이 연구는 일본 문부 과학문화부의 과학 연구 C 보조금(K.N.에 대한 보조금 번호 19K09834)의 지원을 받았습니다.

모든 절차는 인간 복지 윤리위원회가 승인 한 의정서에 따라 수행되었습니다. 모든 환자는 샘플의 연구 사용에 대한 서면 동의서를 제출했으며,이 연구를위한 조직의 수집 및 사용은 테이 쿄 대학의 인간 게놈, 유전자 분석 연구 윤리위원회의 승인을 받았습니다.
1. 레벨 2 생물안전 캐비닛에서 난소암 및 복수 환자의 난소암 줄기세포(OCSC) 분리 및 배양
<ol><li>천자를 통해 얻은 인간 난소 암 복수에서 암 줄기 세포를 분리합니다. 충분한 수의 암 줄기 세포를 채취하기 위해 환자로부터 최소 100-250mL의 복수를 수집하십시오. 추가적으로, 암 줄기 세포 마커 (즉, EpCAM, 칼레티닌, CD133, CD44, CD45, ALDH1, 및 Oct4) 및 난소암 마커 (pAX-8, WT-1)의 발현 프로필을 유세포분석에 의해 평가한다.<ol><li>복수 흡인 후 24시간 이내에 실온에서 10분 동안 300 x g 에서 인간 난소암 복수를 원심분리합니다.</li>
		<li>상청액을 제거하고 OCSC 배지 2mL와 30% 히스토덴츠/인산염 완충 식염수(PBS, pH 7.4) 용액 8mL를 추가합니다.</li>
		<li>OCSC 배지 준비: 스템프로 hESC 보충제, L-글루타민(글루타맥스 배지), 25% BSA, 100μM 2-메르캅토에탄올, 8ng/mL FGF 염기성, 10μM 인슐린 및 20μM Y-27632가 포함된 DMEM⁄F-12.</li>
		<li>15mL 튜브의 1.1.2단계의 세포 용액에 2mL의 OCSC 배지를 조심스럽게 오버레이하고 제동 없이 스윙 버킷 로터에서 실온에서 20분 동안 450 x g 로 원심분리합니다.</li>
		<li>OCSC 층(간기에서 방해받지 않음)을 전사 피펫으로 새 15mL 튜브로 조심스럽게 옮깁니다.</li>
		<li>PBS로 최대 15mL까지 채웁니다. 실온에서 5분 동안 300 x g 으로 원심분리하고 상청액을 제거한다.</li>
		<li>세포 펠릿을 OCSC 배지에 재현탁하고 초저 부착 배양 접시에 시드합니다. 배양물은 5%CO2에서 37°C에서 유지되어야 한다.</li>
		<li>3 일마다 매체를 교체하십시오. 배양 접시를 약 1 분 동안 조심스럽게 서서 상청액의 일부를 버리고 새로운 배지를 첨가하십시오.</li>
	</ol></li>
	<li>CSC의 통과<ol><li>15mL 튜브에 OCSC를 수집하고 실온에서 5분 동안 200 x g 로 원심분리합니다.</li>
		<li>상청액을 제거하고, PBS로 채우고, 실온에서 5분 동안 200 x g 에서 원심분리한다.</li>
		<li>상청액을 제거하고, 단백질 분해 및 콜라겐분해 효소(예: AccuMax)로 구성된 세포 분리 용액 1mL를 추가하고, 37°C에서 10분 동안 배양한다.</li>
		<li>피펫팅으로 잘 혼합하고 37°C에서 5분 동안 배양합니다. 세포가 단일 현탁액에 있는지 확인하십시오.</li>
		<li>피펫팅으로 잘 혼합하고 PBS로 채우고 실온에서 300 x g 에서 5분 동안 원심분리합니다.</li>
		<li>상청액을 제거하고 세포 펠렛을 OCSC 배지에 재현탁시켜 초저 부착 배양 접시에 후속 시딩하고 5%CO2에서 37°C에서 유지한다.</li>
	</ol></li>
</ol>2. HUEhT-1 내피 세포 배양
<ol><li>HUEhT-1 세포의 통과<ol><li>HUEhT-1 배양 접시로부터 배지를 제거하고, 세포를 PBS로 세척한다.</li>
		<li>0.025% 트립신 1mL를 넣고 실온에서 3분간 배양합니다.</li>
		<li>내피 세포 성장 배지 2 5mL를 추가하고 15mL 튜브에 세포를 모은 다음 실온에서 5분 동안 200 x g 에서 원심분리합니다.</li>
		<li>상청액을 제거하고, 세포 펠렛을 HUEhT-1 배지에 재현탁하고, 콜라겐이 코팅된 배양 접시에 세포를 시드한 다음, 5%CO2에서 37°C에서 유지하였다.</li>
		<li>3 일마다 매체를 교체하십시오.</li>
	</ol></li>
</ol>3. HUEhT-1 세포를 이용한 튜브 형성 분석을 위한 NICO-1 공동배양 플레이트의 제조
<ol><li>NICO-1을 조립하고 세포외 기질계 하이드로겔(Matrigel Matrix)로 코팅한다.<ol><li>제조업체의 지침에 따라 NICO-1의 한쪽을 조립하고 얼음 위에 보관하십시오.</li>
		<li>NICO-1의 표면을 300 μL의 차가운 PBS로 덮은 다음 완충액을 제거합니다.</li>
		<li>냉각된 세포외 기질 기반 하이드로겔 300μL를 추가하고 37°C에서 60분 동안 배양합니다.</li>
		<li>평형을 이루려면 필터를 100% 에탄올에 담근 다음 13mm ICCP 필터(0.6μm)를 PBS로 1분 동안 세척합니다.</li>
		<li>본체 부품 A(오른쪽 챔버) 및 B(왼쪽 챔버)를 포함한 NICO-1을 O 링 및 평형 필터와 함께 조립합니다.</li>
	</ol></li>
</ol>4. HUEhT-1 세포 및 CSC를 NICO-1 시스템에 파종
<ol><li>세포 단층을 트립신화하고 세포를 2% 우태아 송아지 혈청으로 내피 세포 성장 배지에 재현탁하여 HUEhT-1 세포 현탁액을 준비합니다.</li>
	<li>1.2 mL의 세포 현탁액(1.5 x 105 세포)을 각 세포외 기질 기반 하이드로겔 코팅 웰에 추가합니다.</li>
	<li>5일 동안 배양한 OCSC 1.5mL를 다른 웰에 추가합니다.</li>
	<li>NICO-1을 5%CO2 중 37°C에서 인큐베이션하고; 튜브 형성은 가지의 수를 통해 측정 된 세포 외 기질 기반 하이드로 겔에서 현미경 및 네트워크 형성으로 관찰 할 수 있습니다.</li>
</ol>

