이 작품에서 잭슨 주립 대학 및 미국 부서 방어, 아이디어 상 11-1-0205의 환경 건강을 위한 RCMI 센터를 통해 건강의 국가 학회 (NIMHD-G12MD007581)에 의해 지원 되었다.

참고:이 프로토콜은 McArdle 외 에 설명 11. 시간 표시 막대를 그림 1에 제공 됩니다.
1입니다. 문화 접시의 준비
<ol><li>유리 아래쪽의 hydrophobicity 낮은 15 분 1 mL의 1 M 염 산 (HCl)와 10 m m 유리 바닥을 포함 하는 35 m m 문화 접시 세척.</li>
	<li>인산 염 버퍼 식 염 수 (PBS) 모든 HCl을 제거의 1 mL로 두 번 문화 접시 세척.</li>
	<li>70% 에탄올의 1 mL로 두 번 문화 접시 세척.</li>
	<li>셀 특정 문화 매체의 1 mL와 함께 문화 접시 린스 (여기, 사용 α-MEM 10% 열 보충 비활성화 FBS와 1% 비 본질적인 아미노산). 참고: 대우 문화 접시 문화 매체를 포함 하는 다음 날까지 37 ° C에서 가습기와 CO2 인큐베이터에 저장할 수 있습니다.</li>
	<li>35 m m 문화 접시의 잘 처리 유리 하단에 100 %confluency 세포 (MSCs 및 MCF-7 공동 문화)를 성장. 공동 문화 35000 MSC 세포와 24 h MCF7 셀 75000.</li>
</ol>2입니다. 콜라겐 젤의 준비
<ol><li>접촉 시 콜라겐의 중 합을 방지 하기 위해 냉동 실에 콜라겐 젤 pipetting 사용 micropipette 팁을 저장 합니다. 참고: 이것은 가장 중요 한 단계 이다.</li>
	<li>볼륨을 확인 사용 되 나 콜라겐의 농도에 따라 쥐 꼬리 콜라겐의. 이 분석 결과 대 한 콜라겐 젤 100 µ L를 준비 합니다. 참고: 쥐 꼬리 콜라겐 나 재고의 높은 농도 더 작동 합니다. 10 배 따라 콜라겐을 희석 하는 데 사용할 Dulbecco의 수정이 글의 중간의 볼륨을 결정 합니다.</li>
	<li>난 (3.8 µ g/mL 증권), 10 x Dulbecco의 수정 독수리의 매체 및 1 x 세포 배양 매체 2% 태아 둔감 한 혈 청 및 2.4 mg/mL의 콜라겐의 최종 농도에 적절 한 chemoattractant 보충 얼음 쥐 꼬리 콜라겐에 결합.</li>
	<li>나트륨 중 탄산염 해결책 콜라겐 젤 중화의 혼합물 4.5%를 추가 합니다. 참고: 나트륨 중 탄산염 솔루션 추가의 볼륨은 다른 시 약의 정확한 pH에 따라 달라질 수 있습니다. 이 pH 중립 해결책을 보장 하거나 산도 표시기를 사용 하 여 문화 매체에 스트립으로 혼합물을 테스트 하려면 좋습니다.</li>
	<li>바닥에 유리-잘 접시의 바닥 문화 내의 셀 콜라겐 혼합물의 80 µ L.</li>
	<li>37 ° c.에서 가습기와 CO2 배양 기에서 30 분 동안 유해를 콜라겐 젤 허용</li>
	<li>감소 된 혈 청 (2%)와 세포 매체의 2 mL와 함께 콜라겐 젤 커버 하 고 37 ° c 24, 48 또는 72 h에 대 한 가습기와 CO2 인큐베이터에서 세포를 품 어. 주의: 접시는 콜라겐 젤 유리 바닥에서 분리 하지 않도록 주의 처리 되어야 합니다.</li>
</ol>3. 콜라겐의 준비의 예 젤 3.8 µ g /mL의 쥐 꼬리 콜라겐 재고를 사용 하 여
<ol><li>얼음, 쥐 꼬리 콜라겐의 114.5 µ L, DMEM, x 10의 16.6 µ L, 문화 매체를 포함 하는 chemoattractant의 33.1 µ L를 결합 한다.</li>
	<li>나트륨 중 탄산염의 14.0 µ L로 콜라겐 혼합을 무력화.</li>
	<li>2.5 단계에서 계속 합니다.</li>
</ol>4. 셀의 이미징
<ol><li>부드럽게 요리에서 미디어를 제거 합니다.</li>
	<li>부드럽게 1 mL의 PBS로 젤 린스.</li>
	<li>15 분 동안 4 %paraformaldehyde 1 mL와 함께 셀을 수정 합니다.</li>
	<li>두 번 1 mL의 PBS로 세포 세척.</li>
	<li>DAPI 솔루션 (100 ng/mL) 실 온에서 20 분에 대 한 셀을 얼룩.</li>
	<li>Confocal 현미경을 사용 하 여 다른 위치에서 400 µ m z-스택 이미지를 16 µ m 단계에서 각 위치의 셀을 이미지. 사용 하는 현미경 confocal 영상 하얀 빛, 아크 여기 소스를 사용 하 여 수 있도록 단위 스캔 디스크가 있다. 0.75의 숫자 조리개와 활용 20 X 렌즈를 사용 합니다.</li>
	<li>이미지 reconstitute<ol>
<li>주어진된 젤 (약 25 이미지)에 대 한 이미지를 열고 이미지 스택 스택 이미지 → 스택 → 이미지를 클릭 하 여 만듭니다. 첫 번째 이미지는 젤 아래쪽에 대응 (z = 0 µ m), 두 번째 이미지는 z 방향에서 첫 번째 단계에 대응 (z = 16 µ m), 세 번째 이미지는 z 방향에서 두 번째 단계에 대응 (z = 32 µ m). 각 이미지 깊이에 각 핵을 평가 하 고 있는 핵 했다 날카로운 초점에 해당 특정 셀에 대 한 침입 깊이 깊이 지정.</li>
	</ol>
</li>
</ol>

