Name: corneal tissue engineering: an in vitro model of the stromal-nerve interactions of the human cornea
Uploaded: 2018-01-24T13:30:00.000+00:00
Duration: 7 min 35 s
Description: Watch this Scientific Journal Video about Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea at JoVE.com

우리는 우리의 진심으 감사 닥터 벤 파울러에 대 한 그의 기술 편 실험을 확장 하 고 싶습니다.

이 프로토콜은 오클라호마 보건 과학 센터의 대학/기관 검토 위원회 (IRB #4509)의 지침을 따릅니다. 프로토콜의 모든 부분 헬싱키의 선언의 신조를 만났다. 각 막 샘플 국가 개발 및 연구 연구소 (NDRI)와 오클라호마 라이온 스 안구 은행에서 얻은 했다.
1입니다. 1 차 셀의 격리
<ol><li>21.5 cm2 무 균 Dulbecco 인산 염 버퍼 식 염 수 (DPBS)의 2 개 mL를 포함 하는 배양 접시에 조직 인간 각 막 조직 샘플을 접수 한 후, 전송 (1 x).</li>
	<li>긁 고 막 돔 얼굴 위로 배치 하 여 각 막 상피를 제거 합니다. 완전 한 제거를 보장 하기 위해, 철저 하 게 45 ° 각도로 면도날을 사용 하 여 상피 층을 다쳤어요. 다음, 각 막 돔 얼굴 다운 플립 하 고 철저 하 게 완전 한 제거를 보장 하기 위해 45 ° 각도로 면도날으로 기질에서 endothelium을 다쳤어요. 워시 DPBS와 실질 조직.</li>
	<li>실질 조직 유지 하는 DPBS를 사용 하 여 모든 시간에 젖은 stromal 조각 10 외과 블레이드를 사용 하는 작은 조각으로 잘라. 미디어 (2 x 2 m m 조직 플라스 크 당 4 또는 5 조각) 없이 T25 문화 플라스 크에 소독 집게와 장소 stromal 조각을 선택 합니다.</li>
	<li>약 30-40 분 동안 37 ° C에서 플라스 크의 밑바닥에 explants 허용. 준수, 일단 천천히 추가 글의 최소 필수 매체 (EMEM) 10% 태아 둔감 한 혈 청 (FBS) 및 1% 포함 된 항생제/Antimycotic는 explants 방해 없이 플라스 크에.</li>
	<li>부드럽게 37 ° c, 5% CO2배양 기에 플라스 크를 배치 합니다. 세포 분리 시작 때까지 explants는 그대로 두고 플라스 크에서 마이그레이션. 약 2-4 주 후, 셀 100% 합류를 도달 한다. 100% 합류 시 조직 문화 미디어를 제거 하 여 세포를 통과 하 고 셀 DPBS 씻어.</li>
	<li>세포를 트립 신을 추가 하 고 37 ° c.에서 그들을 품 어 약 5 분 후 그들은 분리가 확인 가벼운 현미경에서 세포를 확인 하 고 신선한 미디어를 추가 하 여는 베이스를 중단.</li>
	<li>1000 x g.에서 원심 분리는 상쾌한은 펠 렛을 방해 하지 않고 신중 하 게 제거에 대 한 15 mL 원뿔 튜브에 세포 현 탁 액을 전송 하 고 중단 신선한 EMEM 셀 10% FBS 1% 항생제. 셀을 계산 하 고 씨앗 신선한 EMEM와 확장에 대 한 T75 문화 플라스 크에 약 1 x 106 셀 1% FBS 1% 항생제.</li>
</ol>2. 기본 HCFs 문화 및 3D 어셈블리 생성
<ol><li>3D 조립에서 구문을 explants 이전에 성장된 EMEM 10% FBS 1% 항생제/Antimycotic, 통로 및 수 HCFs.</li>
	<li>씨 약 1 x 106 셀/잘 신선한 EMEM의 1.5 mL 함께 3D 구조에서 0.4 µ m 모와 폴 리 카보 네이트 막 삽입에 기본 HCFs의 10% FBS 1% 항생제/Antimycotic 구성의 상단에. 각 구조물의 하단에 같은 미디어의 또 다른 1.5 mL를 추가 합니다.</li>
	<li>셀 시드의 24 시간 후 문화 EMEM 셀 10% FBS 1% 항생제/Antimycotic, 위에 추가 1.5 mL 및 각 구조물의 하단에 1.5 mL에 의해 0.5 m m 2-O-α-D-glucopyranosyl-L-ascorbic 산 (비타민 C) 자극.</li>
	<li>37 ° c, 5% CO2 3 주 동안 인큐베이터에 문화를 유지 하 고 전체 연구 기간 동안 매일 신선한 미디어를 공급. 아니 더 통로 필요 합니다.</li>
</ol>3. 세포 분화 및 공동 문화
<ol><li>3 주 후, 8 x 103 셀/잘 이전 조립된 3D 상단 SH-SY5Y 인간 신경 세포의 10%를 포함 하는 EMEM의 포함 1.5 mL를 구성 하는 것을 추가 FBS, 1% 항생제/Antimycotic, 그리고 0.5 m m 2-O-α-D-glucopyranosyl-L-ascorbic 산 (비타민 C)입니다.<ol>
<li>SH SY5Y 신경 세포 세포 분화를 시작 하기 전에 적어도 24 h에 대 한 각 막 실질 세포의 위에 성장 하는 것을 허용 한다.</li>
	</ol>
</li>
	<li>EMEM 1 %FBS 및 10 µ M의 1.5 mL로 세포를 자극 하 여 SH SY5Y 세포 분화를 시작 구성 위에 Retinoic 산. 구성의 하단에는 EMEM 10% FBS 1% 항생제/Antimycotic 0.5 m m 2-O-α-D-glucopyranosyl-L-ascorbic 산 (비타민 C)의 1.5 mL를 추가 합니다. 참고 Retinoic 산 단계 어둠 속에서 수행 되어야 합니다.<ol>
<li>5 일 동안이 차별화 미디어에 셀 문화를 계속 합니다.</li>
	</ol>
</li>
	<li>세포 분화 단계에 도달, 1.5 mL 혈 청 자유로운 매체 포함 된 2 셀 전환 두뇌 파생 된 neurotrophic 요인 (BDNF)와 1%의 nM 항생제/Antimycotic 구성의 상단에 추가 하 여 EMEM에 추가. 구성의 하단에는 EMEM 10% FBS 1% 항생제/Antimycotic 0.5 m m 2-O-α-D-glucopyranosyl-L-ascorbic 산 (비타민 C)의 1.5 mL를 추가 합니다. 참고 BDNF 단계 어둠 속에서 수행 되어야 합니다.</li>
	<li>표 1에 표시 된 대로 어떤 추가 분석에 대 한 처리 하기 전에 2 추가 일에 대 한 셀을 문화.</li>
</ol>

