ACD is supported by a grant from the National Institute of Health (R01-CA177875). LX is a fellow in the HHMI-funded translational medicine program at UNC Chapel Hill. JVM is supported by a T32 pre-doctoral fellowship from the Integrative Vascular Biology Program at UNC Chapel Hill. We thank Clayton Davis for assistance with confocal microscopy.

다음 프로토콜은 채플 힐 노스 캐롤라이나 대학의 실험 동물 의학의 부에 의해 설립 된 지침에 따라 수행된다. 1. 시작하기 전에 다음 재료 및 시약 준비 <ol><li> 400 ml의 낮은 혈당 보충에 의해 EC 미디어를 준비 (1g / L의 D- 글루코스 또는 LG) 둘 베코의 수정 50 ml의 열 불 활성화 된 소 태아 혈청, 5 ml의 항생제 항진균제 50 ㎖ 뉴 - 세럼 IV, 독수리의 매체 (DMEM) 상업 키트에서 hFGF의, VEGF, hEGF, R3-IGF-1, 헤파린 구성 요소. </li><li> 500㎖의 FACS 완충액 (0.5 % BSA 및 PBS 2 mM의 EDTA)을 제조; 멸균 0.22 μm의 필터 컵을 통해 필터링합니다. </li><li> 소독 또는 보드를 해부 소독. </li><li> 해부 핀, 외과 가위, 및 dissectors을 소독. </li></ol> 2. EC 절연 (1 일 ~ 5 시간) <ol><li> 이산화탄소 또는 다른 방법과 호환 재치 마우스를 안락사H 기관 동물 관리 및 사용위원회 (IACUC) 정책. 참고 : 크기가 다양한 여러 유방 종양 (5 - 직경 15 mm의), 같은 C3-태그와 같은 단일 유전자 변형 마우스의 개발 TEC의 격리를 위해 그들 모두를 수확해야 할 수 있습니다. 하나의 EC 격리를위한 세 가지 1cm 3 종양 동소 마우스에 접목되어 종양, 수영장 두를 사용하는 경우. 여기서 우리는 데모로 유방 종양을 사용하지만, 프로토콜은 다른 유형의 종양에 대해 변형 될 수있다. </li><li> 분무 또는 75 % V / V 에탄올의 충분한 양의 마우스 복부 측면을 닦아 마우스를 소독. </li><li> 멸균 후드 나 클린 벤치에서 무균 기술을 사용하여 가위와 dissectors 일쌍로 종양을 절제. <ol><li> 스트레칭과 해부 보드에 마우스의 사지를 고정. 복막을 열지 않고 가위로 중간 선 복부 절개를합니다. 피부 종양이있는 유선으로 복막 사이에 옆으로 해부.복막을 열지 마십시오. </li><li> 소비세 만 종양 유선에서 조직, 일반 유선 마진을 떠나. 조심스럽게 피부와 근육 등 비 종양 조직을 손질하고, 얼음에 LG-DMEM 30 ml의를 포함하는 원뿔 튜브에 해부 종양을 배치합니다. </li></ol></li><li> 조직 문화 후드에 종양 샘플을 가져와; 2 회 - 멸균 LG-DMEM 1 조직을 씻는다. </li><li> 멸균 조직 문화 배양 접시에 원뿔 튜브에서 전송 종양, 접시에 LG-DMEM 2 ㎖를 추가하고, 조각으로 멸균 가위 &lt;5mm 말하다. </li><li> PBS에서, 디스 파제 1 ㎖를 (주 ​​= 행크의 균형 소금 솔루션에 2 mg을 / ㎖, HBSS를 이하) 콜라게나 제 5 mL를 추가 (재고 = HBSS에 2.5 U / ㎖) 및 데 옥시 리보 뉴 클레아 제 75 μL (주 = 1 ㎎ / ㎖ ) 페트리 접시에. 총 부피는 지금 ~ 10 ㎖이다. </li><li> (미리 설정된 해리 홍보 조직 dissociator 튜브에 페트리 접시에서 콜라겐 / 조직 믹스를 전송하고 두 번 60 초 동안 조직 dissociator에서 실행dissociator에 ogram : 실행 당 1,270 총 라운드). 