저자는 소화 분자 연구 UPENN NIDDK 센터의 분자 병리 및 이미징 코어 및 이미징에 대한 지원은 간 질환 (P30의 DK50306)에게 감사의 말씀을 전합니다. 이 작품은 (RGW에) 건강의 국립 연구소 (R01 DK-092111)에서와 프레드 수잔 Biesecker 소아 간 센터에서 교부금에 의해 및 (SK에) 어린 시절 간 질환 연구 및 교육 네트워크에서 친교에 의해 지원되었다.

달리 명시되지 않는 한 전체 절차는 실온에서 수행된다. 모든 동물의 작업은 지역 기관 동물 관리 및 사용위원회 (IACUC)의 승인을 프로토콜에서 인도적인 조건 하에서 수행되어야한다. 1. 장비의 제조 및 솔루션 <ol><li> 조직 문화 후드와 인큐베이터 (그림 1A)에 근접 마우스 수술 테이블을 설정합니다. 참고 : 필요한 장비는 해부 현미경, 광원, 12.5 cm 긴 직선 홍채 가위, 좋은 팁을 6 인치가 아닌 톱니 모양의 곡선 집게, 4 인치 곡선 팁 (그림 1B)와 톱니 모양의 집게를 포함한다. </li><li> 수술 테이블에 70 % 알코올의 유리 비커에 멸균 악기를 배치합니다. </li><li> 무균 격리 미디어 (표 1)로 3 멸균 60mm 배양 접시를 채우; 수술 테이블에 두 개의 후드 하나, 얼음에 모두 배치합니다. </li><li> 쥐의 꼬리 콜라겐을 놓고,멸균 배 인산염 완충 생리 식염수 (PBS), 멸균, 살균 1 N 수산화 나트륨과 조직 문화 후드에서 담도 상피 세포 (BEC) 미디어 (표 1). 참고 : 콜라겐 얼음에 있어야합니다. </li></ol> 2. 담즙 덕트 분리 및 문화 <ol><li> 의식이 IACUC 가이드 라인 (그림 1C) 당 자궁 전위, 다음까지 이산화탄소에 노출, 생활 0-3 일 사이에서 신생아 쥐를 안락사. 참고 : 이전 마우스는 특정 실험의 필요에 따라 사용할 수 있습니다. </li><li> 앙와위에서 동물을 배치하고 골반 지역의 중간 선을 넘어 작은 수평 절개를합니다. U 모양의 플랩을 형성, 옆으로 최대 복부의 양쪽이 절개를 확장 할 수 있습니다. 복부 장기를 노출하는 플랩을 켭니다. 복부가 열리면,주의 깊게 간, 담관의 더 나은 시각화를 위해 동물의 오른쪽으로 복강 밖으로 창자를 이동. 참고 : 간장의 더 나은 식별을 얻기 위해 흉곽에 가로 절개를 확장 할 필요가있다. </li><li> 해부 현미경을 사용하여, 담관, 간, 창자를 식별합니다. 구조는 매우 섬세하고 있으므로주의를 사용하여, 먼저 담관 (그림 1D)를 확인합니다. 톱니 모양이 아닌 톱니 모양의 집게로, 꼼꼼하게 청소하고 담즙을 둘러싼 지방을 포함하는 결합 조직을 선택합니다. 덕트 청소하게 다른를 개최 한 집게를 사용합니다. </li><li> 담낭을 청소하기 위해 비슷한 방법을 사용합니다. </li><li> 얼음에 격리 미디어의 첫 번째 접시에 청소 덕트 및 담낭을 놓습니다. 미디어에 추가로 결합 조직을 제거하고 담낭에서 담즙을 제거하면서 부드럽게 비 톱니 모양의 집게로 구조를 마사지. </li><li> 격리 미디어의 두 번째 접시에 덕트 및 담낭을 전송합니다. </li><li> 5 동물의 덕트 및 담낭의 분리 후, 전송후드에서 세 번째 페트리 접시에 조각. 참고 : 셀이 5 개 이상 동물로부터 분리 될 경우에, 5 동물의 각 그룹에 대한 새로운 해결책을 이용​​하여 일괄 적으로 수행. </li><li> 조직 문화 후드에 두꺼운 콜라겐 젤을 준비합니다 : 참고 : 어떤 크기의 접시 사용할 수 있습니다. 5 신생아의 각 그룹에 대해 3 ㎖ 콜라겐의 최종 볼륨이있는 60mm 문화 요리는 잘 작동합니다. 이는 갓 절연 덕트 매립시 신선한 준비되어야한다. <ol><li> 얼음에 쥐 꼬리 콜라겐, 살균 배 인산염 완충 생리 식염수 (PBS 배), 멸균 dH보다 2 O 및 멸균 1 N NaOH를 놓습니다. </li><li> 콜라겐의 0.023 배의 볼륨 등을 1 N 수산화 나트륨의 양을 사용하여, dH보다 2 0, 10X PBS, 수산화 나트륨, 콜라겐 1X PBS에 2 ㎎ / ㎖ 콜라겐의 최종 농도와 젤에 필요한의 볼륨을 계산 덧붙였다. 