이 작품은 힐러리 공예, 선생님 더글러스 마이어스 특별한 언급과 제국 BRC, 건강 연구를위한 국립 연구소 및 조치에 대하여 암에 의해 지원됩니다.

절차는 다음과 윤리적 임페리얼 칼리지, 런던에 의해 승인되었습니다. 인간 유방암 세포주에서 차 Mammospheres의 1 세대 참고 : 멸균 문화 후드에서 다음 단계를 수행하십시오. <ol><li> Mammosphere 미디어 2 mM의 L- 글루타민이 보충 된 DMEM / F12, 100 U / ㎖ 페니실린, 100 U / ml의 스트렙토 마이신을 함유하는 준비한다. 10 NG / ml의 재조합 인간 염기성 섬유 아세포 성장 인자 (bFGF; R &amp; D 시스템) 및 1X B27 보충제 20 NG / ml의 재조합 인간 상피 성장 인자 (시그마 EGF)을 첨가하여 사용 직전에 완료 매체를 준비한다. </li><li> (또는 원하는 유방암 세포주 70-80 % 컨 플루 언스 (confluence)에) 부착 MCF-7 또는 MDA-MB-231 세포를 함유하는 플라스크 대기음 매체는 1X PBS에 두 번 세척하고, 세포를 Trypsinize. </li><li> 실온에서 5 분 동안 원심 분리기 (200) XG 세포. mammosphere 미디어의 1-5 ml의 디 캔트 뜨는에 resuspend 세포. 피펫40 μm의 셀 스트레이너 캡 필터 상하 필요한 사용하는 경우, 단일 세포 현탁액을 얻었다. 단일 세포 현탁액 (그렇지 않은 경우, 3 회에 세포 현탁액을 주사기 25 G 바늘을 사용하여) 형성하고 있도록 혈구를 사용한다. </li><li> FACS를 통해 필요한 분리 된 CD44 + CD24- 세포의 일부는 항 CD24 피코 에리 트린 (PE) 및 항 CD44-형광 이소 티오 시아 네이트 (FITC) 단일 클론 항체 (9)를 사용하는 경우. 또한, 자기 활성화 된 셀 정렬 안티 CD44 및 제조업체의 지침에 따라 항 CD24 - 바이오틴 결합 된 마이크로 비즈 (MACS) 시스템을 사용하는 인구를 분리. LD 열을 사용하여 LS 열을 사용하여 긍정적 인 선택, 음의 선택을 수행하고 17 유동 세포 계측법에 의해 모든 격리 세포의 표현형을 확인합니다. </li><li> 트리 판 블루를 사용하여 1 mL 당 생존 세포의 수를 계산합니다. 주 : 세포 생존율에 hemocy 그리드 내의 셀의 총 개수로 나누어 생존 세포의 수로서 계산된다tometer. 트리 판 블루를 차지 세포가 아닌 실행 가능한 것으로 간주됩니다. 다음 절차를 정확하게 생존 세포의 비율을 결정하는데 사용된다. <ol><li> PBS에서 트리 판 블루의 0.4 % 용액을 준비합니다. 세포의 1 ml의 트리 판 블루 원액 0.1 ML을 추가합니다. 혈구를 넣고 낮은 배율 현미경으로 즉시 검사합니다. 총 셀의 개수와 블루 염색 세포의 수를 계산. 가능한 세포 = [1.00 - (파란색 셀의 수 ÷ 총 세포 수)] × 100. </li><li> 배양 1 ㎖ 당 생존 세포의 수를 계산하는 아래의 식을 사용한다. 희석 배수를 보정해야합니다. 104 × 1.1 = 세포 / ㎖ 문화 × 가능한 세포의 수 </li></ol></li><li> 6- 웰 초저 부착 플레이트의 각 웰에 전체 mammosphere 미디어 2 ㎖로 세포의 사전 결정된 양을 재현 탁. 파종 밀도는 일반적으로 500-4,000 세포 / cm 아니라 당이 세포입니다. 선택적으로 사용 싸다w 부착 24 웰 플레이트는 매체의 0.5 ml의 동일한 밀도로 세포를 파종한다. 