빛은 37 ° C에서 75 분 동안 진탕 기에서 진탕 배양한다. </li><li> 필터를 50 ㎖ 원뿔형 튜브 위에 100 ㎛ 세포 스트레이너를 통해서 조직을 분해. FACS 버퍼 5 ㎖와 린스 필터가 남아있는 세포를 씻어. 5 분 동안 280 XG에 스핀 조심스럽게 세포 펠렛을 방해하지 않고 뜨는을 대기음. </li><li> 멸균 수 9 ml의 스톡 RBC 용해 완충액 (10 배)의 1 mL로 희석. 바로 10 용해 완충액 (1X)의 용액과 함께를 Lyse 적혈구는 280 x g에서 5 분간 스핀. 참고 : 약간의 피가 보이는 경우에는이 단계를 건너 뛸 수 있습니다. </li><li> FACS 완충액 10ml에 재현 탁. 트리 판 블루 10 μL와 세포 현탁액의 10 μl를 혼합하고, 혈구를 사용하여 살아있는 세포를 계산합니다. </li><li> ~ 10 7 세포 / 100 μL에서 세포를 재현 탁. 세포 현탁액 100 ㎕ 당 FcRγ 쇄 블록의 10 μl를 추가하고 10 분 동안 얼음에 품어. </li><li> 따라 쥐 방지 마우스 PE - 복합 CD31 항체 추가. 때때로 15 분, 영화 튜브 - 표 1 (10) 얼음에 품어. </li><li> 5 분 동안 280 XG에 튜브와 스핀에 외과 버퍼의 10 ML을 추가합니다. 조심스럽게 상층 액을 제거하고 FACS 버퍼의 5 ㎖로 다시 세포 펠렛을 씻는다. 원심 분리기는 세포 펠렛합니다. 방해 세포 펠렛없이 기음 뜨는. </li><li> . 표 2에 따라 외과 버퍼 및 안티 체육 마이크로 비드를 추가 (10) 얼음에 품어 - 15 분. 가끔 톡 관. </li><li> 5 분 동안 280 XG에 외과 버퍼와 스핀 샘플의 10 ML을 추가; 다시 FACS 완충액 원심 5 ㎖로 한번 세척 하였다. 방해 펠렛없이 대기음 뜨는. </li><li> FACS 버퍼 300 μL에 볼륨을 가져와. G X 280에 35 μm의 셀 스트레이너 덮인 관 5 분을 통해 스핀. 필요한 경우 2 튜브와 더 큰 볼륨을 사용합니다. </li><li> , 후드에서 자기 multistand 및 자기 열을 설정 분리기에 열을 연결하고 FACS 버퍼 2 ㎖로 열을 평형. </li><li> AspiratFACS 완충액 1 ㎖ - E 상등액 및 0.5에서 세포 펠렛을 재현 탁. </li><li> 평형 자기 칼럼을 통해 세포 현탁액을 전달합니다. </li><li> FACS 버퍼 2 ㎖로 열 세 번 세척하고, 15 ML 튜브 (FT 분율)의 흐름을 통해 (FT)를 수집합니다. </li><li> 분리 떨어져 열을 가지고 또 다른 15 ML 튜브 (용출액 부분)에 외과 버퍼의 2 ㎖로 용출. 모든 세포는 열 떨어져 있습니다 보장하기 위해 플런저를 사용합니다. 마다 버퍼 외과의 2 mL로 용출을 두 번 더 반복합니다. </li><li> 5 분 동안 280 XG에서 용출액 스핀. </li><li> 상층 액을 제거하고 EC 미디어 10ml에 펠렛을 재현 탁. </li><li> 마찬가지로 용출액 부분을 분할 (~ 6 ㎖) 10cm 젤라틴 코팅 요리에. 세 1cm 3 종양, 플레이트는 적어도 네 개의 플레이트에서 세포를 용출시켰다. 여러 판에 다른 농도 또는 플레이트가 용출 세포 (예. 0.5 ml의 1 ml의 1.5 ml의, 4 판에 용출액을 3 ml의 씨앗)을 보장하기 위하여t 적어도 하나의 플레이트는 띄엄 띄엄 용출 세포 시딩된다. (- 2.0 X 10 5 부착 된 세포 즉, 약 1.0) 첨부 된 세포의 생장이 ~ 1 %에서 다음날 있는지 확인합니다. 주 : EC 콜로니가 다른 세포 유형에 의해 오염되지 않고 형성 할 수 있도록 세포가 부족한 도금 될 필요가있다. </li><li> 한 10cm 접시에 플레이트 FT 분율은 세포가 O / N을 복구 할 수 있습니다. 다음날 컨 플루 언트 100 % - 플레이트 (80)가 있는지 확인합니다. (-80 ° C) 250 μL 세포 동결 cryotube 미디어 및 액체 질소에 보관 다음날 세포를 동결. 