참고 : 콜라겐의 대체 소스를 사용하는 경우, 중화 젤 형성을위한 제조 업체의 지침을 따르십시오. </li><li> 함께 dH보다 2 0, 1, 혼합0X PBS, 1 N 수산화 나트륨은, 다음도 혼합하기 위해 콜라겐과 피펫을 잘 추가합니다. </li></ol></li><li> 콜라겐 용액을 실온에서 일관성 같은 겔로 두꺼워지면, 곧바로 콜라겐 덕트를 포함. </li><li> 5 % CO 2, 30 분 동안 응고 콜라겐을 활성화하기 위해, 37 ° C에서 인큐베이터에 놓습니다. </li><li> 젤 (흰색 분명에서 색상 변경), BEC 미디어 (표 1) 3.5 mL를 오버레이를 응고 및 37 ° C에서 배양하면, 5 % CO 2. </li><li> BEC 미디어에게 일주일에 3 번을 제거하고 3.5 ml의 신선한 용지로 교체합니다. </li></ol> 두꺼운 콜라겐 젤 3.을 고립 차 Cholangiocytes 주 : 3 주 안에, cholangiocytes (큰 핵이 포함 된 덕트에서 직접 확장과 세포)의 시트 두께의 콜라겐 볼 수 있습니다. 접시에있는 섬유 아세포의 다수가있는 경우, 그것은 폐기되어야한다. 요리의 몇 가지 영역이있는 경우섬유 아세포 성장, 이들은 메스로 잘라 전에 cholangiocytes 분할을 제거 할 수 있습니다. <ol><li> 얇은 콜라겐 젤을 준비합니다 : <ol><li> 무균 PBS로 1 ㎎ / ㎖의 농도로 콜라겐을 희석. 참고 : 100 mm 플레이트 1 ML을 사용; 다른 크기의 요리에 따라 조정합니다. </li><li> 셀 확산기 (큰 접시) 또는 피펫 팁 (작은 접시)로 균등 콜라겐 용액을 퍼지 한 후 10 분 동안 실온에서 떠난다. </li><li> DMEM/F12와 함께 접시를 덮고 37 ° C, 10 분 동안 5 % CO 2에서 배양한다. </li><li> 인큐베이터에서 접시를 제거하고 1X PBS로 세척. 즉시 1X PBS를 대기음. </li><li> 플레이트를 회전하거나 진탕 판을 배치하여 확산 콜라겐의 제 2 박막 층과 코트. 37 ° C, 10 분 동안 5 % CO 2에서 배양한다. </li><li> 콜라겐이 응고 된 후, 커버 DMEM/F12 플레이트와 37 ° C, 30 분 동안 5 % CO 2에서 배양한다. </li></ol></li><li> 일에서 세포를 분리ICK 콜라겐 젤 : <ol><li> 그것은 부동되도록 세포 배양 주걱 또는 세포 스크레이퍼로, 플레이트 떨어져 부드럽게 콜라겐 젤을 다 쳤어요. </li><li> DMEM/F12 10 ㎎ / ㎖로 유형 XI 콜라게나 제를 (자료 목록 참조) 희석하여 콜라게나 솔루션을 준비합니다. 잘 살균 필터 섞는다. </li><li> 두꺼운 콜라겐 세포의 60mm 접시 당 콜라게나 솔루션 (위)의 1 ML을 추가합니다. (3 ㎖이어야 함) BEC 미디어를 제거하지 마십시오. 37 ° C에서 30 분 동안 접시를 품어, 5 % CO 2. </li><li> 세포를 포함하는 솔루션을 제거하고 15 ML 원뿔 튜브에 배치합니다. </li><li> 5 분 2,200 XG에 스핀 다운. 뜨는을 취소하고 DMEM/F12 비슷한 양의 펠렛을 일시 중지 다시. </li><li> 5 분 2,200 XG에서 다시 솔루션을 스핀. 뜨는을 제거합니다. </li><li> 3 0.5 % 트립신의 ML, 37 ° C, 5 분 동안 5 % CO 2 부화에 재현 탁 펠렛. 배양 후, 솔루션을 피펫 아래 cholangiocytes의 시트를 중단 할 수 있습니다. BEC 미디어의 5 ML을 추가합니다5 분 1,500 XG에 차 스핀 솔루션입니다. </li><li> 다음 5 분 동안 1,500 XG에서 솔루션을 회전, DMEM/F12에 뜨는하고,에 resuspend 펠렛을 제거합니다. </li><li> 상층 액을 제거하고 BEC 미디어 (표 1)에서 펠렛을 일시 중지 다시. 이전에 만든 얇은 콜라겐 젤 접시에 세포. </li></ol></li></ol> 세포의 4. 통로 및 저장 참고 : 필요에 따라 얇은 콜라겐 겔에 세포가 분할 할 수 있습니다. <ol><li> 이전에 10 ~ 15 분 동안 37 ° C에서 0.25 % 트립신의 배양에 멸균 PBS로 접시를 씻으십시오. </li><li> BEC 매체 동량으로 중화하고, 5 분 동안 1,500 XG에서 펠렛. DMEM/F12와 펠렛을 씻어 후, 5 분 1,500 XG에서 펠렛. </li><li> 콜라겐 코팅 된 세포 배양 접시에 배포 BEC의 미디어를 Resuspend 세포. 주 : 대안 적으로, 세포는 저장 매체에 재현 탁하고 -80 ° C 냉동고 나 액체 질소 (표 1)에 저장 될 수있다. </li></ol>