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여러 연구 결과 각 막 질환의 더 나은 이해를 개발 하는 데 도움이 뿐만 아니라 수 치료를 발견 하는 다양 한 동물 모델 개발에 집중 되었습니다. 그러나, 이러한 연구에서 인 간에 게 중요 한 가치는 확인 되지는 않았습니다. 날짜 하려면, 다양 한 생체 외에서 모델 개발 고 널리 그들의 놀라운 임상 중요성으로 조사 되었습니다. 우리의 이전 설립 3 차원 생체 외에서 모델은 새로운 시?...

인체 각 막에서는 가장 밀도가 innervated 조직입니다. 신경 터치, 고통, 온도, 다양 한 감각에 대 한 책임은 그리고 또한 상처 치유, 반사, 눈물 분 비5,,67및 생산에 필수적인 역할을 했습니다. 각 막, 실질 신경 줄기 limbal 총에서 발생 하 고 광선으로 주변 각 막 stroma를 입력 합니다. Stromal 신경 조직 콜라겐 lamellae 평행 이며, 그들은 더 작은 성숙기로 그들은 천박한 기질5,8쪽으로 진행으로 분기. 상피 층을 관통 하는 신경 섬유를 추가 하 고 따라서, 신경 분포 널리 퍼져 각 막 상피와 기질. 따라서, 신경 분포는 각 막의 건강 하 고 병에 걸린 상태에서 필수적인 역할을 하고있다. 이 프로토콜에서 우리는 vivo에서 stromal 신경 상호 작용을 모방 하는 종류의 첫 번째 모델 소설 3 차원 생체 외에서 의 발전을 공개. 그것은 가장 설립, 잘 특징이 라인 신경 성장을 공부 하는 데 사용 중 SH-SY5Y 셀 라인이이 연구를 위해 사용 했다. SH SY5Y 셀 라인 두 기판 부착 (S 형) 생산 설명 되었으며 neuroblastic (N 형) 세포 transdifferentiation9를 받을 수 있습니다. 그 결과,이 셀 라인은 N 형 셀의 트리플 연속 subclone 선택에서 파생 된, 비록 그것은 또한 포함 retinoic 산 및 두뇌 파생 된 사용을 통해 신경 세포로 분화를 겪고 수 S 형 세포의 작은 수 科 요소9. 이 당뇨병 성 망막 증 (DM) 및 다른 눈 질병와 관련 된 각 막 합병증의 더 나은 이해로 이어질 수 있는 도구를 제공 합니다. 하 고 눈 질병을 가진 환자에서 신경 세포 배양과 관련 된 어려움 때문에이 3D 생체 외에서 모델 신경 상호 작용을 공부 하 고 각 막 stroma로 신호에 상당한 영향을 제공 합니다.
질병 상태는 종종 매우 큰 규모, 손상 된 삶의 질으로 이어지는 신체의 다양 한 조직 영향을. 눈 dystrophies는 종종 조직의 질병 및 시력의 손실 또는 심지어 영구적인 시력 상실을 리드와 관련 된 일반적인 합병증. 포괄적인 연구는 종종 질병 상태 뿐만 아니라 기저 세포 수준에서 효과의 더 나은 이해를 위해 근본적 이다. 이러한 질병의 효과 연구, 다양 한 생체 외에서 그리고 vivo에서 모델 조직 엔지니어링 응용 프로그램의 도움으로 개발 되었습니다. 각 막 조직 공학 응용 과학10,11,12,,1314, 다양 한 분야에 걸쳐 큰 관심을 얻고 있다 하지만 여전히 주요 제한 중 실제 응용 프로그램을 거부, 감염과 흉터10,11,12,,1314이식 각 막 등. 여러 연구를 성공적으로 개발 하 고 다양 한 생체 외에서 모델3,15,,1617,18, 설립 있다 19,20,21,22,23,,2425,26. 3 차원 생체 외에서 모델은 가장 유망한와 큰 과학적 관심의. 3D 모델은 더 나은 vivo에서 세포 및 생리 적 이벤트 섬유 증 중 중요 하 고 상처 치유15,27,,2829거울 알려져 있습니다. 이러한 생체 외에서 모델 각 막 합병증 등 다른 질병 상태를 치료에 새로운 치료 접근을 찾는 중요 한 역할을. 각 막 기능에 신경 분포의 중요 한 역할에도 불구 하 고 약간의 노력이 했다 각 막 조직 설계 구조2,3내 주변 신경 확산을 촉진. 그러나, 제안 된 3D 생체 외에서 셀 구조는 원하는 조직 기능을 달성 하기 위하여 대상 조직 모방.
당뇨병 각 신경 결함 때문에 여기서 설명 하는 모델에 대 한 명백한 응용 프로그램은, 인간의 생체 외에서 모델 Keratoconus와 Fuchs dystrophies 등 누릴 수 있는 다른 여러 각 막 질환 있다. 우리의 3D 모델이이 전망에서 나온다 고 약 납품을 평가 하기 위해 각 막 직물의 생체 외에서 표현 및 새로운 눈 약의 안전의 개발을 제안.