참고 : 우리는 관심의 각 세포주에 대한 파종 밀도와 문화의 시간을 최적화하는 것이 좋습니다. </li><li> 특히 제 오일의 성장 단계에서, 플레이트를 방해하지 않고 (세포주 및 mammospheres의 크기에 따라) 5-10일 37 ° C, 5 % CO 2에서 초저 부착 6 웰 플레이트를 인큐베이션 . </li></ol> 인간의 유방암 임상 샘플에서 차 Mammospheres의 2 세대 주 : 인간 유방암 조직 유방 종양의 제거를위한 수술을받은 환자로부터 얻을 수있다. 100 U / ml의 페니실린 100 U / ml의 스트렙토 마이신을 포함하는 배지에서 50 ml의 멸균 튜브에 최대 24 시간 동안 얼음에 저장 조직. 멸균 문화 후드에서 다음 단계를 수행합니다. <ol><li> 미디어의 작은 부피와 100mm 조직 배양 접시에 시료를 전송합니다. Remov멸균 가위, scapel 및 핀셋을 사용하여 전자 지방 조직. </li><li> DMEM / F12의 2-3 ML을 추가하고 더 큰 조각이 남아 있지 않을 때까지 살균 scapel 또는 면도날 1mm 3 조각으로 샘플을 말하다. </li><li> 목록 조직편 재현 탁 단백질 분해 효소 (3,000 U / ml의 콜라겐 및 1000 U / ㎖의 히알루로니다 아제)를 함유하는 10 ㎖의 DMEM을 프리 - 가온하고 모든 조직 파편이 소화 될 때까지 회전 진탕하에 37 ℃에서 배양한다. 일반적으로 완전한 소화은 1-3 시간 정도 걸립니다. 종양의 병리학 자에 따라 소화 시간을 조정합니다. (예를 들어 유방 선암 더 취약 짧은 소화 시간이 필요 점액 성 암종에 비해 소화가 일반적으로 어렵다). 혈구에서 20 μl의 서스펜션을 관찰하여 소화 모든 ½ 시간의 정도를 평가합니다. </li><li> 단편 후, 5 분 동안 침강 실 TEM에서 10 분 동안 200 XG에 15 ml의 원추형 폴리 프로필렌 원심 분리 튜브에 상청을 전송하도록 허용아네트. mammosphere 매체 5 ㎖ - 조심스럽게 1에 재현 탁하고 세포 상등액을 가만히 따르다. </li><li> 취득 및 단일 세포 현탁액을 도금하는 세포주를 위해 위의 설명 된 단계 1.1 ~ 1.4을 따릅니다. 필요한 경우 G (25)를 통해 세포가 세포 손상을 제한하기 위해 2 배의 최대 시린지 패스. </li></ol> 3. Mammosphere 효율 (%) 계산 성형 <ol><li> 배양 기간 후, 40 배 배율 현미경 mammospheres (이상 40 μm의) 직경을 계산합니다. 디지털 화상 (5) 임의의 광학 현미경에 디지털 카메라를 사용하여 필드 및 수집 소프트웨어를 이용 mammospheres 크기를 결정한다. 어떤 이미지 분석 소프트웨어를 사용합니다. </li><li> 다음 식을 이용 Mammosphere 형성 효율 (MFE의 %)을 계산한다 MFE (%)는 = (웰당 mammospheres의 #은) / (웰 당 세포의 접종은 #) × 100 </li></ol> 자기 갱신의 평가를위한 Mammospheres 4. 직렬과 Passaging <ol><li&gt; 피펫 15ml를 튜브에 각 웰로부터 mammospheres 함유 매체. PBS로 우물을 세척하고 수집 된 미디어에 추가 할 수 있습니다. 실온에서 10 분 동안 원심 분리기 (115) XG. </li><li> 뜨는을 취소하고 미리 예열 0.5 % 트립신 / 0.2 % EDTA의 500 μL에 펠렛을 재현 탁. 2 ~ 3 분 동안 품어. </li><li> 트립신을 중화 FCS 500 μl를 추가합니다. 5 분 동안 500 XG에 원심 분리기. 구체들을 분해 아래로 mammospheres 미디어, 피펫 최대 100 ㎕에 뜨는에 resuspend 펠렛을 취소하고. </li><li> 혈구와 세포를 세어 세포는 단일 세포 현탁액에있는 경우 결정합니다. 25 G를 통과하지 않으면 단일 세포를 얻기 위해 3 회까지 주사기. 기본 생성에 사용되는 동일한 농도에서 새로운 초저 부착 6 웰 플레이트에 세포를 시드. </li><li> 5-10일 후, 구&gt; 40 μm의 수와 이전에보고 된 공식을 사용하여 구체 형성 효율을 계산한다. </li></ol>