<ol>
	<li>Bray, F., 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=Global+cancer+statistics+2018:+GLOBOCAN+estimates+of+incidence+and+mortality+worldwide+for+36+cancers+in+185+countries.">Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.</a> CA, a Cancer Journal for Clinicians. 68, 394-424 (2018).</li><li>Lengyel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+development+and+metastasis.">Ovarian cancer development and metastasis.</a> American Journal of Pathology. 177 (3), 1053-1064 (2010).</li><li>Lytle, N. K., Barber, A. G., Reya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+fate+in+cancer+growth,+progression+and+therapy+resistance.">Stem cell fate in cancer growth, progression and therapy resistance.</a> Nature Reviews Cancer. 18 (11), 669-680 (2018).</li><li>Dontu, G., 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 and Development. 17 (10), 1253-1270 (2003).</li><li>Lonardo, E., 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=Nodal/Activin+signaling+drives+selfrenewal+and+tumorigenicity+of+pancreatic+cancer+stem+cells+and+provides+a+target+for+combined+drug+therapy.">Nodal/Activin signaling drives selfrenewal and tumorigenicity of pancreatic cancer stem cells and provides a target for combined drug therapy.</a> Cell Stem Cell. 9 (5), 433-446 (2011).</li><li>Ricci-Vitiani, 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+and+expansion+of+human+colon-cancer-initiating+cells.">Identification and expansion of human colon-cancer-initiating cells.</a> Nature. 445 (7123), 111-115 (2007).</li><li>Ohata, 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=Induction+of+the+stem-like+cell+regulator+CD44+by+Rho+kinase+inhibition+contributes+to+the+maintenance+of+colon+cancer-initiating+cells.">Induction of the stem-like cell regulator CD44 by Rho kinase inhibition contributes to the maintenance of colon cancer-initiating cells.</a> Cancer Research. 72 (19), 5101-5110 (2012).</li><li>Ishiguro, 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=Establishment+and+characterization+of+an+in+vitro+model+of+ovarian+cancer+stem-like+cells+with+an+enhanced+proliferative+capacity.">Establishment and characterization of an in vitro model of ovarian cancer stem-like cells with an enhanced proliferative capacity.</a> Cancer Research. 76 (1), 150-160 (2016).</li><li>Singh, S. 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=Identification+of+a+cancer+stem+cell+in+human+brain+tumors.">Identification of a cancer stem cell in human brain tumors.</a> Cancer Research. 63 (18), 5821-5828 (2003).</li><li>Zong, X., Nephew, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+stem+cells:+role+in+metastasis+and+opportunity+for+therapeutic+targeting.">Ovarian cancer stem cells: role in metastasis and opportunity for therapeutic targeting.</a> Cancers (Basel). 11 (7), 934 (2019).</li><li>Liz&#225;rraga-Verdugo, E., 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=Cancer+stem+cells+and+its+role+in+angiogenesis+and+vasculogenic+mimicry+in+gastrointestinal+cancers.">Cancer stem cells and its role in angiogenesis and vasculogenic mimicry in gastrointestinal cancers.</a> Frontiers in oncology. 10, 413 (2020).</li><li>Renaud, J., Martinoli, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+insert+co-culture+system+of+two+cellular+types+in+the+absence+of+cell-cell+contact.">Development of an insert co-culture system of two cellular types in the absence of cell-cell contact.</a> Journal of Visualized Experiments. (113), e54356 (2016).</li><li>Richardson, S. 