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저자는 더 경쟁의 이익이 없다는 것을 선언합니다.

여기에 설명하는 것은 신생아를 포함한 모든 연령대의 마우스 마우스의 간외 담관에서 순수한 cholangiocytes을 분리하는 기술이다. 기술 간외 cholangiocytes은 간내 cholangiocytes 별도로 연구 될 수 있다는 장점을 제공하고, 이러한 세포의 집단 사이의 주요 차이점을 확인하는 연구를 용이하게 할 수있다. 우리는 최근하다는 연구가이 방법에 의해 고립 된 붉은 털 로타 바이러스 8에 감염 간외 chola...

내부 및 간외 cholangiocytes의 기원과 발전은 현저하게 다르다. 간은 복부 foregut의 내배엽 5 게실에서 개발하고 있습니다. 뇌 영역은 간내 담도 나무 5를 생성하는 동안 게실의 꼬리 영역은 간외 담도 트리를 형성한다. 간내 덕트 cholangiocytes는 요정 포털 지역 6 유관 플레이트 주변의 전구 세포에서 파생됩니다. 이러한 세포는 간세포 또는 cholangiocytes 6 하나로 분화 할 수있는 능력이있다. 이 cholangiopathies 특별히 cholangiocytes의 하나의 카테고리를 타겟팅 할 수 있습니다 주어진 중요한 임상 적 의미를 가지고 있습니다. Alagille 증후군은 간내 덕트에 영향을 미치는 예를 들어, 담도 폐쇄증은 처음, 신생아의 간외 덕트에 영향을 미칩니다. 마우스 및 래트에서의 간내 cholangiocytes 및 담관 유닛 절연 다중 디스크립션이있다. 에vestigators 덕트 분리 이후의 부산물에 의해 하나의 세포를 고립, 또는 간 소화 항체에 의해 특정 세포 표면 마커 1-4을 표현 cholangiocytes의 풀다운있다. 기능 및 편광 담관 단위 간 소화 크기 여과 (7)에 의해 고립되었다. 담관 유닛 절연 cholangiocytes 시험관 1,2 ductular 구조를 개발하기 위해 도시되었지만, 자극을 분비하고 분비 유체 7을 설명하기 위해 응답 할 수있다. 이러한 방법의 제한은 소화 효소와 간 관류에 대한 전문 기술 전문 지식과 특별한 장비의 필요성이 (가) 있습니다. 또, 간엽 세포 3 오염의 위험이있다. 구체적 신생아 간외 담관에서 간외 담관 cholangiocytes를 분리 방법은 상술되지 않았다. 이 논문은 신생아를 격리 할 수​​있는 간단한 방법을 설명합니다잘 순도 높은 수준의 성인 간외 담관 세포로의. 이 기술은 내부 및 간외 cholangiocytes과 간외 덕트를 포함 담도 폐쇄증과 같은 질병의 메커니즘에 대한 연구 사이의 차이에 대한 연구를 촉진 할 것이다.