<table><tbody><tr><td>Healthy corneal tissue</td><td>NDRI</td><td></td><td>Samples from donors with no ocular trauma or systemic disease</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Solution (1X)</td><td>Gibco by Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>Sterile forceps</td><td>Fischer Scientific</td><td>13-812-42</td><td>Fisherbrand Dissecting Extra-Fine-Pointed Splinter Forceps</td></tr><tr><td>Single edge razor blades</td><td>Personna</td><td>270100</td><td></td></tr><tr><td>Sterile surgical scalpel blades No.10</td><td>Feather Surgical Blade</td><td>2976#10</td><td></td></tr><tr><td>Eagle&amp;rsquo;s Minimum Essential Medium</td><td>ATCC</td><td>30-2003</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Biologicals</td><td>S11550</td><td>10% FBS is required for media preparation</td></tr><tr><td>Antibiotic-Antimycotic (100X)</td><td>Gibco by Life Technologies</td><td>15240-062</td><td>1% Antibiotic-Antimycotic is required for media preparation</td></tr><tr><td>0.05% Trypsin EDTA(1X)</td><td>Gibco by Life Technologies</td><td>25300062</td><td></td></tr><tr><td>Polycarbonate membrane inserts with 0.4-&amp;mu;m pores</td><td>Corning Costar</td><td>3412</td><td></td></tr><tr><td>2-O-&amp;alpha;-Dglucopyranosyl-L-ascorbic acid (Vitamin C)</td><td>Sigma-Aldrich</td><td>SMB00390-14</td><td>A concentration of 0.5 mM should be used for the study</td></tr><tr><td>Wax block</td><td>VWR</td><td>50-949-027</td><td></td></tr><tr><td>SH-SY5Y Neuroblastoma cells</td><td>ATCC</td><td>SHSY5YATCC CRL-2266</td><td></td></tr><tr><td>Retinoic Acid</td><td>Sigma-Aldrich</td><td>SRP3014-10UG</td><td>Final concentration of 10uM needs to be used</td></tr><tr><td>BDNF</td><td>Sigma-Aldrich</td><td>R2625-100MG</td><td>Final concentration of 2nM needs to be used</td></tr><tr><td>Dimethyl Sulfoxide(DMSO)</td><td>VWR-Alfa Aesar</td><td>67-68-5</td><td>Ultra Pure Grade-Sterile DMSO to be used</td></tr><tr><td>Thermo Scientific Nunc Cell Culture Treated EasYFlasks (T25)</td><td>Fisher Scientific</td><td>12-565-351</td><td></td></tr><tr><td>Thermo Scientific Nunc Cell Culture Treated EasYFlasks (T75)</td><td>Fisher Scientific</td><td>12-565-349</td><td></td></tr></tbody></table>

