Name: isolation and culture expansion of tumor-specific endothelial cells
Uploaded: 2015-10-14T15:00:00.000+00:00
Duration: 10 min 15 s
Description: Watch this Scientific Journal Video about Isolation and Culture Expansion of Tumor-specific Endothelial Cells at JoVE.com

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>

<ol>
	<li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-angiogenesis:+new+concept+for+therapy+of+solid+tumors.">Anti-angiogenesis: new concept for therapy of solid tumors.</a> Ann. Surg. 175 (3), 409-416 (1972).</li><li>Butler, J. M., Kobayashi, H., Rafii, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instructive+role+of+the+vascular+niche+in+promoting+tumour+growth+and+tissue+repair+by+angiocrine+factors.">Instructive role of the vascular niche in promoting tumour growth and tissue repair by angiocrine factors.</a> Nat. Rev. Cancer. 10 (2), 138-146 (2010).</li><li>Franses, J. W., Baker, A. B., Chitalia, V. C., Edelman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+endothelial+cells+directly+influence+cancer+progression.">Stromal endothelial cells directly influence cancer progression.</a> Sci. Transl. Med. 3 (66), 66ra5 (2011).</li><li>Calabrese, C., 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=A+perivascular+niche+for+brain+tumor+stem+cells.">A perivascular niche for brain tumor stem cells.</a> Cancer Cell. 11 (1), 69-82 (2007).</li><li>Beck, B., 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=A+vascular+niche+and+a+VEGF-Nrp1+loop+regulate+the+initiation+and+stemness+of+skin+tumours.">A vascular niche and a VEGF-Nrp1 loop regulate the initiation and stemness of skin tumours.</a> Nature. 478 (7369), 399-403 (2011).</li><li>Aird, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+heterogeneity.">Endothelial cell heterogeneity.</a> Cold Spring Harb. Perspect. Med. 2 (1), a006429 (2012).</li><li>Dudley, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+endothelial+cells.">Tumor endothelial cells.</a> Cold Spring Harb. Perspect. Med. 2 (3), a006536-a006536 (2012).</li><li>Aird, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+heterogeneity+of+tumor+endothelium.">Molecular heterogeneity of tumor endothelium.</a> Cell Tissue Res. 335 (1), 271-281 (2009).</li><li>Voyta, J. C., Via, D. P., Butterfield, C. E., Zetter, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+isolation+of+endothelial+cells+based+on+their+increased+uptake+of+acetylated-low+density+lipoprotein.">Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein.</a> J. Cell Biol. 99 (6), 2034-2040 (1984).</li><li>Zovein, A. C., 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=Fate+tracing+reveals+the+endothelial+origin+of+hematopoietic+stem+cells.">Fate tracing reveals the endothelial origin of hematopoietic stem cells.</a> Cell Stem Cell. 3 (6), 625-636 (2008).</li><li>Beijnum, J. R., Rousch, M., Castermans, K., van der Linden, E., Griffioen, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+fresh+tissues.">Isolation of endothelial cells from fresh tissues.</a> Nat. Protoc. 3 (6), 1085-1091 (2008).</li><li>Xiao, L., Harrell, J. C., Perou, C. M., Dudley, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+stable+molecular+signature+in+mammary+tumor+endothelial+cells+that+persists+in+vitro.">Identification of a stable molecular signature in mammary tumor endothelial cells that persists in vitro.</a> Angiogenesis. 17 (3), 511-518 (2014).</li><li>Beijnum, J. R., 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=Gene+expression+of+tumor+angiogenesis+dissected:+specific+targeting+of+colon+cancer+angiogenic+vasculature.">Gene expression of tumor angiogenesis dissected: specific targeting of colon cancer angiogenic vasculature.</a> Blood. 108 (7), 2339-2348 (2006).</li><li>McDonald, D. M., Choyke, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+angiogenesis:+from+microscope+to+clinic.">Imaging of angiogenesis: from microscope to clinic.</a> Nat. Med. 9 (6), 713-725 (2003).</li><li>Baluk, P., Hashizume, H., McDonald, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+abnormalities+of+blood+vessels+as+targets+in+cancer.">Cellular abnormalities of blood vessels as targets in cancer.</a> Curr. Opin. Genetics Dev. 15 (1), 102-111 (2005).</li><li>Ghosh, K., 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=Tumor-derived+endothelial+cells+exhibit+aberrant+Rho-mediated+mechanosensing+and+abnormal+angiogenesis+in+vitro.">Tumor-derived endothelial cells exhibit aberrant Rho-mediated mechanosensing and abnormal angiogenesis in vitro.</a> Proc. Natl. Acad. Sci. 105 (32), 11305-11310 (2008).</li><li>Amin, D. N., Hida, K., Bielenberg, D. R., Klagsbrun, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+endothelial+cells+express+epidermal+growth+factor+receptor+(EGFR)+but+not+ErbB3+and+are+responsive+to+EGF+and+to+EGFR+kinase+inhibitors.">Tumor endothelial cells express epidermal growth factor receptor (EGFR) but not ErbB3 and are responsive to EGF and to EGFR kinase inhibitors.</a> Cancer Res. 66 (4), 2173-2180 (2006).</li><li>Hida, K., 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=Tumor-associated+endothelial+cells+with+cytogenetic+abnormalities.">Tumor-associated endothelial cells with cytogenetic abnormalities.</a> Cancer Res. 64 (22), 8249-8255 (2004).</li><li>Dudley, A. C., 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=Calcification+of+multipotent+prostate+tumor+endothelium.">Calcification of multipotent prostate tumor endothelium.</a> Cancer Cell. 14 (3), 201-211 (2008).</li><li>Dunleavey, J. M., 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=Vascular+channels+formed+by+subpopulations+of+PECAM1(+)+melanoma+cells.">Vascular channels formed by subpopulations of PECAM1(+) melanoma cells.</a> Nat. Comm. 5, 5200 (2014).</li><li>St Croix, B., 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=Genes+expressed+in+human+tumor+endothelium.">Genes expressed in human tumor endothelium.</a> Science. 289 (5482), 1197-1202 (2000).