Name: modeling ovarian cancer multicellular spheroid behavior in a dynamic 3d peritoneal microdevice
Uploaded: 2017-02-18T15:00:00
Description: Watch this Scientific Journal Video about Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice at JoVE.com

이 작품은 홍콩 연구 보조금위원회에 의해 지원되었다 (보조금 17,122,014, C1013-15G, 719813E 및 17,304,514). AST 웡은 Croucher 수석 연구 활동의받는 사람입니다.

1. 미세 유체 장치 설계 및 제작 <ol><li> 미세 유체 마스터 디자인 <ol><li> 디자인은 컴퓨터 지원 설계 (CAD) 소프트웨어를 사용하여 미세 유체 채널 패턴을 그린다. 주 : 일반적으로, CAD 도면은 포토 마스크를 제조하는 마스크 회사에 전송 될 수있다. 미세 유체 설계는 3 개의 동일한 병렬 채널, 다음과 같은 크기와 각 구성 : 4 mm μm의 250 × mm 25 × (폭 × 높이 × 길이)와 떨어져 2mm를 설정합니다. 두 ...

<ol>
	<li>Jemal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+cancer+statistics.">Global cancer statistics.</a> CA: Cancer J. Clin. 61, 69-90 (2011).</li><li>Burleson, K. 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=Ovarian+carcinoma+ascites+spheroids+adhere+to+extracellular+matrix+components+and+mesothelial+cell+monolayers.">Ovarian carcinoma ascites spheroids adhere to extracellular matrix components and mesothelial cell monolayers.</a> Gynecol. Oncol. 93, 170-181 (2004).</li><li>Chau, W. K., Ip, C. K., Mak, A. S., Lai, H. C., Wong, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-Kit+mediates+chemoresistance+and+tumor-initiating+capacity+of+ovarian+cancer+cells+through+activation+of+Wnt/beta-catenin-ATP-binding+cassette+G2+signaling.">c-Kit mediates chemoresistance and tumor-initiating capacity of ovarian cancer cells through activation of Wnt/beta-catenin-ATP-binding cassette G2 signaling.</a> Oncogene. 32, 2767-2781 (2013).</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, 4311-4320 (2008).</li><li>Tang, M. K. S., Zhou, H. Y., Yam, J. W. P., Wong, A. S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-Met+overexpression+contributes+to+the+acquired+apoptotic+resistance+of+nonadherent+ovarian+cancer+cells+through+a+cross+talk+mediated+by+phosphatidylinositol+3-kinase+and+extracellular+signal-regulated+kinase+1/2.">c-Met overexpression contributes to the acquired apoptotic resistance of nonadherent ovarian cancer cells through a cross talk mediated by phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1/2.</a> Neoplasia. 12, 128-144 (2010).</li><li>Ksiazek, 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=Senescent+peritoneal+mesothelial+cells+promote+ovarian+cancer+cell+adhesion:+the+role+of+oxidative+stress-induced+fibronectin.">Senescent peritoneal mesothelial cells promote ovarian cancer cell adhesion: the role of oxidative stress-induced fibronectin.</a> Am. J. Pathol. 174, 1230-1240 (2009).</li><li>Hafter, R., Klaubert, W., Gollwitzer, R., Vonhugo, R., Graeff, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosslinked+Fibrin+Derivatives+and+Fibronectin+in+Ascitic+Fluid+from+Patients+with+Ovarian-Cancer+Compared+to+Ascitic+Fluid+in+Liver-Cirrhosis.">Crosslinked Fibrin Derivatives and Fibronectin in Ascitic Fluid from Patients with Ovarian-Cancer Compared to Ascitic Fluid in Liver-Cirrhosis.</a> Thromb Res. 35, 53-64 (1984).</li><li>Kenny, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesothelial+cells+promote+early+ovarian+cancer+metastasis+through+fibronectin+secretion.">Mesothelial cells promote early ovarian cancer metastasis through fibronectin secretion.</a> J. Clin. Invest. 124, 4614-4628 (2014).