<table border="0" cellpadding="0" cellspacing="0" style="width:943px;" width="942">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Name of Material/Equipment</td>
			<td style="width:323px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">DMEM/F12 (1:1)</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11320-033</td>
			<td>500ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">FBS</td>
			<td style="width:323px;">Atlanta Biologicals</td>
			<td style="width:119px;">S11150</td>
			<td>25 ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">MEM with non-essential amino acids</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11140-019</td>
			<td>5 ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Insulin-transferrin-selenium</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">51300-044</td>
			<td>5 ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Na Pyruvate</td>
			<td style="width:323px;">Cellgro</td>
			<td style="width:119px;">25-000-CL</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Chemically-defined lipid concentrate</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11905-031</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Penicillin-Streptomycin</td>
			<td style="width:323px;">Cellgro</td>
			<td style="width:119px;">30-002-CI</td>
			<td>5ml, used in BEC media and 500 ul in the isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Gentamicin</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">15750-060</td>
			<td>0.2ml, used in BEC media and 500 ul in the isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Ethanolamine</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">E9508-100ml</td>
			<td>0.13ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">MEM vitamin solution</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11120-052</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Soybean trypsin inhibitor</td>
			<td style="width:323px;">Biowhittaker</td>
			<td style="width:119px;">17-605E</td>
			<td>5ml, used in BEC media. Solvent is PBS, mix to 5mg/ml stock concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">L-glutamine</td>
			<td style="width:323px;">Cellgro</td>
			<td style="width:119px;">25-005-CL</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Bovine pituitary extract</td>
			<td style="width:323px;">Gemini</td>
			<td style="width:119px;">500-102</td>
			<td>1.1ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Dexamethasone</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">D4902</td>
			<td>0.5ml, used in BEC media, Stock conc 393ug/ml dilute with ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">3 3&#39;,5-triiodo-L-thyronine</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">T6397</td>
			<td>0.5ml, used in BEC media, Stock conc 3.4 mg/ml dilute with ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Epidermal growth factor</td>
			<td style="width:323px;">millipore</td>
			<td style="width:119px;">01-101</td>
			<td>0.5ml, used in BEC media, 25ug/ml dilute with DMEM F12+1%BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Forskolin</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">F6886</td>
			<td>5ml, used in BEC media, use at stock concentration of 0.411mg/ml and dilute with DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Fungizone</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">15290-018</td>
			<td>1ml, used in BEC media and 500 ul in the isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Rat-tail collagen</td>
			<td style="width:323px;">BD Biosciences</td>
			<td align="right" style="width:119px;">354236</td>
			<td>variable depending on concentration of collagen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">PBS 10X</td>
			<td style="width:323px;">USB Corporation</td>
			<td align="right" style="width:119px;">75889</td>
			<td>use at 10x, sterlie, used to make collagen, amount used depends on collagen concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">dH2O</td>
			<td style="width:323px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>sterile, used to make collagen, amount used depends on collagen concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">NaOH 10N</td>
			<td style="width:323px;">Fischer Scientific</td>
			<td style="width:119px;">ss255-1</td>
			<td>Dilute to 1N, sterile, used to make collagen, amount used depends on collagen concentration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">collagenase type XI from Clostridium histolyticum</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">C7657</td>
			<td>dilute in DMEM and sterile filter before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">trypsin-EDTA (1x) 0.25%</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">25200-056</td>
			<td>3 ml, incubate max 10 minutes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">trypsin-EDTA (10x) 0.5%</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">15400-054</td>
			<td>3 ml, incubate max 10 minutes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;"></td>
			<td style="width:323px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Dissecting microscope</td>
			<td style="width:323px;">Nikon</td>
			<td style="width:119px;">SMZ645</td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Light source (fiberoptic illuminator)</td>
			<td style="width:323px;">Schott-Fostec</td>
			<td style="width:119px;">Ace EKE LR 92240</td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">12.5 cm straight iris scissors</td>
			<td style="width:323px;">Kent Scientific</td>
			<td style="width:119px;"></td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">6&#34; non-serrated curved forceps with fine tips</td>
			<td style="width:323px;">Electron Microscopy Sciences</td>
			<td style="width:119px;"></td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">4&#34; serrated stainless forceps with fine tips</td>
			<td style="width:323px;">Electron Microscopy Sciences</td>
			<td style="width:119px;"></td>
			<td>Other models acceptable</td>
		</tr>
	</tbody>
</table>