주 : FT 분획 나중 단계에서 및 / 또는 절연 EC 클론 EC 유전자 발현 분석을위한 음성 대조구로 종양 세포 분리를 위해 사용될 수있다. </li><li> 3 일 - 매 2 미디어를 변경합니다. 10 일 - 식민지 7 후 형성하기 시작합니다. 작은 EC의 식민지가 빠르면 접시의 바닥에 미세 팁 마커와 식민지 일 3. 표시로 식별 할 수 있습니다. </li><li> 비 특정 C를 쳤어요멸균을 200㎕ pippet 팁이 식별 된 콜로니 주변의 ELL. </li></ol> 3. 식민지 선택하여 복제의 반지 <ol><li> 3 일 - 매 2 미디어를 변경합니다. 7 일 - EC 순도는 약 5에서 LDL (DII-AC-LDL) 또한 확인할 수 있습니다. 10 ml의 EC 매체 당 LDL의 50 μl를 추가하고 3 부화 - 형광 현미경으로 세포를 확인하기 전에 4 시간. 참고 : 여러 LDL + EC 클론이 7 일 ~에서 관찰 할 수있다. </li><li> 크기가 5 ㎜ - 그들은 3의 직경에 도달하면 EC의 식민지를 수확 시작합니다. (최상의 결과를 위해 작은 LDL + 세포로 포장되어 큰 식민지를 선택합니다.) </li><li> 복제 반지, 프리 코트 0.5 % 젤라틴으로 몇 6 웰 플레이트와 EC를 수확하기 전에. 대기음 젤라틴, 각 웰에 EC 미디어 2 ㎖를 추가하고, 필요할 때까지 인큐베이터에서 접시를 유지한다. </li><li> 다른 세포 유형이 클로닝 링 내에 포획되지 않는다 확인 콜로니의 에지에 비 EC 긁어. </li><li> 사용위상차 현미경 (10X 대물 또는 4X)를 배양 접시의 바닥에 미세한 팁 마커의 개요 EC 콜로니를 포함한 영역. </li><li> PBS 10 mL로 판을 씻고 흡입 할 때 판에 PBS의 매우 얇은 층을 둡니다. (중요 : 소량 (~ 0.5 ㎖) PBS의 복제 링 절차를 수행하는 동안 살아있는 세포를 유지하는 것입니다 또한, 조직 접착제는 접착 물을 필요로한다.) </li><li> 적절한 크기의 복제 링을 선택합니다. 반지를 데리러 해부 포셉 한 쌍을 사용하여 10 μL 피펫 팁으로 균등하게 복제 링에 조직 접착제의 작은 금액을 적용합니다. 참고 : 복제 반지에 조직 접착제 (작은 반지 ~ 0.2 μl를) 최소한의 양을 사용하고 있는지 조직 접착제가 좋은 밀봉을 보장하기 위해 바닥면 주위에 균일하게 확산되어 있는지 확인합니다. 과도한 조직 접착제는 열을 생성하고 세포를 죽일 수있다 필름을 형성한다. </li><li> EC 식민지를 통해 복제 링을 놓습니다. 부드럽게 리 접착제하기 위해 복제 링을 아래로 눌러접시에 겨. 식민지가 반지를 접착하기 전에 건조되지 않도록해야합니다. </li><li> 즉시 복제 링에 효소 세포 분리 솔루션의 25 μl를 피펫과 ~ 1 분을 품어 또는 세포를 느슨하게 부착 될 때까지. </li><li> 미리 예열 EC 미디어를 포함하는 6 웰 플레이트 잘 하나에 복제 링 드롭 현명한에 피펫 세포. (중요 : 세포를 분산 할 수있는 판 흔들지 마십시오; EC 꽉 클러스터에서 성장하는 것을 선호합니다.) 50 복제 반지를 세척 - 가능한 한 많은 세포를 수집하기 위해 100 μL EC 미디어와 같은 6 자에 모든 세척을 전송 . </li><li> 10cm 접시에 일부 식민지 수확하기에 너무 작은 경우, 식민지 더 며칠 동안 성장하자, 10 ml의 신선한 미디어를 추가, 다시 복제 링 절차를 반복합니다. </li><li> 10cm 조직 배양 접시에 그들을 확장하기 전에, 6 웰 플레이트의 3 우물에 세포를 100 % 합류 및 전송 - 80까지 6 웰 플레이트에서 수확 식민지를 성장. 오염 물질의 세포를 긁어. 반복필요한 경우 - 복제 링 절차의 또 다른 라운드 (3.10 3.5 단계). 확장 할 때 - (70 % ~ 60) 상대적으로 합류 세포를 유지합니다. EC는 너무 부족한 도금 경우 성장을 중지 할 수 있습니다. </li><li> 등 외과, 염색, PCR에 의해 EC의 특징은 DIL-AC-LDL이 형광 및 외과 용 PE 또는 다른 형광 항체를 방해 할 수 있습니다. </li></ol>

