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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<li>Chauss, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+state-specific+mitochondrial+dynamic+regulatory+networks+are+revealed+by+global+transcriptional+analysis+of+the+developing+chicken+lens%5BTitle%5D)+AND+%22G3+%28Bethesda%29%22%5BJournal%5D)">Differentiation state-specific mitochondrial dynamic regulatory networks are revealed by global transcriptional analysis of the developing chicken lens.</a> G3 (Bethesda). 4, 1515-1527 (2014).</li>
<li>Leonard, M., Zhang, L., Bleaken, B. M., Menko, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+roles+for+N-Cadherin+linked+c-Src+and+fyn+kinases+in+lens+development%5BTitle%5D)+AND+%22Developmental+dynamics+%3A+an+official+publication+of+the+American+Association+of+Anatomists%22%5BJournal%5D)">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/pubmed?term=((FAK+integrates+growth-factor+and+integrin+signals+to+promote+cell+migration%5BTitle%5D)+AND+%22Nature+cell+biology%22%5BJournal%5D)">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/pubmed?term=((Required+role+of+focal+adhesion+kinase+%28FAK%29+for+integrin-stimulated+cell+migration%5BTitle%5D)+AND+%22Journal+of+cell+science%22%5BJournal%5D)">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/pubmed?term=((The+focal+adhesion+kinase--a+regulator+of+cell+migration+and+invasion%29%5BTitle%5D)+AND+%22IUBMB+life%22%5BJournal%5D)">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/pubmed?term=((Focal+adhesion+kinase+and+its+signaling+pathways+in+cell+migration+and+angiogenesis%5BTitle%5D)+AND+%22Advanced+drug+delivery+reviews%22%5BJournal%5D)">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/pubmed?term=((Regional-specific+alterations+in+cell-cell+junctions%2C+cytoskeletal+networks+and+myosin-mediated+mechanical+cues+coordinate+collectivity+of+movement+of+epithelial+cells+in+response+to+injury%5BTitle%5D)+AND+%22Experimental+cell+research%22%5BJournal%5D)">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/pubmed?term=((Wound+healing--aiming+for+perfect+skin+regeneration%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Scar-free+healing%3A+from+embryonic+mechanisms+to+adult+therapeutic+intervention%5BTitle%5D)+AND+%22Philosophical+transactions+of+the+Royal+Society+of+London.+Series+B%2C+Biological%22%5BJournal%5D)">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/pubmed?term=((Wound+healing+and+inflammation%3A+embryos+reveal+the+way+to+perfect+repair%5BTitle%5D)+AND+%22Philosophical+transactions+of+the+Royal+Society+of+London.+Series+B%2C+Biological%22%5BJournal%5D)">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/pubmed?term=((Wound+healing+in+embryos%3A+a+review%5BTitle%5D)+AND+%22Anatomy+and+embryology%22%5BJournal%5D)">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/pubmed?term=((Wound+repair+and+regeneration%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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><tbody><tr><td>Sodium Chloride (NaCl)</td><td>Fisher Scientific</td><td>S271-3</td><td>Use at 140 mM in TD Buffer</td></tr><tr><td>Potassium Chloride (KCl)</td><td>Fisher Scientific</td><td>P217-500</td><td>Use at 5 mM in TD Buffer</td></tr><tr><td>Sodium Phosphate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma</td><td>S0876</td><td>Use at 0.7 mM in TD Buffer</td></tr><tr><td>D-glucose (Dextrose)</td><td>Fisher Scientific</td><td>D16-500</td><td>Use at 0.5 mM in TD Buffer</td></tr><tr><td>Tris Base</td><td>Fisher Scientific</td><td>BP152-1</td><td>Use at 8.25 mM 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>100 mm 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>35 mm Cell Culture Dish</td><td>Corning</td><td>430165</td><td></td></tr><tr><td>27 G 1 ml 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>Other Items Needed: General dissection instruments,&amp;nbsp; fertile white leghorn chicken eggs, check egg incubator (humidified, 37.7&amp;deg;C), laminar flow hood, binocular stereovision dissecting 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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Developmental Biology

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

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

생체 외 및 생체 내 모델에서 각막 내피 세포 - 중간 엽 전이를 공부하기

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

연구

발생학

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

마이크로 광 및 전기 방사의 조합은 각막 재생을위한 틈새를 장착 멤브레인을 생산하는

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

생체공학

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation results in calcium mobilization events that propagate from cell to cell, which are essential for initiating cellular motility into the wound bed, promoting efficient wound healing. The Trinkaus-Randall lab has developed a methodology for imaging the corneal wound healing response in ex vivo murine globes in real time. This approach involves enucleating an intact globe from a mouse that has been euthanized per established protocols and immediately incubating the globe with a calcium indicator dye. A counterstain that stains other features of the cell can be applied at this stage to assist with imaging and show cellular landmarks. The protocol worked well with several different live cell dyes used for counterstaining, including SiR actin to stain actin and deep red plasma membrane stain to stain the cell membrane. To examine the response to a wound, the corneal epithelium is injured using a 25 G needle, and the globes are placed in a 3D printed holder. The dimensions of the 3D printed holder are calibrated to ensure immobilization of the globe throughout the duration of the experiment and can be modified to accommodate eyes of different sizes. Live cell imaging of the wound response is performed continuously at various depths throughout the tissue over time using confocal microscopy. This protocol allows us to generate high-resolution, publication-quality images using a 20x air objective on a confocal microscope. Other objectives can also be used for this protocol. It represents a significant improvement in the quality of live cell imaging in ex vivo murine globes and permits the identification of nerves and epithelium.