isolation of neonatal extrahepatic cholangiocytes

간 인트라 및 간외 담관이 발달 별개이며, 차동 특정 질환에 의해 영향을받을 수있다. 그러나, 내부 및 간외 cholangiocytes, 신생아와 성인 세포 사이의 차이가 잘 이해되지 않습니다. 간내 담관에서 cholangiocytes의 분리를위한 방법은 잘 1-4 설정됩니다. 이 별개의 cholangiocyte 집단 간의 차이를 이해하고, 간외 덕트를 대상으로 이와 같은 담도 폐쇄증과 같은 질병을 공부에 큰 도움이 될 것이지만, 특히 신생아에서 간외 유관 세포의 분리, 아직 설명되지 않았습니다. 신생아 및 성인 마우스 양쪽 간외 담관 세포를 분리하기 위해 최적화 된 기술은 여기에서 설명한다. 이 기술은 섬유 아세포와 같은 중간 엽 세포에서 최소한의 오염과 순수한 세포 인구를 산출한다. 이 메 톡시D는 철저한 해부와 지방과 섬유 아세포 층을 제거하는 긁기 다음 간외 덕트 및 담낭의 제거를 기반으로합니다. 구조는 실험적인 사용을 위해 다음 트립신 될 수 있으며, 도금 재 단층에 cholangiocytes의 파생물을 할 수 있도록 약 3 주 동안 콜라겐 배양의 두꺼운 층에 포함됩니다.

신생아 마우스 간외 우수한 순도 cholangiocytes, K19의 면역 형광 염색법에 의해 설명되는 것과 같이 (그림 2A와 2B)의 인구의 분리에이 프로토콜의 결과를 사용하는; 우리는 성인 쥐에서 세포를 분리 비슷한 결과를 얻을 수 있습니다. 우리는 두꺼운 콜라겐 겔에 단일 층을 형성하기 위해 갓 고립 담관의 세포 3 주 정도 관찰했다. cholangiocytes는 절연 도관으로부터 세포 시트를 형성하는 두꺼?...

Watch this Scientific Journal Video about Isolation of Neonatal Extrahepatic Cholangiocytes at JoVE.com

Isolation of Neonatal Extrahepatic Cholangiocytes

신생아 생쥐의 간외 담관에서 cholangiocytes을 분리하는 기술을 설명한다. 덕트 꼼꼼하게 해부 한 다음 세포는 두꺼운 콜라겐 젤의 부산물에 의해 격리됩니다. 이 방법은 간외 담관 개발 및 병리 공부를위한 유용한 도구를 제공합니다.

신생아 간외 Cholangiocytes의 분리

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, ...

isolation-of-neonatal-extrahepatic-cholangiocytes

Research

JoVE Journal

Biology

10.2K 조회수.  The Children's Hospital of Philadelphia.  신생아 생쥐의 간외 담관에서 cholangiocytes을 분리하는 기술을 설명한다. 덕트 꼼꼼하게 해부 한 다음 세포는 두꺼운 콜라겐 젤의 부산물에 의해 격리됩니다. 이 방법은 간외 담관 개발 및 병리 공부를위한 유용한 도구를 제공합니다. 

비디오: Isolation of Neonatal Extrahepatic Cholangiocytes

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

호중구 격리 프로토콜

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

연구

생물학

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

판막병 내피 세포의 분리

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

신생아 마우스의 폐 내피 세포의 분리 및 문화

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

의 분리 Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.1 A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer 'epicuticular' layer as well as the blend consisting of the cutin biopolymer and 'intracuticular' waxes.2 Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Sch&auml;ffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) 13C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS 13C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH2O) chemical moieties.
Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state 13C NMR reveals differences in the relative numbers of CHO and CH2O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-&mu;m length scale.

Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.

분리 및 과일 손톱의 Biophysical 연구

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

분리 및 문화 신생아 마우스 심근

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

배아 심실 내피 세포의 분리

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

신생아 쥐의 폐 동맥 평활근 세포의 분리

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

쥐과 밸브 내피 세포의 분리

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.