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The authors have nothing to disclose.

때문에 순수 차 TEC의 문화, 인간 제대 정맥 EC (HUVEC) 13 상업적으로 이용 가능한 EC 라인 또는 기본 EC와 시험관 연구에서 많은 대체 TEC를 얻기에 어려움. 그러나, 정상 조직에서 이러한 EC의 인구는 정상적인 대응에서 크게 차이가 TEC에 대한 프록시 역할을 할 수있다. 예를 들어, TEC는 생체 내에서 표현형 및 기능적 이상이며, 이러한 이상 중 일부는 체외 14-18에서 ?...

내피 세포 (EC)는 고형 종양의 개발 과정에서 필수적이다. 보급과 먼 부위에서 전이의 파종에 휴면 종양에서 혈관 신생 스위치의 시작에서, EC는 종양 성장 1을 유지하기 위해 혈액, 산소를 공급 도관과 영양분을 형성한다. 최근에 제안 된 바와 같이, EC는 관류 독립 펑션이 암 줄기 세포 및 다른 종양 간질 세포 2-5의 성장을 지원하는 틈새를 형성한다. 따라서, 높은 종양 특이 체외 문화에 대한 EC (TEC)는 종양 혈관 신생 및 종양 세포와 혼선을 매개하는 새로운 분자 메커니즘을 밝혀됩니다 루틴 기능 연구를 허용 정제. EC는 기원 6의 조직에 따라 고도의 전문이다. 때문에 다른 종양 유형의 이질적인 성격과 종양 미세 환경에, TEC는 또한 종양 특이 전문화 O를 반영하는 고유의 기능이 표시 될 수 있습니다혈관 F. 예를 들어, 다른 유형의 종양 또는 7,8의 성적으로부터 격리 TEC에서 유전자 발현 서명에 현저한 변화가 존재한다. 그러나, TEC 비-EC,​​ 특히 종양 관련 섬유 아 세포와 종양 세포의 빈번한 공동 정화는 게놈 전체의 발현 분석을 혼동 할 수 있습니다. 이러한 원치 않는 세포 유형은 TEC 배양 시험관 팽창 장기에 의존 연구에서 특히 문제가된다. 여기에 설명이 지속적으로 종양과 다른 조직에서 순수한 EC 문화를 생산하는 고품질의 방법이다. EC의 분수와 공동 정제 비 EC의 제거의 면역 자기 열 농축에 따라, 추가 복제 링 단계 9를 사용하는 순수한 EC 식민지를 촬영합니다. 각 콜로니 비 EC 오염의 발생없이 여러 통로 용 배양에서 확장 될 수있다. 이 방법은 엔도의 연구를위한 이상적인 단일 분리 과정에서 다중 EC 클론을 수득상피 이질성. ZsGreen이 리터 / S / L 기자 마우스 문화 10 ZsGreen이 형광을 유지 &quot;운명 매핑&quot;과 지워지지 표시된 EC를 생성하기위한 유용한 도구입니다 : 또한, Cdh5 CRE는 것을 알 수있다. 프로토콜에 약간의 조정으로,이 방법은 다른 유형의 종양 또는 정상 조직에 적용 할 수 있어야합니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s medium (1 g/L D-glucose) (LG-DMEM)</td>
			<td>Gibco</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 Bullet Kit&#160;</td>
			<td>Lonza</td>
			<td>CC4176</td>
			<td>Not all components used</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (Hyclone)</td>
			<td>Thermo Scientific</td>
			<td>SH30071.03</td>
			<td>Heat inactivated at 56&#176;C for 30 min</td>
		</tr>
		<tr>
			<td>Nu-Serum IV</td>
			<td>Corning</td>
			<td>CB-51004</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBBS)</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS buffer&#160;</td>
			<td>-</td>
			<td>-</td>
			<td>0.5 % BSA and 2 mM EDTA in PBS, filtered through a 0.22 &#956;m filter</td>
		</tr>
		<tr>
			<td>75% v/v ethanol for disinfection</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PE microbeads&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA) fraction V, 7.5%</td>
			<td>Gibco</td>
			<td>15260-37</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell freezing media (Bambanker)</td>
			<td>Wako Chemicals</td>
			<td>302-14681</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type II&#160;&#160;</td>
			<td>Worthington Biochemical</td>
			<td>LS004176</td>
			<td>Make stock concentration 2 mg/ml in HBSS</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I (DNase)</td>
			<td>Worthington Biochemical</td>
			<td>LS002004</td>
			<td>Make stock concentration 1 mg/mL in PBS</td>
		</tr>
		<tr>
			<td>Dil-Ac-LDL</td>
			<td>Biomedical Technologies</td>
			<td>BT-902</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 0.5M, pH 8.0</td>
			<td>Cellgro</td>
			<td>46-034-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymatic cell detachment solution (Accutase)</td>
