이 작품은 NSFC 프로젝트 (81522023, 81530059, 91639113, 81772000, 81371699, 및 81401534)에 의해 재정적으로 지원 되었다.

실험 동물의 사용은 동물 실험 윤리 위원회의 난카이 대학에 의해 승인 하 고 관리 및 실험 동물의 사용에 대 한 가이드에 따라 실시.
1입니다. Electrospun PCL 이식의 제조
참고: 여기, 전기 기술 혈관 이식 조작에 이용 되었다.
<ol><li>각각, 메탄올과 클로 프롬의 혼합물에서 PCL을 용 해 하 여 25 wt %와 10 wt %의 PCL 솔루션을 준비 (1:5 볼륨 비율), 실 온 (RT) 12 시간에서.</li>
	<li>10 mL 유리 주사기를 PCL 솔루션을 로드 합니다.</li>
	<li>21-G 바늘 주사기를 놓습니다.</li>
	<li>컬렉션 악기에 스테인리스 스틸 맨 드릴 (2 mm 직경, 길이 25 c m)를 놓습니다.</li>
	<li>두꺼운 섬유 이식에 대 한 사용 하 여 25 wt %의 PCL 솔루션, 수집기 바늘 끝에서 17 cm의 거리, 8 mL/h의 유량 및 11의 전압 전기 매개 변수로 kV. 얇은 섬유 이식 10 wt %, 수집기에 바늘 끝에서 20 cm의 거리, 2 mL/h의 유량 및 18의 전압의 PCL 솔루션 사용 전기 매개 변수로 kV.</li>
	<li>얻은 이식 하룻밤 잔여 용 매를 제거 하는 진공 상태에서 배치 됩니다 확인 하십시오. 소독 절차 전에 모든 악기 고 무 균 기술을 통해 유지 합니다.</li>
	<li>주입 전에 이식 30 분 75% 에탄올 10 ml에서 그들을 immersing 및 다음 하룻밤 UV 빛에 노출에 의해 소독.</li>
	<li>섬유 및 기 공 크기 측정: 스캐닝 전자 현미경 (SEM) 이미지에 따라 ImageJ 소프트웨어를 사용 하 여 평균 섬유 직경을 계산.</li>
	<li>기계 장비의 테스트:<ol>
<li>면도날을 사용 하 여 길이 3 m m 섹션으로 관 건설 기계를 절단. 건설 기계는 마이크로미터를 사용 하 여 두께 측정 합니다.</li>
		<li>100 북 아의 부하 용량 인장 테스트 기계에 관 건설 기계를 배치</li>
		<li>파열까지 1mm 간 클램프 거리와 풀 10 m m/min의 속도로 경도 건설 기계 클램프. 인장 강도 및 파단 궁극적인 측정 합니다. 초기 선형 영역에서 탄성 계수를 계산 (최대 5% 변형) 응력-변형 곡선의.</li>
	</ol>
</li>
</ol>2. 쥐 복 부 대동맥 이식 모델
참고: 모든 재료와 수술에 사용 되는 계기는 살 균. 수술 동안 거 즈 마스크와 감염을 피하기 위해 멸 균 장갑을 착용 하는 연산자 확인 하십시오. 실내 온도 동물 체온을 유지 하기 위해 27-30 ° C에서 유지 됩니다 확인 하십시오. 진통에 관한 현지 IACUC 지침을 따르십시오.
<ol><li>남성 Sprague Dawley 쥐 혈관 이식의 수신자로 240-270 g의 무게를 사용 합니다. 쥐 수술 하기 전에 24 시간 금식은 확인 합니다. 24 시간 단식 쥐의 목표는 빈 창 자에서 대변 충분히, 그로 인하여 확장 연산자의 수평선.</li>
	<li>쥐의 백 목 잡고 아래쪽으로 머리를 유지, 낮은 복 부의 복 부 구멍으로 springe 바늘을 삽입. 복 주사에 의해 chloral 하이드 레이트 (330 mg/kg)과 마 취에 대 한 쥐를 유도.</li>