<ol>
	<li>Friedl, P., Brocker, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+cell+locomotion+within+three-dimensional+extracellular+matric.">The biology of cell locomotion within three-dimensional extracellular matric.</a> Cell Mol Life Sci. 57, 41-64 (2000).</li><li>Bissell, M. J., Rizki, A., Mian, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+architecture:+the+ultimate+regulator+of+breast+epithelial+function.">Tissue architecture: the ultimate regulator of breast epithelial function.</a> Curr Opin Cell Biol. 15, 753-762 (2003).</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3, 362-374 (2003).</li><li>Yarrow, J. C., Perlman, Z. E., Westwood, N. J., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+cell+migration+assay+using+scratch+wound+healing,+a+comparison+of+image-based+readout+methods.">A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods.</a> BMC Biotechnol. 4, 21 (2004).</li><li>Wolf, 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=Compensation+mechanism+in+tumor+cell+migration:+mesenchymal-amoeboid+transition+after+blocking+of+pericellular+proteolysis.">Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis.</a> J Cell Biol. 160, 267-277 (2003).</li><li>Sahai, E., Marshall, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differing+modes+of+tumour+cell+invasion+have+distinct+requirements+for+Rho/ROCK+signalling+and+extracellular+proteolysis.">Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis.</a> Nat Cell Biol. 5, 711-719 (2003).</li><li>Kam, Y., Guess, C., Estrada, L., Weidow, B., Quaranta, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+circular+invasion+assay+mimics+in+vivo+invasive+behavior+of+cancer+cell+lines+and+distinguishes+single-cell+motility+in+vitro.">A novel circular invasion assay mimics in vivo invasive behavior of cancer cell lines and distinguishes single-cell motility in vitro.</a> BMC Cancer. 8, 198 (2008).</li><li>Staton, C. A., Reed, M. W., Brown, N. J. <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+analysis+of+current+in+vitro+and+in+vivo+angiogenesis+assays.">A critical analysis of current in vitro and in vivo angiogenesis assays.</a> Int J Exp Pathol. 90, 195-221 (2009).</li><li>Poujade, 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=Collective+migration+of+an+epithelial+monolayer+in+response+to+a+model+wound.">Collective migration of an epithelial monolayer in response to a model wound.</a> Proc Natl Acad Sci USA. 104, 15988-15993 (2007).</li><li>Noubissi, F. K., Harkness, T., Alexander, C. M., Ogle, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis-induced+cancer+cell+fusion:+a+mechanism+of+breast+cancer+metastasis.">Apoptosis-induced cancer cell fusion: a mechanism of breast cancer metastasis.</a> FASEB J. 29, 4036-4045 (2015).</li><li>McArdle, T. J., Ogle, B. M., Noubissi, F. 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+In+Vitro+Inverted+Vertical+Invasion+Assay+to+Avoid+Manipulation+of+Rare+or+Sensitive+Cell+Types.">An In Vitro Inverted Vertical Invasion Assay to Avoid Manipulation of Rare or Sensitive Cell Types.</a> J Cancer. 7, 2333-2340 (2016).</li><li>Noubissi, F. K., Ogle, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Cell+Fusion:+Mechanisms+Slowly+Unravel.">Cancer Cell Fusion: Mechanisms Slowly Unravel.</a> Int J Mol Sci. 17, (2016).</li><li>Meyvantsson, I., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+models+in+microfluidic+systems.">Cell culture models in microfluidic systems.</a> Annu Rev Anal Chem. 1, 423-449 (2008).</li><li>Hooper, S., Marshall, J. F., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+migration+in+three+dimensions.">Tumor cell migration in three dimensions.</a> Methods In Enzymol. 406, 625-643 (2006).</li><li>Yang, C., Tibbitt, M. W., Basta, L., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+memory+and+dosing+influence+stem+cell+fate.">Mechanical memory and dosing influence stem cell fate.</a> Nat Materials. 13, 645-652 (2014).</li></ol>