<ol>
	<li>Iliopoulos, D., Hirsch, H. a., Struhl, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+epigenetic+switch+involving+NF-kappaB,+Lin28,+Let-7+MicroRNA,+and+IL6.">An epigenetic switch involving NF-kappaB, Lin28, Let-7 MicroRNA, and IL6.</a> Cell. 139 (4), 693-706 (2009).</li><li>Iliopoulos, D., Hirsch, H. a., Wang, G., Struhl, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+formation+of+breast+cancer+stem+cells+and+their+dynamic+equilibrium+with+non-stem+cancer+cells+via+IL6+secretion.">Inducible formation of breast cancer stem cells and their dynamic equilibrium with non-stem cancer cells via IL6 secretion.</a> Proc Natl Acad Sci U S A. 108 (4), 1397-1402 (2011).</li><li>Visvader, J. E., Lindeman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells:+current+status+and+evolving+complexities.">Cancer stem cells: current status and evolving complexities.</a> Cell stem cell. 10 (6), 717-728 (2012).</li><li>Rosen, J. M., Jordan, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+increasing+complexity+of+the+cancer+stem+cell+paradigm.">The increasing complexity of the cancer stem cell paradigm.</a> Science. 324 (5935), 1670-1673 (2009).</li><li>Rokavec, M., Wu, W., Luo, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL6-mediated+suppression+of+miR-200c+directs+constitutive+activation+of+inflammatory+signaling+circuit+driving+transformation+and+tumorigenesis.">IL6-mediated suppression of miR-200c directs constitutive activation of inflammatory signaling circuit driving transformation and tumorigenesis.</a> Mol Cell. 45 (6), 777-789 (2012).</li><li>Chen, J., Li, Y., 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+restricted+cell+population+propagates+glioblastoma+growth+after+chemotherapy.">A restricted cell population propagates glioblastoma growth after chemotherapy.</a> Nature. 488 (7412), 522-526 (2012).</li><li>Driessens, G., Beck, B., Caauwe, A., Simons, B. D., Blanpain, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+mode+of+tumour+growth+by+clonal+analysis.">Defining the mode of tumour growth by clonal analysis.</a> Nature. 488 (7412), 527-530 (2012).</li><li>Schepers, A. G., Snippert, H. 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=Lineage+tracing+reveals+Lgr5++stem+cell+activity+in+mouse+intestinal+adenomas.">Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal adenomas.</a> Science. 337 (6095), 730-735 (2012).</li><li>Sheridan, C., Kishimoto, H., 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=CD44+/CD24-+breast+cancer+cells+exhibit+enhanced+invasive+properties:+an+early+step+necessary+for+metastasis.">CD44+/CD24- breast cancer cells exhibit enhanced invasive properties: an early step necessary for metastasis.</a> Breast cancer research: BCR. 8 (5), R59 (2006).</li><li>Ginestier, C., Hur, M. H., 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=ALDH1+is+a+marker+of+normal+and+malignant+human+mammary+stem+cells+and+a+predictor+of+poor+clinical+outcome.">ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome.</a> Cell stem cell. 1 (5), 555-567 (2007).</li><li>Liu, S., Cong, Y., 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=Breast+Cancer+Stem+Cells+Transition+between+Epithelial+and+Mesenchymal+States+Reflective+of+their+Normal+Counterparts.">Breast Cancer Stem Cells Transition between Epithelial and Mesenchymal States Reflective of their Normal Counterparts.