</li><li>Bhati, R., 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=Molecular+characterization+of+human+breast+tumor+vascular+cells.">Molecular characterization of human breast tumor vascular cells.</a> Am. J. Pathol. 172 (5), 1381-1390 (2008).</li><li>Johnson, C. S., Chung, I., Trump, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+silencing+of+CYP24+in+the+tumor+microenvironment.">Epigenetic silencing of CYP24 in the tumor microenvironment.</a> J. Steroid Biochem. Mol. Biol. 121 (1-2), 338-342 (2010).</li><li>Gimbrone, M. A., Cotran, R. S., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+vascular+endothelial+cells+in+culture.+Growth+and+DNA+synthesis.">Human vascular endothelial cells in culture. Growth and DNA synthesis.</a> J. Cell Biol. 60 (3), 673-684 (1974).</li><li>Burridge, K. A., Friedman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environment+and+vascular+bed+origin+influence+differences+in+endothelial+transcriptional+profiles+of+coronary+and+iliac+arteries.">Environment and vascular bed origin influence differences in endothelial transcriptional profiles of coronary and iliac arteries.</a> Am. J. Physiol. Heart Circ. Physiol. 299 (3), H837-H846 (2010).</li><li>Zhang, J., Burridge, K. A., Friedman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+differences+between+endothelial+transcriptional+profiles+of+coronary+and+iliac+arteries+revealed+by+microarray+analysis.">In vivo differences between endothelial transcriptional profiles of coronary and iliac arteries revealed by microarray analysis.</a> Am. J. Physiol. Heart Circ. Physiol. 295 (4), H1556-H1561 (2008).</li><li>Paruchuri, 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=Human+pulmonary+valve+progenitor+cells+exhibit+endothelial/mesenchymal+plasticity+in+response+to+vascular+endothelial+growth+factor-A+and+transforming+growth+factor-beta2.">Human pulmonary valve progenitor cells exhibit endothelial/mesenchymal plasticity in response to vascular endothelial growth factor-A and transforming growth factor-beta2.</a> Circ. Res. 99 (8), 861-869 (2006).</li><li>Wylie-Sears, J., Aikawa, E., Levine, R. A., Yang, J. -. H., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitral+valve+endothelial+cells+with+osteogenic+differentiation+potential.">Mitral valve endothelial cells with osteogenic differentiation potential.</a> Arterioscler. Thromb. Vasc. Biol. 31 (3), 598-607 (2011).</li><li>Ginsberg, M., 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=Efficient+direct+reprogramming+of+mature+amniotic+cells+into+endothelial+cells+by+ETS+factors+and+TGF&#946;+suppression.">Efficient direct reprogramming of mature amniotic cells into endothelial cells by ETS factors and TGF&#946; suppression.</a> Cell. 151 (3), 559-575 (2012).</li><li>Sapino, A., 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=Expression+of+CD31+by+cells+of+extensive+ductal+in+situ+and+invasive+carcinomas+of+the+breast.">Expression of CD31 by cells of extensive ductal in situ and invasive carcinomas of the breast.</a> J. Path. 194 (2), 254-261 (2001).</li><li>Maddaluno, L., 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=EndMT+contributes+to+the+onset+and+progression+of+cerebral+cavernous+malformations.">EndMT contributes to the onset and progression of cerebral cavernous malformations.</a> Nature. 498 (7455), 492-496 (2013).</li><li>Cooley, B. C., 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=TGF-&#946;+signaling+mediates+endothelial-to-mesenchymal+transition+(EndMT)+during+vein+graft+remodeling.">TGF-&#946; signaling mediates endothelial-to-mesenchymal transition (EndMT) during vein graft remodeling.</a> Sci. Transl. Med. 6 (227), 227ra34-227ra34 (2014).</li><li>Garcia, J., 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=Tie1+deficiency+induces+endothelial-mesenchymal+transition.">Tie1 deficiency induces endothelial-mesenchymal transition.</a> EMBO Rep. 13 (5), 431-439 (2012).</li><li>Xiao, L., 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=Tumor+endothelial+cells+with+distinct+patterns+of+TGF&#946;-driven+endothelial-to-mesenchymal+transition.">Tumor endothelial cells with distinct patterns of TGF&#946;-driven endothelial-to-mesenchymal transition.</a> Cancer Res. 75 (7), 1244-1254 (2015).</li><li>Kusumbe, A. P., Ramasamy, S. K., Adams, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+angiogenesis+and+osteogenesis+by+a+specific+vessel+subtype+in+bone.">Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone.</a> Nature. 507 (7492), 323-328 (2014).</li><li>Wang, L., 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=Identification+of+a+clonally+expanding+haematopoietic+compartment+in+bone+marrow.">Identification of a clonally expanding haematopoietic compartment in bone marrow.</a> EMBO J. 32 (2), 219-230 (2012).</li><li>Sawamiphak, 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=Ephrin-B2+regulates+VEGFR2+function+in+developmental+and+tumour+angiogenesis.">Ephrin-B2 regulates VEGFR2 function in developmental and tumour angiogenesis.</a> Nature. 465 (7297), 487-491 (2010).</li><li>Chi, J. -. T., 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=Endothelial+cell+diversity+revealed+by+global+expression+profiling.">Endothelial cell diversity revealed by global expression profiling.</a> Proc. Natl. Acad. Sci. 100 (19), 10623-10628 (2003).</li><li>Nolan, D. J., 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=Molecular+signatures+of+tissue-specific+microvascular+endothelial+cell+heterogeneity+in+organ+maintenance+and+regeneration.">Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.</a> Dev. Cell. 26 (2), 204-219 (2013).</li><li>Ingram, D. A., 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=Vessel+wall-derived+endothelial+cells+rapidly+proliferate+because+they+contain+a+complete+hierarchy+of+endothelial+progenitor+cells.">Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells.</a> Blood. 105 (7), 2783-2786 (2005).</li></ol>