저자는 경쟁 관심 있는지 선언.

여기, 희귀 및 그들의 microenvironment에 의존 하는 셀을 포함 하 여 세포 유형의 다양 한 편리한 간단한 반전된 세로 침공 분석 결과 설명 합니다. 이 3D 침공 분석 결과에서 세포는 그들의 원래 microenvironment에 남아 하 고 콜라겐 젤 포함 하는 chemoattractant에 의해 보호 됩니다. 세포 이동 그리고 콜라겐에 침공. 이 분석 결과에서 셀 스테인드 고 쉽게 젤 내 모니터링 될 수 있습니다. 단순 하 고이 분석 결?...

세포 운동 성 세포의 정상적인 발달 및 생리 적 과정 이다. 그러나, 변화 패턴 및를 침공 하는 세포의 능력에 염증 성 질환 및 암 전이1,2,3를 포함 하 여 병 적인 상태와 연결 됩니다. 전이 암에 가장 저조한 이해 측면. 전이, 암 세포 해야 합니다 여분의 세포 매트릭스 (ECM)을 저하를 현지 조직 및 intravasate를 침략. 혈액 순환, 암 세포는 먼 사이트에 유포 됩니다, 일단 extravasate 하 고 2 차 종양을 형성 한다. 따라서, 능력 저하는 ECM 셀, 마이그레이션, 침략 하 고 관련이 그들의 전이성 잠재력. 다른 방법은 분석 하 고 세포의 이동과 침략 기능 척도 개발 되었습니다. 가장 많이 사용 되는 접근 분석 결과, 시간 경과 현미경 검사 법, 그리고 Boyden 챔버 분석 결과 치유 하는 상처를 포함 한다. 상처 치유 분석 결과4 와 시간 경과 현미경 검사 법은 간단 하 고 편리한 분석 셀 마이그레이션에 예비 통찰력을 줄 것 이라고. 그러나, 둘 다는 세포 생체 내에존재 하는 3D 환경을 반영 하지 않는 2D 분석 실험입니다. 연구 결과 세포 2D 한5,6에 비해 3D 환경에 다르게 반응으로 나타났습니다. 또한, 상처 치유 분석 실험은 재현 하 고 양적 결과7,,89를 어렵습니다. 셀 제외 영역 분석 결과, 분석 결과 치유 하는 상처의 3D 수정 이면 더 순수 관련 분석 결과 이지만 하이브리드 셀 셀 퓨전 이벤트10,11 인 등 수에 낮은 셀에 적합 하지 않습니다. .
Boyden 챔버 분석 결과 세포 연구에 대 한 관련 microenvironments를 제공 하는 3 차원 분석 결과가 이다. 그러나, 그것은 그들의 원래 microenvironment에서 제거 하 고 예금 Boyden 분석 결과 시스템 마이그레이션 및 chemoattractant 포함 매체를 향해 아래쪽으로 침략의 위 상공으로 셀 필요 합니다. 이 3D 접근 취약 그들의 microenvironment 또는 제한 번호10,,1112셀의 마이그레이션 및 침략 기능 분석에 적합 하지 않다. 셀 마이그레이션 및 침략을 분석 하는 새로운 접근은 미세 그라데이션 챔버 시험13. 적합 하 고 마이그레이션 및 침략에 신뢰할 수 있는, 비록이 분석 결과 더 비싼 이며 전형적인 실험실에 대 한 기술적으로 어려울 수 있습니다. 여기 그들의 microenvironment에 민감한 세포를 포함 한 많은 세포 종류와 호환은 숫자에 제한 된 간단한 반전된 세로 침공 분석 결과 설명 합니다. 이 분석 결과 후 퍼 그 외 여러분 에 의해 제안 된 프로토콜에서 적응 시켰다 14.이 분석 결과에서 셀 그들의 원래 microenvironment 및 그들의 이동에 남아 있고 chemoattractant11를 포함 하는 콜라겐 젤에서 위쪽으로 이동 침입 모니터링. 이 분석 결과 재현성 및 최적화 되었으며 35 m m 문화 접시에. 그것은 다른 세포 문화 크기, 다른 행렬 또는 접착, 및 다른 chemoattractants의 강도 포함 하 여 다른 시험 조건에 적응 시킬 수 있었다. 이 분석 결과 약 발견 과정에서 초기 심사를 위한 생체 외에서 플랫폼으로 될 수 있습니다.

<table border="0" cellpadding="0" cellspacing="0" style="width:880px;" width="880">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">MatTek P35G-1.5-10C</td>
			<td style="width:269px;">MatTek Corporation, Ashland, MA</td>
			<td style="width:151px;">P35G-1.5-10-C</td>
			<td style="width:232px;">mattek glass bottom dishes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat tail collagen I</td>
			<td>Corning, Corning, NY, USA</td>
			<td align="right">354236</td>