</li><li>Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+tumor+vasculature:+An+emerging+concept+in+antiangiogenic+therapy.">Normalization of tumor vasculature: An emerging concept in antiangiogenic therapy.</a> Science. 307, 58-62 (2005).</li><li>Chang, S. 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=Tumor+cell+cycle+arrest+induced+by+shear+stress:+Roles+of+integrins+and+Smad.">Tumor cell cycle arrest induced by shear stress: Roles of integrins and Smad.</a> Proc. Natl. Acad. Sci. USA. 105, 3927-3932 (2008).</li><li>Rutkowski, J. M., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+driving+force+for+change:+interstitial+flow+as+a+morphoregulator.">A driving force for change: interstitial flow as a morphoregulator.</a> Trends Cell Biol. 17, 44-50 (2007).</li><li>Rizvi, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+induces+epithelial-mesenchymal+transition,+cellular+heterogeneity+and+biomarker+modulation+in+3D+ovarian+cancer+nodules.">Flow induces epithelial-mesenchymal transition, cellular heterogeneity and biomarker modulation in 3D ovarian cancer nodules.</a> Proc. Natl. Acad. Sci. USA. 110, E1974-E1983 (2013).</li><li>Ip, C. 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=Stemness+and+chemoresistance+in+epithelial+ovarian+carcinoma+cells+under+shear+stress.">Stemness and chemoresistance in epithelial ovarian carcinoma cells under shear stress.</a> Sci. Rep. 6, 26788 (2016).</li><li>Avraham-Chakim, 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=Fluid-flow+induced+wall+shear+stress+and+epithelial+ovarian+cancer+peritoneal+spreading.">Fluid-flow induced wall shear stress and epithelial ovarian cancer peritoneal spreading.</a> PloS one. 8, e60965 (2013).</li><li>Burkhalter, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peritoneal+mechanobiology+and+metastatic+success+in+epithelial+ovarian+cancer.">Peritoneal mechanobiology and metastatic success in epithelial ovarian cancer.</a> Faseb Journal. 26, (2012).</li><li>Lane, W. O., 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=Parallel-plate+flow+chamber+and+continuous+flow+circuit+to+evaluate+endothelial+progenitor+cells+under+laminar+flow+shear+stress.">Parallel-plate flow chamber and continuous flow circuit to evaluate endothelial progenitor cells under laminar flow shear stress.</a> Journal of visualized experiments : JoVE. , (2012).</li><li>Botta, G. P., Manley, P., Miller, S., Lelkes, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+assessment+of+three-dimensional+cell+aggregation+in+rotating+wall+vessel+bioreactors+in+vitro.">Real-time assessment of three-dimensional cell aggregation in rotating wall vessel bioreactors in vitro.</a> Nat. Protoc. 1, 2116-2127 (2006).</li><li>Ismadi, M. Z., 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=Flow+characterization+of+a+spinner+flask+for+induced+pluripotent+stem+cell+culture+application.">Flow characterization of a spinner flask for induced pluripotent stem cell culture application.</a> PloS one. 9, e106493 (2014).</li><li>Yu, W., 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+microfluidic-based+multi-shear+device+for+investigating+the+effects+of+low+fluid-induced+stresses+on+osteoblasts.">A microfluidic-based multi-shear device for investigating the effects of low fluid-induced stresses on osteoblasts.</a> PloS one. 9. 9, e89966 (2014).</li></ol>