			<td>Sigma-Aldrich</td>
			<td>A6964-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin, 2 % in water, tissue culture grade</td>
			<td>Sigma-Aldrich</td>
			<td>G1393-100ML</td>
			<td>Dilute in PBS to make 0.5 % gelatin solution</td>
		</tr>
		<tr>
			<td>Mouse FcR Blocking Reagent&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral protease (Dispase)</td>
			<td>Worthington Biochemical</td>
			<td>LS02104</td>
			<td>Make stock concentration 2.5 U/mL in HBSS</td>
		</tr>
		<tr>
			<td>PE-rat anti-mouse CD31 antibody</td>
			<td>BD Pharmingen</td>
			<td>553373</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis buffer (BD Pharm Lyse)</td>
			<td>BD Pharmingen</td>
			<td>555899</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4 %&#160;</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mm tissue culture dishes</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes (sterile)</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes (sterile)&#160;</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td>Corning</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-dissociator tubes (gentleMACS) C tubes)&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Separator&#160; (MidiMACS)</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 100 &#956;m&#160;</td>
			<td>Corning</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Cloning rings (assorted sizes)</td>
			<td>Bel-Art Products</td>
			<td>378470000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotubes</td>
			<td>Thermo Scientific</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting board</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize or disinfect with 75% v/v ethanol before use&#160;</td>
		</tr>
		<tr>
			<td>Dissecting forceps and scissors</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize before use&#160;</td>
		</tr>
		<tr>
			<td>Dissecting pins 2&#34;</td>
			<td>-</td>
			<td>-</td>
			<td>Sterilize before use&#160;</td>
		</tr>
		<tr>
			<td>FACS tubes with 35 &#956;m filter cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter cup (Stericup, 0.22 &#956;m)</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-tip marker</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Multistand</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue adhesive (Vetbond)</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>Or a centrifuge with similar capacity for 15 mL and 50 mL conical tube centrifugation</td>
		</tr>
		<tr>
			<td>Tissue culture hood</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue dissociator (gentleMACS)</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-235</td>
			<td>Preset program &#34;m_impTumor_01&#34; used for tissue dissociation&#160;</td>
		</tr>
		<tr>
			<td>Liquid nitrogen freezer</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate or rotary shaker</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast light microscope</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture expansion of tumor-specific endothelial cells

