이 작품은 만페이 스즈키 당뇨병 재단 (YT), 보스턴 아동 병원 OFD / BTREC / CTREC 교수 진로 개발 보조금, 보스턴 아동 병원 안과 재단, BCH 파일럿 상, BCH 맨튼 센터 펠로우십의 보조금에 의해 지원되었다, 리틀 기린 재단 (ZF), 독일 연구 재단 (DFG; BC [CA1940/1-1]), NIH R24EY024868, EY017017, R01EY01717-13S1, EY030904-01, BCH IDDRC (1U54HD)

설명된 모든 동물 실험은 보스턴 아동 병원의 기관 동물 관리 및 사용 위원회에 의해 승인되었습니다 (ARCH 프로토콜 번호 19-04-3913R).
1. 준비
<ol><li>페니실린/스트렙토마이신(10000 U/mL)과 5mL 및 10mL의 상용 보충제를 500mL의 완전한 클래식 매체에 추가합니다. Aliquot 50 mL 의 배지 는 처음에. 참고: 오염을 방지하기 위해 배지를 재고로 되돌리지 마십시오.</li>
	<li>얼음에 완전한 클래식 매체의 알리쿼트를 넣습니다.</li>
	<li>70%의 에탄올을 사용하여 해부하는 현미경, 집게 및 가위를 청소하십시오.</li>
	<li>2개의 세포 배양 접시 (10cm), 해부 현미경에 하나, 얼음에 하나 준비; 각 접시에 완전한 클래식 매체 10 mL을 넣습니다.</li>
</ol>2. 실험 단계(그림 1)
<ol><li>산후 (P) 주위에 C57BL/6J 마우스를 희생 (P) 20 75-100 mg/kg 케타민과 7.5-10 mg/kg 자일라진 주입을 사용 하 여. 해부 전에 얼음에 완전한 고전적인 매체에 눈을 유지합니다.</li>
	<li>눈에 결합 조직 (근육과 지방 조직) 및 시신경을 제거합니다.</li>
	<li>마이크로 가위를 사용하여 0.5mm 후방을 각막 limbus로 원주 절단합니다. 각막/홍채 복합체, 유리체 및 렌즈를 제거합니다.</li>
	<li>시신경을 향해 절단 가장자리에 수직으로 1mm 절개를 하고 1mm 너비의 할례 밴드를 자릅니다. 복합체의 중앙 및 주변 영역을 분리합니다. 집게를 사용하여 RPE/choroid/sclera 복합체에서 망막을 벗깁니다.</li>
	<li>주변 초보 밴드를 얼음에 완벽한 클래식 매체로 유지하십시오. 다른 눈을 분리하고 두 번째 밴드를 잘라 과정을 반복합니다.</li>
	<li>원형 밴드를 6 ~동등한 사각형 조각 (~1mm x 1mm)으로 자른다. 참고: 가장자리를 만지지 마십시오.</li>
	<li>제조의 지시에 따라 기저막 추출물(BME)을 해동합니다. 24 개의 잘 조직 배양 플레이트의 각 우물의 중심에 30 μL / 웰BME를 추가합니다. BME의 액적가장자리가 가장자리를 건드리지 않고 플레이트 바닥에 볼록돔을 형성해야 합니다. 참고 : 냉장고에서 하룻밤 BME를 해동하십시오. BME는 해동 후 언제든지 얼음위에 있어야 합니다.</li>
	<li>BME의 중간에 조직을 배치합니다. 참고 : 코로이드 축출을 평평하게하지 마십시오; 일반적으로, 조직이 BME 내에서 확장하자. 조직의 방향 (경피측 위 또는 아래) 실험 결과에 영향을 미치지 않습니다.</li>
	<li>37°C에서 10분 동안 플레이트를 배양하여 젤이 고화하게 합니다.</li>
	<li>전체 클래식 중간/웰의 500 μL을 추가합니다.</li>
	<li>격일(500 μL)의 클래식 매체를 변경합니다. 코로이드 발아는 현미경으로 3 일 후에 관찰 될 수있다. 참고: 성장 인자 치료를 위해 4 시간 동안 조직을 굶어. 성장 인자 감소 매체에서 시험 화합물을 희석 (1:200 대신 부스트 1:50).</li>
</ol>3.SWIFT-코로이드 전산화 정량화 방법17 (도 2)
참고: 성장하는 선박이 적용되는 영역을 측정하는 전산화 된 방법이 사용되었습니다. ImageJ 소프트웨어에 대한 매크로 플러그인은 정량화 하기 전에 필요합니다(자세한 내용은 추가 정보 참조).
<ol><li>ImageJ로 코로이드 발아 이미지를 열고"이미지 | 유형| 회색눈금으로 8비트"</li>
	<li>"이미지| 조정 | 밝기/콘트라스트(Ctrl/shift/C)" 및 콘트라스트를 최적화합니다.</li>
	<li>마법 지팡이 기능을 사용하여 새싹 중앙에 존재하는 코로이드 조직을 이미지에서 윤곽을 그리고 제거합니다(바로 가기 키"F1")(그림2A, B). 참고: 마법 지팡이의 허용 오차 속도를 20-30%로 설정합니다.</li>
	<li>무료 선택도구(그림 2C)로이미지의 배경을 제거합니다. "이미지| 조정 | 임계값(Ctrl/shift/T)". 임계값 함수를 사용하여 배경 및 주변부(그림2D)에대해 미세 혈관 새싹을 정의합니다.</li>
	<li>"F2"를클릭하면 요약이 나타납니다. "저장"을 클릭하여 선택한 영역의 이미지를저장합니다. 향후 참조를 위해 원본 이미지와 동일한 폴더에 저장합니다.</li>
	<li>샘플 그룹을 측정한 후 데이터 분석을 위해 기록된 샘플을 복사합니다. 참고:"분석| 스케일 바가 있는 이미지를 사용하여 "배율"을 설정합니다.</li>
</ol>