</a> Stem cell reports. 2 (1), 78-91 (2014).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255 (5052), 1707-1710 (1992).</li><li>Pastrana, E., Silva-Vargas, V., Doetsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eyes+wide+open:+a+critical+review+of+sphere-formation+as+an+assay+for+stem+cells.">Eyes wide open: a critical review of sphere-formation as an assay for stem cells.</a> Cell stem cell. 8 (5), 486-498 (2011).</li><li>Dontu, G., Abdallah, W. 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=In+vitro+propagation+and+transcriptional+profiling+of+human+mammary+stem+/+progenitor+cells.">In vitro propagation and transcriptional profiling of human mammary stem / progenitor cells.</a> Genes Dev. , 1253-1270 (2003).</li><li>Ponti, D., Costa, 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=Isolation+and+in+vitro+propagation+of+tumorigenic+breast+cancer+cells+with+stem/progenitor+cell+properties.">Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties.</a> Cancer Res. 65 (13), 5506-5011 (2005).</li><li>Grimshaw, M. J., Cooper, 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=Mammosphere+culture+of+metastatic+breast+cancer+cells+enriches+for+tumorigenic+breast+cancer+cells.">Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells.</a> Breast Cancer Res. 10 (3), R52 (2008).</li><li>Pham, P. V., Phan, N. L. 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=Differentiation+of+breast+cancer+stem+cells+by+knockdown+of+CD44:+promising+differentiation+therapy.">Differentiation of breast cancer stem cells by knockdown of CD44: promising differentiation therapy.</a> J Transl Med. 9 (1), 209 (2011).</li><li>Manuel Iglesias, J., Beloqui, I., 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=Mammosphere+formation+in+breast+carcinoma+cell+lines+depends+upon+expression+of+E-cadherin.">Mammosphere formation in breast carcinoma cell lines depends upon expression of E-cadherin.</a> PloS one. 8 (10), e77281 (2013).</li><li>Coles-Takabe, B. L. K., Brain, I., 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=Don&#8217;t+look:+growing+clonal+versus+nonclonal+neural+stem+cell+colonies.">Don&#8217;t look: growing clonal versus nonclonal neural stem cell colonies.</a> Stem Cells. 26 (11), 2938-2944 (2008).</li><li>Stingl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+analysis+of+mammary+gland+stem+cells.">Detection and analysis of mammary gland stem cells.</a> J Pathol. 217 (2), 229-241 (2009).</li><li>Kreso, A., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+cancer+stem+cell+model.">Evolution of the cancer stem cell model.</a> Cell stem cell. 14 (3), 275-291 (2014).</li><li>Al-Hajj, M., Clarke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+and+solid+tumor+stem+cells.">Self-renewal and solid tumor stem cells.</a> Oncogene. 23 (43), 7274-7282 (2004).</li><li>Yu, F., Yao, H., 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=let-7+regulates+self+renewal+and+tumorigenicity+of+breast+cancer+cells.">let-7 regulates self renewal and tumorigenicity of breast cancer cells.</a> Cell. 131 (6), 1109-1123 (2007).</li></ol>