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><tbody><tr><td>Antibiotic-Antimycotic&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A5955</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;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&amp;nbsp;</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 &amp;deg;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&#039;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&amp;nbsp;</td><td></td><td></td><td>0.5 % BSA and 2 mM EDTA in PBS, filtered through a 0.22 &amp;mu;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&amp;nbsp;</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&amp;nbsp;&amp;nbsp;</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&amp;nbsp;</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 %&amp;nbsp;</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)&amp;nbsp;</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)&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-093-237</td><td></td></tr><tr><td>Cell Separator&amp;nbsp; (MidiMACS)</td><td>Miltenyi Biotech</td><td>130-042-302</td><td></td></tr><tr><td>Cell strainer 100 &amp;mu;m&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>Dissecting forceps and scissors</td><td></td><td></td><td>Sterilize before use&amp;nbsp;</td></tr><tr><td>Dissecting pins 2&quot;</td><td></td><td></td><td>Sterilize before use&amp;nbsp;</td></tr><tr><td>FACS tubes with 35 &amp;mu;m filter cap</td><td>Corning</td><td>352235</td><td></td></tr><tr><td>Filter cup (Stericup, 0.22 &amp;mu;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 &quot;m_impTumor_01&quot; used for tissue dissociation&amp;nbsp;</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