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저자는 재정 적 공개가 없습니다. 전산화 된 방법은 저자를 통해 교육 기관에 무료로 사용할 수 있습니다.

천로이드 발아 분석체는 NEovascular AMD9,10,18,19,20에서연구를 돕습니다. 초로이드 이질은 쥐뿐만 아니라 쥐와인간(17,21)으로부터분리될 수 있다. choroid 축출에는 EC, 대식세포 및 pericytes17이포함됩니다. 이 분석에서 ch...

Choroidal 혈관 신생 이소화 조절은 신생 혈관 노화 관련 황반 변성 (AMD)1과관련이 있습니다. choroid는 망막 색소 상피 (RPE) 아래에 존재하는 미세 혈관 침대입니다. 코로이드의 혈류감소는 AMD2의진행과 관련이 있는 것으로 나타났다. 혈관 내피, RPE, 대식세포, 회낭체 및 기타 세포 사이의 복잡한 관계는 조직3,4,5의항상성에 대한 책임이 있다. 따라서, 재현 가능한 분석 모델링 choroidal 마이크로 환경은 neovascular AMD의 연구에 대 한 중요 한.
전 생체 혈관 신생 분석 및 체외 내피 세포 배양은 생체 내 미생물 의 행동, 신약 테스트 및 병인의 연구를 보완 할 수 있습니다. 인간 망막 미생물 내피 세포(HRMEC), 인간 배꼽 정맥 내피 세포(HUVEC), 격리된 원발성 동물 뇌 또는 망막 IC와 같은 내피 세포는 종종 안구 혈관 신생 연구6,7,8을위한 체외 연구에서 사용된다. 특히 HRMEC는 내피증, 이주, 관 형성 및 혈관 누설을 평가하여 체외 외 혈관 신생혈관화(CNV)9의 모델로 널리 사용되어 내정간섭6,10을평가한다. 그러나, 배양에 있는 EC는 choroid에서 찾아낸 그밖 세포 모형과의 상호 작용의 부족 때문에 CNV의 모형으로 제한되고 이 소집에서 이용되는 대부분의 EC는 choroid에서 유래하지 않기 때문에. 마우스 코로이드 EC는 문화권에서 분리하고 유지하기가 어렵습니다.
대동맥 고리 분석은 매크로 혈관 증식의 모델로 널리 사용된다. 대동맥 각출소에서 혈관 새싹은 EC, 백혈구 및대식세포(11)를포함한다. 대동맥 고리 분석모델은 큰 혈관 혈관신생을 잘12,13,14로모델한다. 그러나, 대동맥 고리로서 코로이드 외혈관화의 모델로서 한계가 있어 특징적인 초로이드 미생물 혈관 환경이 결여된 대색조직이며, 큰 혈관으로부터의 새싹은 미생물 병리학에 관여하는 모세관 네트워크로부터 새싹과 다를 수 있다. 최근 그룹은 전 생체 망막 분석15,16을발표했다. 망막 신혈관 질환에 적합하지만 AMD에서 볼 수 있듯이 코로이드 신혈관화에는 적합하지 않습니다.
마우스 RPE, 코로이드 및 경마 성 이식 조직을 이용한 코로이드 발아 분석체는 CNV를 더 잘 모델링하기 위해 개발되었다. 조직은 마우스 (또는 다른 종) 눈(17)로부터쉽게 분리 될 수 있습니다. 이 분석법은 약리학적 화합물의 프로 및 항 혈관 신생 잠재력의 재현 가능한 평가 및 유전자 변형 마우스로부터 조직을 사용하여 코로이드 신생 혈관화에서 특정 경로의 역할의 평가 및 제어18을허용한다. 이 코로이드 발아 분석체는 많은 후속 간행물9,10,18,19,20에서참조되었다. 여기서, 이 분석법의 사용에 관여하는 방법이 입증된다.