	<li>쥐는 근육과 꾸준히 호흡을 완화 했다 함으로써 적절 한 anesthetization를 확인 합니다. 운영 현미경 쥐 부정사 위치에 놓습니다.</li>
	<li>바 셀 린 안과 수 의사 연 고 마 취에서 건조를 방지 하기 위해 눈에 적용 됩니다. 수술 전에 꼬리 정 맥 주입에 의해 heparinized 생리 염 분 해결책 (50 UI/mL)와 anticoagulation (UI/100kg)을 관리 합니다.</li>
	<li>면도칼 블레이드를 사용 하 여 전방 복 벽에 털을 면도 하십시오 그리고 요오드 솔루션 및 의료 알코올 솔루션을 사용 하 여 피부를 청소.</li>
	<li>수술가 위로 중간 개복 술 절 개를 수행 하 고 절 개 길이, 약 4-5 m 인지 확인 다음 복 부 구멍을 노출.</li>
	<li>철회 하 고 우선적으로 생리 식 염 수를 적신 거 즈로 내장을 포장.</li>
	<li>신중 하 게 복 부 대동맥 해 부.</li>
	<li>식별 하 고 9-0 monofilament 나일론 봉합을 사용 하 여 모든 작은 가지를 선.</li>
	<li>격리 된 섹션을 클램프 (길이 최대 1 c m) 2 개의 혈관 클램프를 사용 하 여 대동맥의. 대동맥 20-30 분 채워 남아 있을 수 있습니다.</li>
	<li>Anastomotic 사이트를 만들 마이크로-가 위를 사용 하 여 두 개의 클램프 사이의 복 부 대동맥 transect</li>
	<li>잔여 혈액을 제거 하 heparinized 염 (UI/50ml) 솔루션을 사용 하 여 대동맥의 두 끝을 플러시.</li>
	<li>마이크로-가 위를 사용 하 여 adventitia 벗기다.</li>
	<li>9-0 monofilament 나일론 봉합을 사용 하 여 숫자의 8 봉합 패턴 2 m m 내경 및 쥐의 복 부 대동맥에 길이 1 cm 이식 anastomose.</li>
	<li>첫째, 9, 3, 12, 및 근 위 측에 6 시 위치 순서 대로 4 개의 anastomoses을 구성 다음 두 봉합 사이 4 바늘 컷된 가장자리 anastomose. 근 위 봉합을 마치고 같은 방법으로 원심 측을 봉합. 참고: 모든 스티치는 네이티브 쪽은 약간은 이식에 포함 된 보장 합니다.</li>
	<li>이식, 흘러 혈액을 있도록 원심 클램프 다음 인접 클램프를 제거.</li>
	<li>멸 균 면봉 또는 스폰지 작은 거 즈를 사용 하 여 출혈을 막으려고 봉합 끝을 누릅니다. 약 3 분, hemostasis 때까지 누릅니다.</li>
	<li>복 부 구멍으로 내장을 반환 합니다.</li>
	<li>플러시 gentamicin (320 U/mL)와 따뜻한 생리 식 염 수 솔루션을 사용 하 여 복 부 구멍.</li>
	<li>각각 근육과 피부 층에 3-0 나일론 봉합 사를 사용 하 여 복 벽을 바느질.</li>
	<li>깨끗 하 고 건조 감 금 소에 쥐를 놓고 동물 체온; 유지 케이지 난방 패드를 넣어 마 취에서 회복 하기 위해 쥐를 기다립니다. Sternal recumbency를 유지 하기 위해 충분 한 의식 회복 될 때까지 동물에 참석.</li>
	<li>후 의식 회복, 음식과 물을 함께 단일 케이지에 쥐를 넣어. 수술 후 감염을 방지 하기 위해 상처에 요오드를 적용 합니다. 그것은 완벽 하 게 복구 될 때까지 다른 동물의 회사에는 쥐를 반환 합니다.</li>
	<li>미리 정해진된 시간 지점에서 기관 지침에 따라 쥐 안락사</li>
</ol>