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=Intervertebral+disc+cell-mediated+mesenchymal+stem+cell+differentiation.">Intervertebral disc cell-mediated mesenchymal stem cell differentiation.</a> Stem Cells. 24 (3), 707-716 (2006).</li><li>Plotnikov, E. 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=Cell-to-cell+cross-talk+between+mesenchymal+stem+cells+and+cardiomyocytes+in+co-culture.">Cell-to-cell cross-talk between mesenchymal stem cells and cardiomyocytes in co-culture.</a> Journal of Cellular and Molecular Medicine. 12 (5), 1622-1631 (2008).</li><li>Sheng, 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=A+critical+role+of+IFN-gamma+in+priming+MSC-mediated+suppression+of+T+cell+proliferation+through+up-regulation+of+B7-H1.">A critical role of IFN-gamma in priming MSC-mediated suppression of T cell proliferation through up-regulation of B7-H1.</a> Cell Research. 18 (8), 846-857 (2008).</li><li>Csaki, C., Matis, U., Mobasheri, A., Shakibaei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-culture+of+canine+mesenchymal+stem+cells+with+primary+bone-derived+osteoblasts+promotes+osteogenic+differentiation.">Co-culture of canine mesenchymal stem cells with primary bone-derived osteoblasts promotes osteogenic differentiation.</a> Histochemistry and Cell Biology. 131 (2), 251-266 (2009).</li><li>Aguirre, A., Planell, J. A., Engel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+bone+marrow-derived+endothelial+progenitor+cell/mesenchymal+stem+cell+interaction+in+co-culture+and+its+implications+in+angiogenesis.">Dynamics of bone marrow-derived endothelial progenitor cell/mesenchymal stem cell interaction in co-culture and its implications in angiogenesis.</a> Biochemical and Biophysical Research Communications. 400 (2), 284-291 (2010).</li><li>Proffen, B. L., Haslauer, C. M., Harris, C. E., Murray, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+from+the+retropatellar+fat+pad+and+peripheral+blood+stimulate+ACL+fibroblast+migration,+proliferation,+and+collagen+gene+expression.">Mesenchymal stem cells from the retropatellar fat pad and peripheral blood stimulate ACL fibroblast migration, proliferation, and collagen gene expression.</a> Connective Tissue Research. 54 (1), 14-21 (2013).</li><li>Goers, L., Freemont, P., Polizzi, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-culture+systems+and+technologies:+taking+synthetic+biology+to+the+next+level.">Co-culture systems and technologies: taking synthetic biology to the next level.</a> Journal of the Royal Society &amp; Interface. 11 (96), 20140065 (2014).</li><li>De Palma, M., Biziato, D., Petrova, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+tumour+angiogenesis.">Microenvironmental regulation of tumour angiogenesis.</a> Nature Reviews Cancer. 17, 457-474 (2017).</li><li>Burger, 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=Incorporation+of+bevacizumab+in+the+primary+treatment+of+ovarian+cancer.">Incorporation of bevacizumab in the primary treatment of ovarian cancer.</a> New England Journal of Medicine. 365, 2473-2483 (2011).</li><li>Goel, H., Mercurio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+targets+the+tumour+cell.">VEGF targets the tumour cell.</a> Nature Reviews Cancer. 13, 871-882 (2013).</li><li>Yu, 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=Interaction+between+bevacizumab+and+murine+VEGF-A:+a+reassessment.">Interaction between bevacizumab and murine VEGF-A: a reassessment.</a> Investigative Ophthalmology and Visual Science. 49 (2), 522-527 (2008).</li></ol>