<table>
	<tbody>
		<tr>
			<td>AnaSed (Xylazine)</td>
			<td>AKORN</td>
			<td>59339-110-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Basal membrane extract (BME) Matrigel</td>
			<td>BD Biosciences</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>NEST</td>
			<td>704001</td>
			<td>10cm</td>
		</tr>
		<tr>
			<td>Complete classic medium with serum and CultureBoost</td>
			<td>Cell systems</td>
			<td>4Z0-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol 200 Proof</td>
			<td>Pharmco</td>
			<td>111000200</td>
			<td>use for 70%</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>ZEISS</td>
			<td>Axio Observer Z1</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>GIBCO</td>
			<td>15140</td>
			<td>10000 U/mL</td>
		</tr>
		<tr>
			<td>Tissue culture plate (24-well)</td>
			<td>Olympus</td>
			<td>25-107</td>
			<td></td>
		</tr>
		<tr>
			<td>VetaKet CIII (Ketamine)</td>
			<td>AKORN</td>
			<td>59399-114-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo choroid sprouting assay of ocular microvascular angiogenesis

노화와 관련된 황반 변성의 현저한 특징인 병리학적인 천동 혈관신생은 시력 장애와 실명으로 이어집니다. 인체망막 내피 세포(EC) 증식 분석(HRMEC) 또는 분리된 1차 망막 IC를 이용한 증식 분석은 생체외 모델에서 망막 혈관신생을 연구하는 데 널리 사용된다. 그러나, 순수한 뮤린망 내피 세포를 격리하는 것은 기술적으로 도전적이고 망막 EC는 choroidal 내피 세포 및 다른 세포/세포 상호 작용과 다른 증식 반응을 가질 수 있다. 초로이드 미생물 증식의 모델로서 매우 재현 가능한 전 생체 균 발아 분석이 개발되었다. 이 모형은 choroid 혈관분기 (EC, 대식세포, pericytes) 및 망막 안료 상피 (RPE)사이 상호 작용을 포함합니다. 마우스 RPE/choroid/scleral 이병은 성장 인자 감소 기막 추출물(BME)(일 0)에서 분리되고 배양됩니다. 배지는 격일로 변경되고 코로이드 발아는 6일째에 정량화된다. 개별 choroid explant의 이미지는 반전된 상 현미경으로 촬영되고 발아 영역은 이 실험실에서 개발된 ImageJ 소프트웨어에 반자동 매크로 플러그인을 사용하여 정량화됩니다. 이 재현 가능한 ex vivo choroidal 발아 분석은 야생 유형 및 유전자 변형 마우스 조직을 사용하여 choroidal 마이크로 혈관 증식에 관여하는 경로를 평가하기 위해 잠재적 인 치료 및 마이크로 혈관 질환 연구에 대한 화합물을 평가하는 데 사용할 수 있습니다.