			<td>Collagen gel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydrochloric acid</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178 USA</td>
			<td>H1758</td>
			<td>HCl</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline</td>
			<td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td>
			<td align="right">10010023</td>
			<td>PBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10X Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178 USA</td>
			<td>D2429</td>
			<td>10X DMEM</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium bicarbonate</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178 USA</td>
			<td>S5761</td>
			<td>NaHCO3</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal Fluorescent microscope</td>
			<td>Olympus America, Center Valley, PA, USA</td>
			<td>Olympus IX81</td>
			<td>Confocal&#160; microscope</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Defined Fetal Bovine Serum</td>
			<td>GE Healthcare Life Sciences HyClone Laboratories, Utah, USA</td>
			<td>SH30070.03</td>
			<td>FBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ti:Sapphire multi-photon laser scanning microscope</td>
			<td>Prarie Technologies, Sioux Falls, SD, USA</td>
			<td>40x NA 0.8 objective</td>
			<td>MPLSM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaris software</td>
			<td>Bitplane Inc. Zurich, CH</td>
			<td>Imaris 7.5.2</td>
			<td>Imaris software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td style="width:269px;">Sigma-Aldrich, St. Louis, MO 63178, USA</td>
			<td>D9542</td>
			<td>DAPI</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td>
			<td align="right">50980487</td>
			<td>PFA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#945;-minimum essential medium</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178, USA</td>
			<td>M0894</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MDA-MB-231</td>
			<td>ATCC; Manassas, VA, USA</td>
			<td>HBT-22</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MSC</td>
			<td>Trivedi and Hematti, 2007</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20X lens UPLAPO</td>
			<td>Olympus America, Center Valley, PA, USA</td>
			<td>36-064</td>
			<td>Olympus UPLSAPO 20X Objective</td>
		</tr>
	</tbody>
</table>

moving upwards: a simple and flexible in vitro three-dimensional invasion assay protocol