저자는 공개 할 것이 없습니다.

제시된 프로토콜은 시험관 내 설정에서 OCSC의 종양 미세 환경을 모방하는 방법을 설명합니다. 이 방법의 주요 구성 요소는 간접 Transwell 공동 배양 시스템인 NICO-1 시스템을 사용하여 얻은 재현성이 높은 공동 배양 모델을 구성합니다. 현재 이용 가능한 많은 공동 배양 모델은 공동 배양 된 세포 집단 12,13,14,15,16,17,18에 대한 직접적인 세포-세포 접촉의 효과를 조사합니다.<s...

난소암은 전 세계적으로 여성에게 8번째로 흔한 악성 종양으로, 매년 약 300,000건의 새로운 진단과 약 180,000명의 사망이발생합니다1. 초기 진단에서 난소 암은 종종 심각한 증상을 나타내며 환자의 약 75 %가 이미 III-IV 기에 있습니다. 이에 따라 부인암2 중 5년 생존율이 <30%, 사망률이 가장 높으며, 난소암의 치료 효율은 용적폐 수술의 성공적 성취, 항암에 대한 내성, 초기 치료 후 재발 등의 임상적 요인에 크게 의존한다.
난소암 조직은 계층적으로 구성되어 있으며 모든 종양 구성 요소가 동등하게 자손을 생성할 수 있는 것은 아닙니다. 자가 재생하고 이질적인 종양 세포 집단을 생성할 수 있는 유일한 세포는 암 줄기 세포(CSC)를 나타내는 것으로 간주됩니다3. CSC 자가 재생 및 종양 개시는 지지적 틈새를 유지하기 위한 목적으로 종양 미세환경을 리모델링하기 위한 혈관신생의 촉진을 동반한다. 그러나 이전 모델은 다중 계대 배양 후 스페로이드의 파괴로 인해 임상 샘플에서 파생된 CSC 배양의 재현성이 제한적이기 때문에 시험관 내 분석에 활용할 수 없었습니다. 보다 최근에, 환자로부터 CSC를 배양하기 위한 실험적 방법이여러 응용 4,5,6,7을 위해 개발되었다. 특히, 배양된 줄기세포는 무혈청 배지로 초저 부착판에서 스페로이드를 형성하여 성장하는 CSC의 특성을 이용함으로써, 다계통 분화 가능성이 있는 정상 종양세포에서는 발현되지 않는 줄기세포 표면 마커를 발현하도록 유도한다(8,9).
최근 데이터에 따르면 복막에서 전파로 시각화 된 휴면 난소 (O) CSC의 지속성은 재발 성 종양10으로 재생되는 것과 관련이 있습니다. 따라서 OCSC의 분자 및 생물학적 특징을 이해하면 이러한 세포를 효과적으로 표적화하고 박멸하여 잠재적인 종양 완화를 초래할 수 있습니다. 특히, 혈관신생11에서 CSCs 역할의 세포 및 분자 기계론적 특징에 대해서는 알려진 바가 거의 없다. 따라서, 본 프로토콜에서는 임상 환경에서 전이 부위에서 CSC 및 내피 세포의 종양 미세 환경을 모방 할 수있는 공동 배양 모델을 사용하여 내피 세포의 혈관 신생 특성을 조사하기 위해 시험관 내 설정에서 환자 유래 OCSC를 사용했습니다. 궁극적으로 신생혈관형성은 종양 성장과 전이를 지원하는 데 필요한 중요한 과정을 구성하기 때문에 그 메커니즘을 더 잘 이해하면 전이 부위에서 OCSC에 대한 새로운 표적 요법을 개발할 수 있습니다.
여기에서는 CSC를 둘러싼 신생혈관화 단계를 시간 경과 방식으로 시각화하는 프로토콜을 제시합니다. 이 프로토콜의 장점은 3D 공동 배양 시스템인 NICO-1을 사용하여 완전히 재현 가능한 조사를 허용하여 내피 세포 혈관신생 동안 OCSC 유래 종양 개시 능력이 환자에 미치는 영향을 관찰할 수 있다는 것입니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.025% Trypsin&#160;</td>
			<td>Thermo</td>
			<td>R001100</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Pipet</td>
			<td>Thermo</td>
			<td>170356N</td>
			<td></td>
		</tr>
		<tr>
			<td>1250 &#181;L Pipet tip</td>
			<td>QSP</td>
			<td>T112XLRS-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tube</td>
			<td>Nunc</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L Pipet tip</td>
			<td>QSP</td>
			<td>T110RS-NEW</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Thermo (Gibco)</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Pipet</td>
			<td>Thermo</td>
			<td>170366N</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tube</td>
			<td>Corning</td>
			<td>430290</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuMAX</td>
			<td>Innovative Cell Technologies</td>
			<td>AM105</td>
			<td></td>
		</tr>