하루에 빛나는 choroid 의 성장의 비교
우리는 BME에 내장 된 sclera로 choroid를 해부하고 6 일 동안 배양(그림 1). 3일부터 6일까지 C57BL/6J 마우스에서 의초로이드 발아는 현미경으로 검사되었고 IMAGEJ에서 반자동 정량화 방법인 SWIFT-Choroid로 정량화되었다. 대표적인 경우, 축출면적(축출자체를 제외한 각성으로부터 연장된 혈관)은 3일째 0.38mm2(그...

Watch this Scientific Journal Video about An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis at JoVE.com

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

이 프로토콜은 마이크로 혈관 증식의 전 생체 모델인 초로이드 발아 분석체를 제시합니다. 이 분석체는 코로이드 마이크로 혈관의 증식에 관여하는 경로를 평가하고 야생 유형 및 유전자 변형 마우스 조직을 사용하여 약물 치료를 평가하는 데 사용할 수 있습니다.

안구 미세 혈관 신생의 전 비보 코로이드 발아 분석

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

an-ex-vivo-choroid-sprouting-assay-ocular-microvascular

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Biology

5.5K 조회수.  Boston Children's Hospital, Harvard Medical School. 이 프로토콜은 마이크로 혈관 증식의 전 생체 모델인 초로이드 발아 분석체를 제시합니다. 이 분석체는 코로이드 마이크로 혈관의 증식에 관여하는 경로를 평가하고 야생 유형 및 유전자 변형 마우스 조직을 사용하여 약물 치료를 평가하는 데 사용할 수 있습니다.

비디오: An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

크리스토퍼 휴즈 : Angiogenesis 연구를위한 체외 모델 (인터뷰)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

연구

생물학

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Angiogenesis 연구에 최적화된 섬유소 젤 비드 분석

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

텐든의 전 생체내 기계 중

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

쥐 장간막의 Angiogenesis 분석

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

에 대한 내피 세포의 관 형성의 분석<em&gt; 체외</em&gt; 혈관 신생 연구

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

<em&gt; 예 생체 내</em&gt; 문화 시스템 갑상선 개발을 연구하는

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

은 C-FOS 단백질 면역 조직 검색 : 특정 생리적 반응에 관여 중앙 진학의 마커로서 유용한 도구<em&gt; 생체</em&gt; 및<em&gt; 전의 VIVO</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

Ex Vivo 세포 생리학 연구에 대 한 망막 Arterioles의 절연

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique tissue important for proper function of the synovial joint and is constituted by a dense extracellular matrix (ECM), rich in proteoglycan and type II collagen. Chondrocytes are the only cell type present within cartilage and are widespread and relatively low in number. Altered external stimuli and cellular signalling can lead to changes in ECM composition and deterioration, which are important pathological hallmarks in diseases such as osteoarthritis (OA) and rheumatoid arthritis.
Ex vivo cartilage models allow 1) profiling of chondrocyte mediated alterations of cartilage tissue turnover, 2) visualizing the cartilage ECM composition, and 3) chondrocyte rearrangement directly in the tissue. Profiling these alterations in response to stimuli or treatments are of high importance in various aspects of cartilage biology, and complement in vitro experiments in isolated chondrocytes, or more complex models in live animals where experimental conditions are more difficult to control.
Cartilage explants present a translational and easily accessible method for assessing tissue remodeling in the cartilage ECM in controllable settings. Here, we describe a protocol for isolating and culturing live bovine cartilage explants. The method uses tissue from the bovine knee, which is easily accessible from the local butchery. Both explants and conditioned culture medium can be analyzed to investigate tissue turnover, ECM composition, and chondrocyte function, thus profiling ECM modulation.

Here, we present a protocol describing isolation and culturing of cartilage explants from bovine knees. This method provides an easy and accessible tool to describe tissue changes in response to biological stimuli or novel therapeutics targeting the joint.

소 무릎 이식에서 연골 리모델링의 전 생체 외 조직 문화 모델

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

정수리 상피 세포 활성화에 관여하는 통로를 분석하는 전 비보 분석으로 구체 아웃스승

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...