갓 절연 종양 특이 내피 세포 (TEC)는 종양 혈관 신생의 분자 메커니즘을 탐구 및 암에 대한 새로운 혈관 신생 억제제를 개발하기위한 시험 관내 모델로서 기능 할 수있다. 그러나 쥐의 내피 세포 (EC)의 체외 확장에 장기적으로 인해 비 EC와 표현형 문화 드리프트 (내피 - 투 - 중간 엽 전환) 및 오염에 도전한다. 이것은 쉽게 문화의 공동 정제 섬유 아 세포 또는 종양 세포에 의해 outcompeted된다 TEC 특히 사실이다. 여기서, 콜로니를 선택하여 시험 관내 확장에 결합 면역 자기 농축 활용 고음질 분리 방법이 설명된다. 이 접근법은 기질 또는 종양 세포를 오염 완전히 무료 EC 순수한 분획을 생성한다. 또한 그 혈통 추적 Cdh5의 CRE 주심 : 프로토콜 사용의 ZsGreen 리터 / S / L 기자 마우스는, 여기에 기술, 세포를 확인하는 유용한 도구입니다이 마우스에서 분리 된 EC의 식민지로 순도는 문화의 내구성과 화려한 ZsGreen이 형광을 보여줍니다.

EC는 대부분의 성인 조직 (11)의 전체 세포 인구의 작은 부분을 나타냅니다. 그것은 완전히 세포 외 기질 (ECM) 및 결합 조직에서 EC의 최대 방출을 보장 단일 세포 현탁액으로 수확 된 조직을 소화하는 것이 중요하다. 우리의 경험에 의하면, CD31 매개 면역 자기 선택은 풍부한하지만 순수한 EC의 분수를 제공하며, 따라서, 또 다른 중요한 단계는 클로닝 링 (도 1)를 사용하여 EC 콜로...

Watch this Scientific Journal Video about Isolation and Culture Expansion of Tumor-specific Endothelial Cells at JoVE.com

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

종양 특이 내피 세포의 분리 및 문화 확장

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

isolation-and-culture-expansion-of-tumor-specific-endothelial-cells

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JoVE Journal

Medicine

11.1K 조회수.  University of North Carolina at Chapel Hill. We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models. 

비디오: Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

인간 치과 펄프의 줄기 세포의 분리, 특성화 및 비교 차별화 두 가지 방법을 사용하여 영구 치아에서 파생

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

연구

의학

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

환자 유래 세포 배양 및 CD133의 절연<sup&gt; +</sup흑종에서&gt; Putative 암 줄기 세포

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

인간의 태아 췌장 세포 클러스터의 분리, 문화, 및 이미징

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

분리, 냉동 보존 및 문화 임상 응용 프로그램에 대한 인간 양막 상피 세포

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

다중 매개 변수 형광 활성화 된 셀 정렬하여 인간의 림프 내피 세포의 분리

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

개 동맥과 정맥에서 차 내피 세포의 분리 및 문화

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

심장 수술 후 혈청 샘플의 영향을 건강한 자원 봉사자와 그들의 철새 잠재력에서 내피 전구 세포의 분리

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

준비 및<em&gt; 체외</em&gt; 자화 특성의 내피 세포를 미르 변성

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

3D 세포 배양 및 비 anticoagulated 전 혈을 사용 하 여 내 피 세포의 항 응 혈 약 그리고 항 염증 제 속성에 대 한 평가

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

근육의 불균형: 테스트 및 기능 편심 햄 스트링 운동 인구 강도 훈련

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...