		<tr>
			<td>BioCoatTM Collagen I 60mm Dish</td>
			<td>Corning</td>
			<td>356401</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>KUBOTA</td>
			<td>2800</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 6 Well Clear Flat Bottom Ultra Low Attachment Multiple Well Plates</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium 2&#160;</td>
			<td>PromoCell</td>
			<td>C-22011&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>WAKO</td>
			<td>057-00456</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-Basic</td>
			<td>Thermo (Gibco)</td>
			<td>PHG0021</td>
			<td></td>
		</tr>
		<tr>
			<td>Histodenz</td>
			<td>SIGMA</td>
			<td>D2158</td>
			<td></td>
		</tr>
		<tr>
			<td>HUEhT-1 cell</td>
			<td>JCRB Cell Bank</td>
			<td>JCRB1458</td>
			<td></td>
		</tr>
		<tr>
			<td>ICCP Filter 0.6 &#181;m</td>
			<td>Ginrei Lab.</td>
			<td>2525-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin, human</td>
			<td>SIGMA (Roche)</td>
			<td>11376497001</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>PerkinElmer</td>
			<td>ARVO MX-flad</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Matrix</td>
			<td>Corning</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Yokogawa</td>
			<td>CQ-1</td>
			<td></td>
		</tr>
		<tr>
			<td>NICO-1</td>
			<td>Ginrei Lab.</td>
			<td>2501-02</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPlate-96</td>
			<td>PerkinElmer</td>
			<td>6005290</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 Pipet</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 Pipet</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thermo (Gibco)</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro hESC SFM</td>
			<td>Thermo (Gibco)</td>
			<td>A1000701</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Pipet</td>
			<td>FALCON</td>
			<td>357575</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>WAKO</td>
			<td>253-00513</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluating the angiogenetic properties of ovarian cancer stem-like cells using the three-dimensional co-culture system, nico-1

암 줄기 세포 (CSC)는지지 틈새 시장에 상주하여 인접한 기질 세포, 혈관 및 세포 외 기질로 구성된 미세 환경을 구성합니다. 내피 발달에 참여하는 CSC의 능력은 종양 발생 및 종양 전이의 메커니즘에 대한 일반적인 이해에 직접적으로 기여하는 중요한 특성을 구성합니다. 이 연구의 목적은 난소암 줄기세포(OCSC)의 종양 개시 능력을 조사하기 위한 재현 가능한 방법론을 확립하는 것입니다. 본 발명에서는 시험관 내 공동 배양 모델 NICO-1을 사용하여 내피 세포와 OCSC 사이의 신생혈관형성 메커니즘과 내피 세포의 형태학적 변화를 조사했습니다. 이 프로토콜은 OCSC를 둘러싼 신생 혈관 단계를 시간 경과 방식으로 시각화 할 수 있습니다. 이 기술은 종양 전이에서 OCSC의 혈관 신생 특성에 관한 통찰력을 제공 할 수 있습니다.