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genesis+and+growth+of+extracellular-vesicle-derived+microcalcification+in+atherosclerotic+plaques.">Genesis and growth of extracellular-vesicle-derived microcalcification in atherosclerotic plaques.</a> Nat Mater. 15 (3), 335-343 (2016).</li><li>Tara, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Well-organized+neointima+of+large-pore+poly(L-lactic+acid)+vascular+graft+coated+with+poly(L-lactic-co-epsilon-caprolactone)+prevents+calcific+deposition+compared+to+small-pore+electrospun+poly(L-lactic+acid)+graft+in+a+mouse+aortic+implantation+model.">Well-organized neointima of large-pore poly(L-lactic acid) vascular graft coated with poly(L-lactic-co-epsilon-caprolactone) prevents calcific deposition compared to small-pore electrospun poly(L-lactic acid) graft in a mouse aortic implantation model.</a> Atherosclerosis. 237 (2), 684-691 (2014).</li></ol>

저자 아무 충돌 금융 관심 있다.

셀 침투는 중생에 대 한 중요 하 고16 vivo에서혈관 이식의 개장 이다. 제한 된 셀 침투는 종종 이식 벽으로 세포의 이동 방해는 이식의 상대적으로 작은 구멍 관련이 있습니다. 이 문제를 해결 하려면 우리는 큰 기 공 구조 electrospun PCL 혈관 이식 준비 하 수정된 방법을 개발. 세부 사항에, 기 공 크기는 쉽게 처리 매개 변수에 의해 제어 될 수 있는 섬유 두께의 증가 함께 증가...

합성 중합체에서 만든 혈관 이식 심장 혈관 질병 (CVDs)의 치료를 위한 병원에 널리 활용 됩니다. 불행히도, 작은 직경의 혈관 이식의 경우 (D < 6 m m) 사용할 수 없습니다 종종 이르게 혈전 증, 내 증식, 그리고 다른 감소 된 혈액 흐름 속도 의해 발생 하는 낮은 patency 성공적인 제품 있다 합병증1.
조직 공학 장기 patency 및 항상성 비 계 기반 혈관 재생 및 재건에 따라 실현 하는 대체 전략을 제공 합니다. 자세히, 혈관 이식, 3 차원 서식 파일로 제공할 수 있는 기계적인 지원 및 혈관 조직 및 기능의 영향 세포, 세포 접착, 이동, 확산를 포함 하 여 재생 하는 동안 구조 지도 기질2분 지금까지, 다양 한 합성 폴리머 혈관 조직 공학에서 응용 프로그램에 대 한 평가. 이러한 고분자 중 poly(ε-caprolactone) (PCL)가 집중적으로 조사 좋은 셀 호환성 및 느린 저하 몇 개월에서 2 년3에 이르기까지. 쥐 대동맥 모델4,,56, PCL 혈관 이식 전기 처리 우수한 구조적 무결성 및 patency, 뿐만 아니라 지속적으로 증가 세포 침입과에서 neovascularization 전시는 최대 6 개월까지 이식 벽입니다. 그러나, 불리 한 조직 세포와 모세 혈관의 석 회화, 회귀를 포함 하 여 리 모델링 또한 관찰 되었다 더 긴 timepoints에서 최대 18 개월.
Cellularization 혈관 이식의 조직 재생을 결정 하 고7을 remolding 핵심 요소입니다. 전기, 다양 한 기법으로는 널리 고용 나노 섬유 구조8혈관 이식의 준비에 대 한. 불행히도, 상대적으로 작은 기 공 구조는 종종 후속 조직 재생을 제한 하는 electrospun 혈관 이식에 부족 한 세포 침투를 끈다. 이 문제를 해결 하려면 다양 한 기술은 시도 기 공 크기와 전반적인 다공성,9,10, 수집 장치, 레이저 방사선11에 의해 치료 후의 수정 leaching 소금/폴리머를 포함 하 여 증가 , 등등. 사실, electrospun 이식 (를 포함 하 여 섬유 직경, 기 공 크기 및 다공성)의 구조는 밀접 하 게 관련이 처리 조건12,13. 중 전기, 섬유 직경 폴리머 솔루션, 흐름 속도, 전압 등의 농도 같은 매개 변수를 변경 하 여 쉽게 제어할 수 있습니다. 14 , 15, 그러므로, 숨 구멍 크기와 다공성 향상 되었습니다 따라와.
우리는 최근 macroporous 구조 (5-7 µ m의 직경 및 30-40 µ m의 모 섬유) 수정된 PCL electrospun 이식을 했다. Vivo에서 쥐 복 부 대동맥을 대체 하 여 이식 3 개월 후 수술16patency, 좋은 endothelialization, 부드러운 근육 중생의 높은 비율을 보였다. 더 중요 한 것은, 아니 불리 한 조직의 석 회화 및 셀 회귀를 포함 하 여 개장 이식의 1 년 후에 관찰 될 수 있었다.