3D 침입 분석을 개발 하 고, 그들의 microenvironment의 무결성을 영향을 주지 않고 세포를 연구 하는 도전 남아 있다. 전통적인 3D Boyden 챔버 필요한 셀은 원래 문화 위치에서 전치 하 고 새로운 환경에 이동와 같은 분석 실험. 뿐만 아니라이 microenvironment, 내부는 세포질 과정을 방해 하지만 그것은 종종 세포의 손실에 결과. 이러한 문제는 드문, 또는 그들의 microenvironment에 매우 민감한 세포와 거래 할 때 특히 도전. 여기, 우리 둘 다 문제를 방지 하는 3 차원 침공 분석 결과의 개발을 설명 합니다. 이 분석 결과에서 세포는 잘 작은 내 도금 고는 ECM 매트릭스는 chemoattractant를 포함 하는 셀 위에. 이 없는 셀 변위를 요구 하 고 행렬에 위쪽으로 침략을 셀 수 있습니다. 이 분석 결과에서 셀 침공으로 세포 형태 콜라겐 젤 내 평가 될 수 있습니다. 이 분석 결과 사용 하 여, 우리는 희귀 하 고 민감한 세포;의 침략 적 용량 특징 유방암 세포 MCF7 고 엽/multipotent 사이의 융합에서 발생 하는 하이브리드 세포 줄기/기질 세포 (MSCs).

우리 유리 아래쪽와 쥐 꼬리 콜라겐 35 m m 문화 접시 사용 개발 하 고 생체 외에서 3D 침공 최적화 나 프로토콜을 분석. 우리는 유 방 암 세포 MCF7 및 MSC 보였다이 분석 결과 사용 하 여 셀 48 h 후 각각 0.04 ± 1.8 µ m 및 41.3 ± 76.0 µ m의 평균 거리에 콜라겐 젤으로 침략 수 때 IGF1 (18.6 ng/mL) chemoattractant (그림 2)로 사용 되었다. 더 중요 한 것은, 우리는 ?...

Watch this Scientific Journal Video about Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol at JoVE.com

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

이 프로토콜 셀 셀 microenvironment를 유지 하면서 3 차원 설정의 마이그레이션 및 침입 기능을 계량 하는 데 사용할 수 있는 반전된 세로 침공 분석 결과를 설명 합니다. 이 분석 결과 희소 한 또는 민감한 세포에 적합 합니다.

위쪽으로 이동: 간단 하 고 유연한 생체 외에서 3 차원 침공 분석 실험 프로토콜

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

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JoVE Journal

Cancer Research

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

6.0K 조회수.  University of Minnesota - Twin Cities. 이 프로토콜 셀 셀 microenvironment를 유지 하면서 3 차원 설정의 마이그레이션 및 침입 기능을 계량 하는 데 사용할 수 있는 반전된 세로 침공 분석 결과를 설명 합니다. 이 분석 결과 희소 한 또는 민감한 세포에 적합 합니다.

비디오: Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

마약 검사에 대 한 높은 처리량 3 차원 종양 Spheroids의 생성

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

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Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

실시간 탐지의 체 외 종양 세포 Apoptosis에 CD8에 의해 유도 된+ T 세포 종양 침투 골수성 세포의 면역 진압 기능 연구

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

자동된 라이브 셀 이미징 사용 하 여 간단한 마이그레이션/침공 워크플로

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

다리에 대 한 3 차원 뼈 세포 외 매트릭스 모델

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

3 차원 세 포스 페 로이드 침입 분석에서 완전 한 인간 종양 기반 매트릭스

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

종양 대식 세포 상호 작용을 연구 하는 시험관 내 분석

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

3D 스페로이드 모델은 시험관 내 암 관련 섬유아세포 분화 및 침입을 위한 보다 생리적 시스템으로

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

환자 전립선암 뼈 전이 표본 및 이종이식에서 파생된 3차원(3D) 오르가노이드의 설립 및 분석

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

대장암 줄기세포를 연구하는 스페로이드의 3차원 모델

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

콜라겐 기반 스캐폴드를 사용한 신경모세포종의 3차원 체외 생체모방 모델

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...