스페로이드에 대한 장기간 안정적인 배양을 수행하기 위해 수술 또는 천자 중 진행성 난소암 환자로부터 얻은 복수액을 수집했습니다. 여기에서는 CSC1 및 CSC2라고 하는 난소 CSC의 장기 스페로이드 배양 사례를 제시합니다. 두 세포주 모두 동일한 진단 및 조직 학적 프로파일을 가지고 있습니다. OCSC를 둘러싼 내피 세포의 신생 혈관을 유도하는 데 필요한 내피 세포와의 상?...

Watch this Scientific Journal Video about Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1 at JoVE.com

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

난소암 줄기세포(OCSC)는 암 시작, 재발, 치료 내성 및 전이를 담당합니다. OCSC 혈관 틈새는 OCSC의 자가 재생을 촉진하여 화학 내성을 유발하는 것으로 간주됩니다. 이 프로토콜은 시험관 내에서 재현 가능한 OCSC 혈관 틈새 모델을 구축하기 위한 기초를 제공합니다.

3차원 공동배양시스템을 이용한 난소암 줄기유사세포의 혈관신생적 특성 평가 NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

evaluating-angiogenetic-properties-ovarian-cancer-stem-like-cells

Research

JoVE Journal

Biology

2.5K 조회수.  Teikyo University School of Medicine. 난소암 줄기세포(OCSC)는 암 시작, 재발, 치료 내성 및 전이를 담당합니다. OCSC 혈관 틈새는 OCSC의 자가 재생을 촉진하여 화학 내성을 유발하는 것으로 간주됩니다. 이 프로토콜은 시험관 내에서 재현 가능한 OCSC 혈관 틈새 모델을 구축하기 위한 기초를 제공합니다.

비디오: Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

안녕하세요 - C : Genomes의 3 차원 구조를 공부하는 방법.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

연구

생물학

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

마우스 Meiotic 셀 유동세포계측법의 정제

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

세 가지 차원 혼합 셀 회전 타원체 공동 문화에서 Mammary 상피 세포의 분리

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

공장 Vasculature의 3 차원 구조 및 기능을 시각화하기 위해 높은 해상도로 계산 된 단층 촬영을 사용하여

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

담배 과도 식 시스템을 사용하여 MAPK 신호 전달 통로의 공간 개편 이미징

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

세포 - 세포 연락이없는 두 세포 유형의 삽입 공동 배양 시스템 개발

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

수동 선명도 삭제 마우스 난소의 혈관 구조의 3 차원 재구성

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-지우기: 조직 지방 조직의 3 차원 이미징 방법 삭제

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

단세포 분해능 3차원 알래기 알코노이드

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The Quadruple&#160;checkerboard, also called the Q-checkerboard, aims at maximizing the number of possible combinations that can be obtained between four drugs in one experiment to minimize the time and work needed to accomplish the same results with other protocols. This protocol is based on the simple micro dilution technique where the drugs are diluted and combined together in several 96-well plates.
In the first set of 96-well plates, Muller-Hinton broth is added followed by the first required drug (e.g., Cefotaxime here) to serially dilute it. After the first step is done, another set of 96-well plates is used to dilute the second drug (e.g., Amikaci), which will be transferred by removing a specific volume of drug 2 and put in the corresponding wells in the first set of 96-well plates that contains drug one. The third step is done by adding the required concentrations of the third drug (e.g., Levofloxacin), to the appropriate plates in the initial set containing combination of drug 1 and 2. The fourth step is done by adding the required concentrations of the fourth drug (e.g., Trimethoprim-sulfamethoxazol) into the appropriate plates in the first set. Then, E. coli ESBL bacterial inoculum will be prepared and added.
This method is important to evaluate all the possible combinations and has a wider range of possibilities to be tested furthermore for in vivo testing. Despite being a tiring technique requiring a lot of focus, the results are remarkable and time saving where a lot of combinations can be tested in a single experiment.

This protocol describes how to study all possible combinations that can be obtained between four drugs in one single experiment. This method is based on the standard 96-well plate micro dilution assay and the calculation of fractional inhibitory concentrations (FICs) to evaluate the results.