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...