corneal tissue engineering: an in vitro model of the stromal-nerve interactions of the human cornea

조직 공학 인간 각 막 교체 각 막 시력 손실1에서 고통을 전세계 약 10 백만 명에 대 한 높은 수요로 인해 상당한 인정을 받고 있다. 가능한 인간 각, 3 차원 (3D) 조직에 중요 한 진행에 대 한 수요를 해결 하기 위해 공학이 했다2,,34. 이러한 각 막 모델 에서부터 간단한 단층 시스템 3D 전체 두께 각 막 등가물2로 이어지는 다층된 모델.
그러나, 생체 외에서 질병 모델의 맥락에서 3 차원 조직 설계 각 막의 사용 날짜 부족 유사 다층된 3 차원 각 막 조직 구조, 기능, 및 서로 다른 세포 유형 (즉, 신경, 네트워킹을 공부 상피와 기질, 내 피)2,3. 또한, 생체 외에서 각 막 조직 모델에 대 한 수요는 제약 제품에 대 한 동물 실험을 줄이기 위해 시도에서 증가 했다. 따라서, 더 정교한 모델은 인간의 생리 적 요구 사항, 시스템 일치 하는 데 필요한 그리고 더 환자 인구에 관련 된 모델의 개발은 절대적으로 필요 하다. 각 막에 여러 세포 유형 영향을 받는 질환과 dystrophies Keratoconus, 당뇨병 각 Fuchs, 등을이 모델에 포함 된 기본 인간 각 막 섬유 아 세포 (HCFs) 건강 한 기증자와에서 뉴런에서의 3D 공동 문화 모델 SH SY5Y 셀 라인입니다. 인간의 각 막 조직 내에서 2 개의 세포 유형 사이 상호 작용을 조사 하기 위해 처음으로 수 있습니다. 우리는이 모델 잠재적으로 관련 된 신경 손상 하는 각 막 질환의 신경 기질 상호 작용 하는 근본적인 메커니즘을 해 부 수 믿습니다. 이 3D 모델 vivo에서 각 막 직물의 기본 해 부 및 생리 적 특성을 반영 하 고 수 도구로 사용 될 미래에는 각 막 결함 조사로 동물 테스트 하기 전에 다양 한 에이전트의 효능을 심사.

그림 1 은 작업 모델 3D 생체 외에서 단계별 대표 이미지입니다. 첫 번째 단계에서 세포 인간 각 막에서 격리 됩니다. 다음, 그들은 폴 리카 보 네이트 막에 성장 하 고 자기 secreted 3 차원 매트릭스를 비타민 C와 자극. 이 3D 구조 시스템 유도는 다층 세포 vivo에서의 합성-stromal 매트릭스 처럼. 그 후, 신경 세포는 신경 세포 분화를 시작 하 여...

Watch this Scientific Journal Video about Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea at JoVE.com

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

이 프로토콜에는 새로운 3 차원 생체 외에서 모델을, 어디 각 막 실질 세포와 분화 된 신경 세포 교양 함께 시험에는 두 종류의 세포 상호 작용의 이해를 돕기 위해 설명 합니다.