<table>
	<tbody>
		<tr>
			<td>Poly(&#949;-caprolactone) (PCL) pellets (Mn=80,000)</td>
			<td>Sigma</td>
			<td>704067</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Tianjin Chemical Reagent Company</td>
			<td>1060</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol</td>
			<td>Tianjin Chemical Reagent Company</td>
			<td>1083</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Tianjin Chemical Reagent Company</td>
			<td>A1007</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Tianjin Fengchuan Company</td>
			<td>2296</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Alfa Aesar</td>
			<td>A16046</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley rats</td>
			<td>Laboratory Animal Center of the Academy of Military Medical Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal saline</td>
			<td>Hebei Tiancheng Pharmaceutical company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloral hydrate</td>
			<td>Tianjin Ruijinte chemical company</td>
			<td>2223</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium Injection</td>
			<td>Tianjin Biochem Pharmaceutical company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin Sulfate Injection</td>
			<td>Jiangsu Lianshui Pharmaceutical company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-&#945;-SMA primary antibody</td>
			<td>Abcam</td>
			<td>ab7817</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-smooth MYH primary antibody</td>
			<td>Abcam</td>
			<td>ab683</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti-rat elastin antibody</td>
			<td>Abcam</td>
			<td>ab23748</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-von Willebrand factor primary antibody</td>
			<td>Abcam</td>
			<td>ab6994</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (Alexa Fluor 488)</td>
			<td>Invitrogen</td>
			<td>ab150117</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (Alexa Fluor 488)</td>
			<td>Invitrogen</td>
			<td>ab150077</td>
			<td></td>
		</tr>
		<tr>
			<td>5% normal goat serum</td>
			<td>Zhongshan Golden bridge</td>
			<td>ZLI9022</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and eosin (H&#38;E)</td>
			<td>Beijing leagene biotech</td>
			<td>DH0006</td>
			<td></td>
		</tr>
		<tr>
			<td>Masson&#39;s trichrome</td>
			<td>Beijing leagene biotech</td>
			<td>DC0032</td>
			<td></td>
		</tr>
		<tr>
			<td>Verhoeff-van Gieson (VVG)</td>
			<td>Beijing leagene biotech</td>
			<td>DC0059</td>
			<td></td>
		</tr>
		<tr>
			<td>Von Kossa</td>
			<td>Beijing leagene biotech</td>
			<td>DS0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical sutures needles with thread,3-0 silk</td>
			<td>Shanghai Jinhuan medical supplies company</td>
			<td>G3002b</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical sutures needles with thread,9-0 silk</td>
			<td>Shanghai Jinhuan medical supplies company</td>
			<td>H901</td>
			<td></td>
		</tr>
	</tbody>
</table>

implantation of electrospun vascular grafts with optimized structure in a rat model

여기, 우리는 macroporous PCL 혈관 이식 조작 및 복 부 대동맥 교체의 쥐 모델을 사용 하 여 평가 프로토콜을 설명 하는 프로토콜을 제시. Electrospun 혈관 이식 자주는 이식으로 세포 침투를 제한 하 고 재생성 하 고 개장 하는 신 동맥의 방해는 상대적으로 작은 구멍을가지고 있다. 이 연구에서는 두꺼운 섬유 (5-6 µ m)와 더 큰 모 (~ 30 µ m) PCL 혈관 이식 수정된 처리 기술을 사용 하 여 조작 했다. 이식의 장기 성능 쥐 복 부 대동맥 모델에 주입에 의해 평가 되었다. 초음파 분석은 이식 남아 동맥 또는 이식의 12 개월 후에 발생 하는 협 착 증 없이 특허를 보여주었다. Macroporous 구조는 셀 ingrowth를 개선 하 고 따라서 3 개월에서 재생성 조직 승진. 더 중요 한 것은, 12 개월 후 이식 벽 내 석 회화 등 불리 한 개장의 아무 표시도 없었다. 따라서, 처리 하는 수정 된 macroporous와 electrospun PCL 혈관 이식 장기 이식에 대 한 동맥 대체 될 잠재적인 개최.