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this article.

기본 및 보조 mammospheres의 성공적인 평가는 세포가 최소한의 구 집합으로, 단일 클론에서 양식을 mammospheres 충분히 낮은 밀도에서 도금되는에 의존합니다. 그러나 너무 낮은 밀도가 너무 적었 mammospheres 통계적 처리의 효과를 구별하기 위해 형성 할 수있다. 파종 밀도가 그들의 영역 형성의 효율에 상당히 다를 수 있기 때문에 사용 된 각각의 세포주에 대해 최적화되어야한다 (낮은 E-cadherin의 표현?...

줄기 같은 암 줄기 세포가 이끄는 종양 세포 계통의 존재는 크게 종양 이질성에 대한 우리의 이해에 추가했다. 종양의 일부 표현형 다양성 유전자 구별 클론의 클론 생장에서 발생 않지만, 실질적인 성분은 후생 유전 학적 차이에 기인하는 것으로 나타난다 : 암 세포가 특정 유전자의 활성화 또는 억제를 통해 줄기, 전구 및 분화 상태 사이 (가역적 때때로) 전환 할 수 식 프로그램 1-3. 이것은 현재 조절 성 요소를 제공하는 암, 간질 또는 면역 인접 셀로부터 측 분비와 함께 그 결과의 분비 신호와 세포에서 발현되는 유전자 발현 프로그램을 반영 셀 고유 또는 외인성 인자를 반영하고있다 같은 정도로서 microenviromental 조건 저산소증 2,4,5. 혁신적인 혈통 추적 방법이지만이들 생체 내 틈새 (6)에 추정되는 암 줄기 세포를 연구 할 수있는 능력 전진 - 8 구 - 형성 분석법 적어도 사용 분석 조건 하에서, 줄기 세포처럼 행동하도록 유방암 세포 잠재력을 추정 인기 편리한 접근 남아있다. 그것은 종종 더 mesenchymal- 상피 대응하도록 제안되어 막 마커 CD44 및 CD24 9 및 효소 ALDH (알데히드 탈수소 효소)의 활동 수준 (10), 마커의 발현에 의해, 정화 암 줄기 세포 대 향적 방법과 함께 사용 -like 암 줄기 세포 각각 11. 구 형성 방법이 제 상피 성장 인자 (EGF) (12)의 첨가 비 점착성, 무 혈청 조건에서 단일 클론으로부터 추정되는 줄기 세포의 성장을 가능 neurosphere 분석법 개발되었다 나중에 유용 정상 및 암에 적용되는 유방 조직. 뼈 선의의 줄기 세포는 생체 내에서 활성화를 선호 할 요인의 정확한 조합을 발생하지 않습니다, G0 단계에서 내립니다. mammosphere 분석 대신 유사 분열 부문 태세 이미 13을 분할하거나 세포의 성장을 할 수 있습니다. 이들 전구 세포가 아닌 세포 진정한 정지되지만, 분석에 사용 된 EGF 및 염기성 섬유 아세포 성장 인자 (bFGF)와 유사 분열 증식 세포 단계 일 수있다. 그럼에도 불구하고, 그들은 활성화 줄기 세포 - 관련 시그널링의 범위 (14) 경로를 포함하고있다. 또한, 그 형성 속도는 마우스 이종 이식편에서 2,15,16을 제한 희석 분석에서의 역가 측정들이 찍은 된 조직의 종양 발생에 관한 &lt;/ SUP&gt;. 여기서 우리는 단일 세포를 분리 및 인간 유방암 세포주 및 유방암의 임상 샘플 모두로부터 mammospheres 차를 생성하기 상세한 프로토콜을 제공한다. 우리는 또한 자기 갱신을 평가하는 주요 mammospheres의 일련 구절을 수행하는 방법, 및 구 다른 시드 밀도에서 비교를 (그림 1의 방식을 참조) 할 수 있습니다 효율성을 형성 계산하는 방법에 대해 설명합니다.