Research

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

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Bryostatin의 1/Ionomycin에 의해 종양 반응 T 세포의 생체내 전 확장 및 일반 감마 체인 크린 시토킨 제제

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

연구

면역학 및 감염병학

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

정상적인 대 장 암과 대 장 암-향상 된 프로토콜에서 인간의 내 피 세포의 고립

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

암 연구학

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

인간 탯 줄 혈액에서 내 피 조상 세포의 고립

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

발생학

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

수술 조직에서 기본 인간 대장 종양 세포의 분리 및 견인 세포 계측법 부드러운 탄성 기판에 직접 배양

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

생체공학

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

분리 및 성인 인간 내피 콜로니 형성 전구 세포의 대규모 확장

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

생물학

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

배아 전뇌에서 내피 세포의 분리 및 문화

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

신경과학

A Patient-Derived Xenograft Model for Venous Malformation

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional veins. The purpose of this article is to carefully describe the establishment of a murine xenograft model that mimics human VM and is able to reflect patient heterogeneity. Hyper-activating non-inherited (somatic) TEK (TIE2) and PIK3CA mutations in endothelial cells (EC) have been identified as the main drivers of pathological vessel enlargement in VM. The following protocol describes the isolation, purification and expansion of patient-derived EC expressing mutant TIE2 and/or PIK3CA. These EC are injected subcutaneously into the back of immunodeficient athymic mice to generate ectatic vascular channels. Lesions generated with TIE2 or PIK3CA-mutant EC are visibly vascularized within 7&#8210;9 days of injection and recapitulate histopathological features of VM patient tissue. This VM xenograft model provides a reliable platform to investigate the cellular and molecular mechanisms driving VM formation and expansion. In addition, this model will be instrumental for translational studies testing the efficacy of novel drug candidates in preventing the abnormal vessel enlargement seen in human VM.

We present a detailed protocol to generate a murine xenograft model of venous malformation. This model is based on the subcutaneous injection of patient-derived endothelial cells containing hyper-activating TIE2 and/or PIK3CA gene mutations. Xenograft lesions closely recapitulate the histopathological features of VM patient tissue.

정맥 기형에 대한 환자 유래 제노이식 모델

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional ...

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 CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

Clonal 확장 CD133 + 간 줄기 세포의 분리

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.