PCL 이식 했다 explanted 3 개월 및 12 개월에서 post-operatively 및 hematoxylin에 오신 (H & E), 표준 조직학 기술에 의해 trichrome Masson, 분석 Verhoeff 반 Gieson (VVG), 폰 Kossa, 면역 형광 α-SMA를 위한 얼룩 MYH, vWF, 그리고 엘라 스 틴입니다. 조직학 이미지 똑바로 현미경을 사용 하 여 찍은 그리고 면역 형광 이미지 ﬂuorescence 현미경을 사용 하 여 찍은.
<p class="jove_content" fo:keep-together.within-pag...

Watch this Scientific Journal Video about Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model at JoVE.com

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

쥐 모델에 최적화 된 구조를 가진 Electrospun 혈관 이식 술의 이식

여기, 우리는 PCL 두꺼운 섬유와 큰 모 공, 혈관 이식 조작 및 복 부 대동맥 교체의 쥐 모델에서 vivo에서 성능을 평가 하기 위해 프로토콜을 설명 하는 수정 된 전기 방법을 제시.

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta ...

implantation-electrospun-vascular-grafts-with-optimized-structure-rat

Research

JoVE Journal

Biology

7.9K 조회수.  Nankai University. 여기, 우리는 PCL 두꺼운 섬유와 큰 모 공, 혈관 이식 조작 및 복 부 대동맥 교체의 쥐 모델에서 vivo에서 성능을 평가 하기 위해 프로토콜을 설명 하는 수정 된 전기 방법을 제시.

비디오: Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation.

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.

The skilled reaching scale divides the movement by a forelimb in a reach for food act into composite elements each of which are evaluated with a three-point scale. The rating scale is described for a normal rat and can be applied toward evaluating neurological motor disorders.

쥐에 도달 전문 Forelimb의 구조 : 운동 평가 척도

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and ...

연구

생물학

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

랫 중심에 엔지니어링된 조직의 주입

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

배아 림브 스킨 Vasculature에 대한 전체 마운트 Immunohistochemical 분석 : 엠브료에 혈관 분기 Morphogenesis를 공부하는 모델 시스템

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

Mycoplasma의 최적화된 PCR 기반의 감지

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

정제 된 야생형과 돌연변이 체 CFTR 단백질의 기능의 재구성 및 채널 활동 측정

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby the permeability of the endothelium to blood cells, plasma macromolecules, and water can be adapted according to the physiological need. In certain diseases, cytokines and growth factors are released that target the endothelial barrier to transiently increase vascular permeability; however, their prolonged presence may cause chronic vascular hyperpermeability and thereby tissue-damaging edema. The Miles assay is an in vivo technique that allows researchers to study vascular hyperpermeability through the proxy measurement of vascular leakage. Here, we provide a detailed protocol on how to perform this procedure in the mouse, which is the most widely used model organism to study mammalian physiology and pathology. The procedure involves the intravenous injection of Evans blue dye to label the circulating albumin followed by multiple intradermal injections of permeability-inducing agents and vehicle control solutions into opposing flanks of the mouse. Consequently, Evans blue dye gradually leaks into the dermis, where it accumulates and can be extracted for quantification as leakage induced by the permeability-inducing agent relative to the vehicle. The Miles assay can be performed in wild type or genetically modified mouse models and may be combined with drug administration to study molecular mechanisms that regulate vascular permeability and identify agents/targets capable of inducing or blocking hyperpermeability.

Here, we present a protocol to measure the vascular leakage induced by intradermal administration of permeability promoting agents into the murine skin. This technique can be used to determine the ability of molecules to promote or inhibit vascular leakage or to study the molecular mechanisms that regulate vascular permeability.

마일 분석 결과 함께 피부에 혈관 Hyperpermeability 유도 요원 평가

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

체 외에서 세포 마이그레이션 자동화 된 광학 카메라의 테스트에 대 한 스크래치 분석 결과 최적화

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

페리 이식 단계 인간 배아에서 트로포 블라스트 세포의 단일 세포 수집

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

다중 파라미터 관류 조절을 통한 원발성 쥐 간세포의 분리

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...