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			<td>233-FB-025</td>
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			<td height="22" style="height:22px;width:625px;">1x B27 supplement&#160;</td>
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			<td height="21" style="height:21px;">&#160;0.5% trypsin-0.2%EDTA;</td>
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			<td>59418C</td>
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			<td height="21" style="height:21px;">Fetal Calf Serum</td>
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			<td>02-00-850</td>
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			<td height="21" style="height:21px;">&#160;Low attachment 6 well plates</td>
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			<td height="21" style="height:21px;">Collagenase type 1A</td>
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			<td height="21" style="height:21px;">Hyaluronidase</td>
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			<td height="21" style="height:21px;width:625px;">Sterile scalpel</td>
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			<td height="21" style="height:21px;width:625px;">Sterile micro-dissecting scissors</td>
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			<td>S3146</td>
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mammosphere formation assay from human breast cancer tissues and cell lines

건강한 조직 많은 혈액 및 고형 악성 종양과 유사하게이 더욱 분화 된 자손을 발생주기 동안 자기 - 갱신 줄기 같은 암세포의 서브 세트로, 계층 적으로 구성하는 것으로 생각된다. 이해 및 향상된 화학 치료 및 비 - 조혈 종양 부피에 비해 전파 저항을 가질 수있다 유방암 이러한 암 줄기 세포를 표적으로는, 중요한 연구 분야가되고있다. mammosphere 분석이 또한 널리 향적 sphere-를 식별하는 능력을 위해 사용되었다 : CD44, CD24, 및 ALDH 활성 포함 마커 전향 면역 결핍 생쥐에 주입 할 때 향상된 발암 표시 세포를 분리하는 (FACS)를 정렬 형광 활성화 된 세포를 사용하여 평가할 수있다 하나의 줄기 세포와 같은 세포 클론 개발을 형성한다. 여기에서 우리는 세포 라인 또는 기본 환자 샘플, 자신의 계대 및 구 F를 추정하는 계산에서 mammospheres의 적절한 배양 방법을 간략하게 설명orming 효율 (SFE). 먼저 우리는 적절한 계획과 mammosphere 실험의 해석에 주요 고려 사항과 함정에 대해 설명합니다.

다른 샘플이나 다른 치료를 실시 그 시드 초기 세포 정상화 후 형성 mammospheres&gt; 40 μm의 수에 따라 다를 수 있습니다. 세중에서 재배 각각의 치료를위한 mammosphere 형성 효율 (MFE)를 계산합니다. 이것은 다른 시드 밀도 실험을 비교 할 수 있습니다. 데이터는 가장 이상적 양성 및 음성 대조군으로, 막대 그래프에 표시하고, 삼중 웰 걸친 표준 편차를 표시한다. 지속적으로 60-80% 합류 사이에 비 부착 ?...

Watch this Scientific Journal Video about Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines at JoVE.com

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

인간 유방암 조직과 세포주에서 Mammosphere 형성 분석

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

mammosphere-formation-assay-from-human-breast-cancer-tissues-cell

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33.0K 조회수.  Imperial College London. Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

비디오: Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

인간 Neuroendocrine 종양 치료의 연구를위한 3 차원 모델로 인간 Neuroendocrine 종양 세포 라인

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

연구

의학

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

세포 배양을위한 인간 감소 Mammoplasty과 유방 절제술의 조직의 처리

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

기본 Preneoplastic 유방 상피 세포의 시간 경과 영상은 유방암의 유전자 변형 마우스 모델에서 파생

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

개인 신장 세포 암에서 단백질 표현 변화를 탐험하기 위해 역 위상 단백질 배열 (RPPA)의 사용

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

설치류 비뇨 부비동 간엽 및 인간 줄기 세포의 조직 재조합을 사용하여 인간의 전립선 상피의 형성

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

유방암 세포에서 에스트로겐 조절 마이크로 RNA의 프로파일

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

3 차원 (3D) 모델을 사용하여 유방암 세포 침윤의 정량화

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

인간 세포주에서 Chemoresistant 난소 암 줄기 세포에 대한 심화 학습

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

<em&gt; 생체 내</em&gt; 모델은 인간의 유방암에서 호르몬 행동을 연구하기 위해

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

배양 인간의 대퇴골 조직 외식하는 방법은 전이성 틈새의 유방암 세포 집락을 연구하기 위해

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...