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

The present work describes a novel experimental protocol that utilizes a 3D printed holder to enable high-resolution live cell imaging of enucleated globes. Through this protocol, the cellular calcium signaling activity in wounded corneal epithelium from ex vivo globes can be observed in real time.

새로운 3D 프린팅 홀더를 사용한 온전한 생체 외 구체의 라이브 셀 이미징

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation ...

생화학

Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of transparency due to fibrotic scarring. The tissue integrity of injured embryonic corneas is intrinsically restored with no detectable scar formation. Given its accessibility and ease of manipulation, the chick embryo is an ideal model for studying scarless corneal wound repair. This protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. First, eggs are windowed at early embryonic ages to access the eye. Second, a series of in ovo physical manipulations to the extraembryonic membranes are conducted to ensure access to the eye is maintained through later stages of development, corresponding to when the three cellular layers of the cornea are formed. Third, linear cornea wounds that penetrate the outer epithelial layer and the anterior stroma are made using a microsurgical knife. The regeneration process or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure. Studies to date using this model have revealed that wounded embryonic corneas display activation of keratocyte differentiation, undergo coordinated remodeling of ECM proteins to their native three-dimensional macrostructure, and become adequately re-innervated by corneal sensory nerves. In the future, the potential impact of endogenous or exogenous factors on the regenerative process could be analyzed in healing corneas by using developmental biology techniques, such as tissue grafting, electroporation, retroviral infection, or bead implantation. The current strategy identifies the embryonic chick as a crucial experimental paradigm for elucidating the molecular and cellular factors coordinating scarless corneal wound healing.

The present protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. The regenerating or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure.&#160;

배아 상처 병아리 각막의 흉터없는 조직 재생 조사

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

각 막에 대 한 모델로 눈 버와 각 막 상피 마모 상처 치유

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

의학

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

Ex Vivo 막 기관 문화 모델에 대 한 상처 치유 연구

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

An Epithelial Abrasion Model for Studying Corneal Wound Healing

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases.

각막 상처 치유를 연구하기위한 상피 마모 모델

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

각막 상피의 기계적 및 화학적 손상에 대한 Ex Vivo 및 In Vivo 동물 모델

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

Human blood-derived topical therapies have been a boon to clinicians in recent decades. Autologous serum (AS) and platelet-rich plasma (PRP) are enriched in epitheliotropic growth factors that are essential in corneal wound healing. Unlike AS, PRP is based on a differential centrifugation system, yielding more platelet-derived growth factors. Autologous conditioned serum (ACS) not only preserves the preparation of AS and PRP, but also focuses on immune-modulating properties, which are important in inflammatory diseases.
The lack of standardized protocols and high preparation costs are limitations for the clinical application of ACS. This video experiment demonstrates a standard operating procedure for preparing modified autologous conditioned serum (mACS) eye drops. First, glycerol was added into heparin syringes as the blood cell stabilizer during hypoxic incubation. To activate the blood cells, a 4 h incubation at 37 &#176;C was initiated. Then, the blood samples were centrifuged at 3,500 &#215; g for 10 min at room temperature. After filtration of the supernatant through a 0.22 &#181;m filter, the mACS eye drops were fully prepared.
A tentative try-out of the therapeutic effect of mACS showed that it may have competitive advantages over conventional AS in the corneal wound healing in ex vivo mouse eyes. The AS used in this study was prepared according to published studies and the clinical practice in our hospital. Therefore, the efficacy of mACS on ocular surface diseases could be evaluated in future research through in vivo animal studies and clinical trials.

Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

This article describes a protocol to simplify the process and render the preparation of autologous conditioned serum (ACS) less expensive. No special syringes or surface-coated glass beads are needed. Moreover, the modified ACS (mACS) has competitive advantages over conventional autologous serum in the corneal wound healing of murine eyes ex vivo.

변형된 자가 조절 혈청의 생산 및 쥐 각막 상피에서의 치유 가능성에 대한 생체 외 평가

Human blood-derived topical therapies have been a boon to clinicians in recent decades. Autologous serum (AS) and platelet-rich plasma (PRP) are enriched ...

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

All animal organs, from the skin to eyes to intestines, are covered with sheets of epithelial cells that allow them to maintain homeostasis while protecting them from infection. Therefore, it is not surprising that the ability to repair epithelial wounds is critical to all metazoans. Epithelial wound healing in vertebrates involves overlapping processes, including inflammatory responses, vascularization, and re-epithelialization. Regulation of these processes involves complex interactions between epithelial cells, neighboring cells, and the extracellular matrix (ECM); the ECM contains structural proteins, regulatory proteins, and active small molecules. This complexity, together with the fact that most animals have opaque tissues and inaccessible ECMs, makes wound healing difficult to study in live animals. Much work on epithelial wound healing is therefore performed in tissue culture systems, with a single epithelial cell-type plated as a monolayer on an artificial matrix. Clytia hemisphaerica (Clytia) provides a unique and exciting complement to these studies, allowing epithelial wound healing to be studied in an intact animal with an authentic ECM. The ectodermal epithelium of Clytia is a single layer of large squamous epithelial cells, allowing high-resolution imaging using differential interfering contrast (DIC) microscopy in living animals. The absence of migratory fibroblasts, vasculature, or inflammatory responses makes it possible to dissect the critical events in re-epithelialization in vivo. The healing of various types of wounds can be analyzed, including single-cell microwounds, small and large epithelial wounds, and wounds that damage the basement membrane. Lamellipodia formation, purse string contraction, cell stretching, and collective cell migration can all be observed in this system. Furthermore, pharmacological agents can be introduced via the ECM to modify cell:ECM interactions and cellular processes in vivo. This work shows methods for creating wounds in live Clytia, capturing movies of healing, and probing healing mechanisms by microinjecting reagents into the ECM.