각 막 조직 공학:는 생체 외에서 의 모델 인간 각 막의 실질 신경 상호 작용

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

corneal-tissue-engineering-an-vitro-model-stromal-nerve-interactions

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

8.8K 조회수.  University of Oklahoma Health Sciences Center. 이 프로토콜에는 새로운 3 차원 생체 외에서 모델을, 어디 각 막 실질 세포와 분화 된 신경 세포 교양 함께 시험에는 두 종류의 세포 상호 작용의 이해를 돕기 위해 설명 합니다.

비디오: Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

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

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 ...

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 ...

의학

Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting factor(s) for success of deep anterior lamellar and penetrating keratoplasty. These are surgical procedures that involve replacing part or all of the diseased corneal layers, respectively, by donated tissue, the graft, taken from a recently deceased individual. However, means to evaluate stromal quality of corneal grafts in eye banks are limited and lack the capability of high-resolution quantitative assessment of disease indicators. Full-field optical coherence microscopy (FF-OCM), permitting high-resolution 3D imaging of fresh or fixed ex vivo biological tissue samples, is a non-invasive technique well suited for donor cornea assessment. Here we describe a method for the qualitative and quantitative analysis of corneal stroma using FF-OCM. The protocol has been successfully applied to normal donor corneas and pathological corneal buttons, and can be used to identify healthy and pathologic features on both the macroscopic and microscopic level, thereby facilitating the detection of stromal disorders that could compromise the outcome of keratoplasty. By improving the graft quality control, this protocol has the potential to result in better selection (and rejection) of donor tissues and hence decreased graft failure.

We describe use of full-field optical coherence microscopy as a method for high quality assessment of corneal donor stroma. This protocol can be used to identify features indicative of health or disease, and is aimed at improving the screening and selection of donor tissues, and hence the results of keratoplasty.

각막 이식편의 기질 특징에 대한 조직학과 유사한 분석을 위한 전체 필드 광학 간섭 현미경

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting ...

면역학 및 감염병학

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

분할 각막 단추를 사용하여 돼지 각막 내피 내피 기관 문화 모형

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

각막 내피 세포 손실의 비 침습적, 레이저 보조 실험 모델의 개발

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

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, ...

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

Descemet's Stripping Only is an experimental procedure wherein patients with central corneal guttae resulting from Fuchs Endothelial Corneal Dystrophy have Descemet's membrane stripped for peripheral cells to regenerate the endothelial layer. We present novel methodology simulating DSO in dystrophic human corneas ex vivo with accelerated healing stimulated by eFGF1 (NM141).

조작된 섬유아세포 성장 인자 1에 의해 자극된 가속화된 치유를 통해서만 Descemet의 스트리핑의 인간 각막 배양 모델

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the ...

축삭 재생 연구를 위한 각막 감각 섬유 손상 프로토콜

Three Strategies to Induce Neurotrophic Keratitis and Nerve Regeneration in Murine Cornea

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation provides sensation and trophic function to the eye and contributes to preserving corneal integrity. The pathological disruption of this innervation is termed neurotrophic keratitis. This can be triggered by injury to the eye, surgery, or disease. In this study, we propose three different protocols for inflicting damage on the innervation in ways that recapitulate the three types of cases generally encountered in the clinic.
The first method consists in making an abrasion of the epithelium with an ophthalmic burr. This involves the removal of the epithelial layer, the free nerve endings, and the subbasal plexus in a manner similar to the photorefractive keratectomy surgery performed in the clinic. The second method&#160;only targets the innervation by sectioning it at the periphery with a biopsy punch, maintaining the integrity of the epithelium. This method is similar to the first steps of lamellar keratoplasty and leads to a degeneration of the innervation followed by regrowth of the axons in the central cornea. The last method damages the innervation of a transgenic mouse model using a multiphoton microscope, which specifically localizes the site of cauterization of the fluorescent nerve fibers. This method inflicts the same damage as photokeratitis, an overexposure to UV light.
This study describes different options for investigating the physiopathology of corneal innervation, particularly the degeneration and regeneration of the axons. Promoting regeneration is crucial for avoiding such complications as epithelium defects or even perforation of the cornea. The proposed models can help test new pharmacological molecules or gene therapy that enhance nerve regeneration and limit disease progression.

저자 스포트라이트: 혁신적인 모델을 통한 각막 신경 분포 연구의 발전 

Here, we propose three different methods of damaging the sensory fibres innervating the cornea. These methods facilitate the study of axon regeneration in mice. These three methods, which are adaptable to other animal models, are ideal for the study of corneal innervation physiology and regeneration.

쥐 각막의 신경 영양성 각막염 및 신경 재생을 유도하는 세 가지 전략

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation ...