This study was supported by a grant from the Baden-W&#252;rttemberg Stiftung (Adult Stem Cells Program II) awarded to C.L. We thank Dr. Martina Konantz for critical input and review of the manuscript. We thank Emmanuel Traunecker and Toni Krebs from the DBM FACS Facility (University Hospital Basel) for assistance with FACS sorting.

OVCAR-3 인간 난소 암 세포의 1 세대 안정적 렌티가 SOX2 규제 지역 리포터 구조를 포함하는 형질 도입 <ol><li> 10,21 바와 같이 SOX2 조절 영역을 인식하는 리포터 구조체와 함께 HEK 293T 패키징 세포주를 형질 감염에 의해 렌티 바이러스 입자를 생성한다. 참고 : 기자가 추가로 구성 앞서 tdTomato 형광 단백질의 ProteoTuner 쉴드 시스템의 불안정 도메인이 포함되어 있습니다. Shield1을함으로써 형광 단백질 (22)을 분해하는 프로 테아 좀을 방지 불안정 도메인에 결합한다. </li><li> 24 시간의 시간주기 동안 렌티 바이러스 입자 OVCAR-3 세포를 형질 도입. 그 후, 바이러스 상청액을 제거하고 완전 배지에 인산 완충 생리 식염수 (PBS)와 함께 배양 된 세포를 세척 (RPMI 100 U / ㎖ 페니실린 / ㎖ 스트렙토 마이신 100 μg의, 10 % FBS가 보충 된). </li><li> 48 시간 후, 10 ㎍ / ㎖의 퓨로마이신은 배양에 첨가하고, 적절하게 형질 도입 된 세포의 선택을 허용하기 위해 5 일간 유지 하였다. </li></ol> 셀 정렬 및 도금 2. 준비 <ol><li> 1 Shield1을 추가 : 셀 정렬하기 전에 1,000 희석 24 시간을. 음성 대조군으로 Shield1을 처리 (그림 1)하지 않고 안정적으로 형질 OVCAR-3 세포를 사용합니다. 플라스크에서 대기음 매체는 3 분 동안 0.05 % 트립신 - EDTA로 세포를 1X PBS로 세포를 씻어를 Trypsinize. </li><li> 5 분 - (25 ° C 15) RT 300 XG에서 세포 수, 세포를 원심 분리 카운트 (위 참조) 완전 배지를 사용하여 트립신을 중지. </li><li> 가만히 따르다 뜨는에 resuspend 세포 신중에 0.5-1 ml의 멸균 PBS. </li><li> 단일 세포 현탁액을 얻기 위해 40 μm의 셀 스트레이너 캡 필터를 사용한다. </li><li> 1 mL 당 500 만 세포에 세포 수를 조정합니다. </li><li> , MEGM는 성장 인자, 사이토 카인 및 보조 식품으로 보충 (100 μL와 매체 초저 첨부 파일 96 웰 플레이트를 구체 준비B-27, heparine 나트륨; 또는 성장 인자, 사이토 카인, 및 보충, B-27, 또는 1 % 메틸 셀룰로오스 무첨가 heparine 나트륨이 보충 된 DMEM / F12는) 또한 표 1 참조. 선택적으로, 가능한 오염의 위험을 최소화하기 위해 100 U / ㎖ 페니실린 및 100 ㎍ / ml의 스트렙토 마이신의 농도로 배지에 항생제를 추가한다. </li><li> 정렬 RFP + 이상에서 제조 된 96 웰 플레이트에 RFP- 세포, 잘 당 1 셀 (하나의 셀 기반 분야 분석) 각각 아니라 100 세포 (다중 셀 기반 분야 분석). 100 μ 노즐, 칼집 압력이 20psi, 수율 마스크 0, 순도 마스크 (32), 위상 마스크 16 : 단일 셀 모드, 정렬 설정을 사용하여 (자료 참조) 종류에 시판 셀 소터를 수행합니다. </li><li> 현미경 (단일 세포 기반 분석의 경우) 세포를 포함하는 우물을 점수에 의해 다중 셀 기반 분석을 위해 (개인 우물에서 세포 수를 계산하여 효율성을 도금 평가; 그림 2 </strong&gt;). </li><li> 매체 분야의 표준 조건에서 세포를 품어 37 ° C에서 5 % CO 2 (조성물 단계 2.6 참조). 매일의 bFGF (20 NG / ㎖) 및 EGF (20 NG / ㎖)을 보충합니다. </li><li> 일주일이 지난 뒤, 집적 리포터 시스템으로부터의 형광 신호를 검출하기 위해 4 배 또는 10 배 배율 및 형광 현미경으로 표준 현미경을 사용하여 종양 신생 분야의 숫자를 카운트. &quot;작은&quot;구 100 μm의 (그림 3) - 직경 50 &quot;큰&quot;구체로 100 ㎛를 초과하는 직경 분야 및 구체를 계산합니다. 당신이 진짜 구체를 계산하고 클러스터를 셀 수 없습니다 있는지 확인하십시오. 참고 : 단일 세포 기반 분석에서 구 형성이 문화의 10 (7 대) 일 후 현미경으로 득점하기가 쉽습니다. </li><li> RFP의 +의 구 형성 세포 및 단일 세포 기반 분석에서 각각 RFP- 세포 (각 실험 한 96 웰 플레이트) 또는 멀티 셀 바의 비율을 계산쇼 등에 의해 제시된 나오지 분야 분석 (각각의 실험에 대해 잘 하나). (16) 주 : 구 형성 세포 (%) = (구체의 개수) / (셀 시드 수)의 비율은 100 X </li></ol> 분야 3. 직렬과 Passaging <ol><li> 실온에서 10 분 동안 300 XG에 적​​절한 멸균 튜브와 원심 분리기에 각 웰의 내용을 놓습니다. 다중 셀 기반 분야 분석을 위해, 함께 잘 하나의 구체를 수집합니다. PBS 긴 원심 분리기 2 분에 5 회 - 잘 3을 씻으십시오. 하나의 셀 기반 분야 분석의 경우, 개별 분야를 수집합니다. 때문에 세포의 낮은 숫자로, 원심 분리 및 세척 단계 1.5 ml의 튜브를 사용합니다. </li><li> 0.05 % 트립신 - EDTA의 200 μL에 뜨는에 resuspend 펠렛을 제거합니다. </li><li> 최적의 세포 분리를 달성하기 위해, 부드러운 진탕을 5 분 동안 37 ℃에서 세포 현탁액을 배양한다. 낮은 세포 사멸 속도로 셀 라인 트립신 시간을 최적화 : 더 큰 경우구 부드럽게 100 μL 피펫 팁을 사용하여 볼 수 씹다입니다. 경우 큰 분야는 여전히 존재하는 또 다른 3 분 트립신과 부화 후 분쇄 단계로 진행합니다. 하나의 경우 셀 기반 분석은 트립신 단계 동안 살아있는 세포의 최적 세포 수율의 시간을 최적화해야합니다. </li><li> , 트립신을 비활성화 단일 세포 분석, 추가로 2 분 동안 원심 분리기의 경우, 10 분 동안 300 XG에 500 μL에게 완전한 매체와 원심 분리기를 추가합니다. </li><li> 구체 매체에 조심스럽게 뜨는에 resuspend 세포를 제거합니다. </li><li> 단일 세포 현탁액을 얻기 위해 40 μm의 셀 스트레이너 캡 필터를 사용한다. </li><li> RFP의 + 및 다중 - 셀 bassed RFP- 세포 내 분석을 사용하는 경우, 유동 세포 측정기 분석을 통해 재 부유 한 후 각 웰에서 형광 세포의 비율을 평가. </li><li> 위에서 설명한 바와 같이 제조 한 세포의 직렬 replating 분석, 씨앗 아니라 당 1 셀 새로운 초저 첨부 파일 96 웰 플레이트에 들어. 한 개인 영역에서 약 20 개인 우물을 씨앗. 다중 셀 기반의 주 분야 replating 분석법, 종자 세포는 새로운 96- 웰 플레이트에 주요 분야 중 하나 웰로부터 수득하고 현미경으로 세포 수를 카운트 다음날. </li><li> 단계 2.11에 기재된 식을 이용하여 이차 분석 분야에서 구 - 형성 세포의 비율을 평가한다. </li></ol> 4. 결과 분석 <ol><li> 독립적 인 3 회 반복 실험을 수행의 결과를 분석하고 양면 학생의 t- 검정 통계 분석을 위해 정규 분포 값과 달리 맨 - 휘트니 - 테스트를 분석하는 데 사용할. </li></ol>

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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=Non-small+cell+lung+cancer+cells+expressing+CD44+are+enriched+for+stem+cell-like+properties.">Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties.</a> PLoS One. 5 (11), e14062 (2010).</li><li>Bertolini, 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=Highly+tumorigenic+lung+cancer+CD133++cells+display+stem-like+features+and+are+spared+by+cisplatin+treatment.">Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment.</a> Proc Natl Acad Sci U S A. 106 (38), 16281-16286 (2009).</li><li>Ma, L., Lai, D., Liu, T., Cheng, W., Guo, L. <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-like+cells+can+be+isolated+with+drug+selection+in+human+ovarian+cancer+cell+line+SKOV3.">Cancer stem-like cells can be isolated with drug selection in human ovarian cancer cell line SKOV3.</a> Acta Biochim Biophys Sin (Shanghai). 42 (9), 593-602 (2010).</li><li>Wang, H., Zhang, Y., Du, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+and+breast+cancer+spheres+are+similar+in+transcriptomic+features+and+sensitive+to+fenretinide).">Ovarian and breast cancer spheres are similar in transcriptomic features and sensitive to fenretinide).</a> Biomed Res Int. 2013, 510905 (2013).</li><li>Du, Y. 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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=Identification+of+cancer+stem+cell-like+cells+from+human+epithelial+ovarian+carcinoma+cell+line.">Identification of cancer stem cell-like cells from human epithelial ovarian carcinoma cell line.</a> Cell Mol Life Sci. 67 (22), 3915-3925 (2010).</li><li>Zhang, 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=Identification+and+characterization+of+ovarian+cancer-initiating+cells+from+primary+human+tumors.">Identification and characterization of ovarian cancer-initiating cells from primary human tumors.</a> Cancer Res. 68 (11), 4311-4320 (2008).</li><li>Liang, D., 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=The+hypoxic+microenvironment+upgrades+stem-like+properties+of+ovarian+cancer+cells.">The hypoxic microenvironment upgrades stem-like properties of ovarian cancer cells.</a> BMC Cancer. 12, 201 (2012).</li></ol>

The authors have nothing to disclose.

구체 배양 암 줄기 세포는 잠재적 인 분석 및 인간 종양 세포 15,25,26 다양한 줄기 세포와 같은 농축 널리 사용되는 방법이다. 이러한 배양 조건 하에서, 자기 갱신 능력이 부족한 암세포 분화 결국 세포 사멸을 거칠 것으로 예상된다. 그들은 초기에 세포 클러스터 또는 특히 차 내 분석 분야, 심지어 종양을 형성 할 수 있지만, 그들로 인해 자기 특성 갱신 부족 직렬 replating시 구 형성능을 유...

There is increasing evidence that ovarian carcinomas are comprised of heterogeneous mixtures of cells and harbor so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for disease initiation, maintenance and relapse after conventional cytotoxic therapies1-3. Therefore, the development of molecular strategies targeting ovarian CSCs is an important goal and promises to improve the therapy of ovarian cancer patients.
A pre-requisite for the understanding of the molecular features of CSCs is their reliable isolation from the non-CSCs. However, identification of ovarian CSCs appears challenging. While CD133 expression and aldehyde dehydrogenase (ALDH) activity4,5 have been reported to mark ovarian CSCs, some data indicate that these markers are unstable6. Consistently, in ovarian cancer, other than for example in breast carcinoma7, expression of ALDH1 associates with favorable outcome8 and expression of the proposed stem cell marker CD44 variant has no prognostic value9. More recently, we have shown that expression of the embryonic stem cell protein SOX2 confers stemness to ovarian carcinoma cells10 and high SOX2 expression associates with clinically aggressive ovarian and breast carcinomas11,12. Therefore, in this report we use a lentiviral reporter construct containing a red fluorescence protein (RFP) whose expression is controlled by a SOX2 regulatory region, as a method to isolate putative ovarian CSCs.
By definition, CSCs can both self-renew and differentiate, giving rise to all tumor cell types. Putative CSC populations need to be analyzed in functional assays performed in vivo. For obvious reasons, in human cells such functional tests are confined to xenograft assays, comprising mostly transplantation of human tumor cells into immuno-compromised mice10,13.
An alternative in vitro method was offered by Brent Reynolds and Sam Weiss who firstly reported the so-called neurosphere assay as a surrogate assay evaluating stem potential in neural cells14. Dontu and colleagues later confirmed the use of this assay for evaluation of stem cell potential in breast cells15,16. Here, human mammary cells were plated in different numbers in serum-free medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), B-27 and heparin and cultured under non-adherent conditions for seven to ten days before sphere formation was scored by microscopy. Following this protocol with some adjustments in cell numbers, growth medium and supplements, several groups have explored in vitro stem cell potential from several cancer types such as breast17, brain18, pancreas19 and colon20 tumors. In ovarian carcinoma, we have recently reported feasibility of the spheres assay and compared its results to those collected in in vivo murine xenograft models10. We found that overexpression of the stem cell protein SOX2 enhanced both in vitro sphere formation as well as in vivo tumorigenicity of human ovarian carcinoma cells10. However, the frequency of sphere-initiating cells was higher than the frequency of tumor-initiating cells measured in vivo10 suggesting that either the sphere assay may lead to false positive results due to technical reasons or, alternatively, the in vivo assay may be inefficient and result in false negative results.
In this report, we analyze multi cell-based ovarian spheres assays in more detail, review the different protocols available in the literature and compare them to a single cell-based assay. We show that the single cell-based assay provides more accurate and reproducible results than multi cell-based assays, which can be highly influenced by the density of plated cells unless methylcellulose is added to the cultures to immobilize cells. However, also in single cell-based assays, in vitro sphere-initiating potential is observed at higher frequency than in vivo tumor-initiating potential.

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			<td style="width:112px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:189px;">Comments/Description</td>
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			<td height="21" style="height:21px;">low-Attachment-plate&#160;</td>
			<td>Corning</td>
			<td style="width:119px;">3474</td>
			<td></td>
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			<td height="21" style="height:21px;">MEGM</td>
			<td>Lonza</td>
			<td>CC-3151</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Insulin</td>
			<td>Lonza</td>
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			<td>SingleQuots&#8482; Kit</td>
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			<td height="21" style="height:21px;width:216px;">Hydrocortison</td>
			<td>Lonza</td>
			<td style="width:119px;">CC-4136</td>
			<td>SingleQuots&#8482; Kit</td>
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			<td height="21" style="height:21px;width:216px;">EGF</td>
			<td>Lonza</td>
			<td style="width:119px;">CC-4136</td>
			<td>SingleQuots&#8482; Kit</td>
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			<td height="21" style="height:21px;">EGF</td>
			<td>Sigma</td>
			<td>E9644</td>
			<td>end concentration: 20 ng/ml</td>
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		<tr height="21">
			<td height="21" style="height:21px;">FGF</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td>end concentration: 20 ng/ml</td>
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		<tr height="21">
			<td height="21" style="height:21px;">B-27</td>
			<td>Invitrogen/ Gibco</td>
			<td>17504-044</td>
			<td>end concentration: 1x</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Heparin-Natrium-25000 IE</td>
			<td>Ratiopharm</td>
			<td style="width:119px;">N68542.02</td>
			<td>dilution 1:1000</td>
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			<td height="21" style="height:21px;">Pen/Strep</td>
			<td>Gibco</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FCS&#160;</td>
			<td>Gibco</td>
			<td style="width:119px;">10500-064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>Gibco</td>
			<td style="width:119px;">21875-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA&#160;</td>
			<td>Gibco</td>
			<td style="width:119px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s PBS (1x)</td>
			<td>Gibco</td>
			<td style="width:119px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shield1&#160;</td>
			<td>Clontech</td>
			<td style="width:119px;">632189</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM/F12</td>
			<td>Gibco</td>
			<td style="width:119px;">21041-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM/F12 (powder)</td>
			<td>Gibco</td>
			<td style="width:119px;">42400-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methyl cellulose</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">M0387</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Puromycin dihydrochloride</td>
			<td>applichem</td>
			<td style="width:119px;">A2856</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cell sorter&#160;</td>
			<td style="width:112px;">BD</td>
			<td style="width:119px;"></td>
			<td>Aria III cell sorter&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS analyser</td>
			<td style="width:112px;">BD</td>
			<td style="width:119px;"></td>
			<td>accuri c6 flow cytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microscope</td>
			<td style="width:112px;">Olympus&#160;</td>
			<td style="width:119px;"></td>
			<td>IX50 Osiris</td>
		</tr>
	</tbody>
</table>

evaluation of stem cell properties in human ovarian carcinoma cells using multi and single cell-based spheres assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

기존의 구체 분석에서 RFP + OVCAR-3 세포 대 RFP- 세포의 20 %의 거의 40 %는 주 분야 분석 (그림 4A)의 개별 종양 분야에 상승했다. 또한, RFP + 세포에 의해 형성 한 구체 RFP- 세포에 의해 형성되는 것보다 크기가 더 컸다. 단일 세포 기반 분석에서 도금시 RFP + 세포는 또한 상기 결과를 확인하고, 이상 세포 RFP- 분야 형성. 그러나, 다중 셀 기반 분석 (도 4A, B) ?...

Watch this Scientific Journal Video about Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays at JoVE.com

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

다중 및 단일 세포 기반 분야 분석 실험을 사용하여 인간의 난소 암 세포의 줄기 세포 특성의 평가

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

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9.5K 조회수.  University Hospital Basel. In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.