이 분석은 접착 분석, 중피 통관 분석, 약물 검사, 제한 등의 다양한 생화학 및 세포 기반 분석과 통합,하지만 할 수있는 유연하고 생리 학적으로 관련 모델을 제공합니다. 이것은 암 진행에 복강 내 미세 환경의 영향의 평가에 적용 할 수있다. 그러나, 여러 실험 조건은 프로젝트의 목적에 따라 최적화 할 수있다 (예를 들어, 채널, 공동 배양 시간 등 당 시드 HPMCs 암 타원체의 수). 이러...

<html> 난소 암은 부인과에서 가장 치명적인 암이며 널리 보급 복막 악성 복수 형성 한 것을 특징으로한다. 이 광범위한 복막 전이 주요 임상 도전을 나타내고 빈약 한 임상 결과와 연결되어 있습니다. 혈액을 통해 전이 가장 단단한 암과는 달리, 난소 암은 주로 복강 내에서 발신. 종양 세포는 전이 (2)의 과정에서와 같은 다세포 응집체 / 타원체 존재한다. 현탁 배양은 줄기 난소 암 / 종양 세포를 풍부하게 개시 할 수 있다는 사실이 상기 스페 로이드는 모두 종양의 공격성과 연관 chemoresistance 3,4- 향상 될 수 있음을 시사한다. 아마도 다른 분자 메커니즘 5가 2D 및 3D 문화 사이의 약물 반응에 차이가 있습니다. _content는 &quot;&gt;는 mesothelium에 필수적인 상호 작용은 난소 종양 진행에 대한 기본 미세 환경을 구성한다.이 중피 세포를 피브로넥틴은 유비쿼터스 성분 인 세포 외 기질 (ECM)에 거짓말. 증가 중피 세포 유래 피브로넥틴의 표현 사이의 링크를 종양의 진행을 도시하고있다. 피브로넥틴 악성 복수 (6, 7)에 풍부하게 존재한다. 난소 암 세포는 또한 초기 난소 암 전이 (8)을 촉진하기 위해 중피 세포에서 파이브로 넥틴의 분비를 유도 할 수있다. 증거 신흥 전단 응력을 포함한 기계적 자극, 세포 형태, 유전자 발현을 조절하고, 따라서, 종양 세포를도 9,도 10,도 11의 표현형 수 있음을 보여준다. 악성 복수로 개발 및 t 동안 축적umor 진행성 난소 종양 세포는 유체 유동하고, 생성 된 전단 응력에 노출된다. 그룹의 수는 우리가 포함 골격 변형, 상피 간 간엽 전환 및 암 stemness 12, 13, 14, 15을 포함하여, 난소 암의 진행에 대한 전단 응력의 영향을 보여준다. 따라서, 생리 학적으로 관련 미세 종양 복막 전이의 조사를 위해 중요하다. 그러나, 현재의 체외 배양 유체 시스템은 일정한 낮은 생리 학적 관련성 전단 응력 16, 17, 18, 19를 모방하고 제어하는 방법에 제한이있다. 체외에서 기존 여전히 제한되어 세포 또는 기계적 환경 중 하나에 초점을 맞추고 접근적절한 생리 학적 관련성과 복강 내 미세 환경의 복잡성을 흉내 낸. 여기서, 종래의 전략의 한계를 극복하고, 암전의 복강 구획 연구를 진행하는 복막의 새로운 모델을 설계하기 위해서는, 제어 된 유체 유동을 가진 3 차원 미세 기반 플랫폼을 설계 하였다. 이 모델에서, 난소 암 타원체 연속 유체 흐름 (도 1a) 아래에 미세 유체 칩 차 인간 복막 중피 세포와 공동 배양 하였다. 중피 세포를 피브로넥틴에 도금했다. 비 - 부착 성 난소 암 타원체는 시린지 펌프에 의해 관류 연속 유동 매질과 미세 유체 채널로 접종 하였다. 3 차원 환경과 동적 기계적 힘 모두 전이성 캐스케이드 매우 중요한 요인이다. 이 플랫폼은 복잡한 CEL의 관점에서 복강 미세 조사를 위해 사용될 수있다뿐만 아니라 동적 기계적 큐에 관해서적인 다공질 공동 배양 상호 작용.

<table>
	<tbody>
		<tr>
			<td>Silicon wafer</td>
			<td>University wafer</td>
			<td>#1196</td>
			<td>100mm</td>
		</tr>
		<tr>
			<td>SU-8 2075 photoresist&#160;</td>
			<td>Microchem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 developer&#160;</td>
			<td>Microchem</td>
			<td>108-65-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane</td>
			<td>Sigma</td>
			<td>448931</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184</td>
			<td>Dow Corning</td>
			<td>1673921</td>
			<td>Polydimethylsiloxane (PDMS) + curing agent kit</td>
		</tr>
		<tr>
			<td>Biopsy punch&#160;</td>
			<td>Miltex</td>
			<td>33-31AA</td>
			<td>1 mm diameter</td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>SCI</td>
			<td>BB31695-PE/5</td>
			<td>0.86mm (inner diameter)</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Terumo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Longer precision pump&#160;</td>
			<td>&#160;LSP01-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Invitrogen</td>
			<td>31100-035</td>
			<td>Add 2.2g/L sodium bicarbonate</td>
		</tr>
		<tr>
			<td>MCDB 105 Medium</td>
			<td>Sigma</td>
			<td>M6395</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30068.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin&#160;</td>
			<td>Invitrogen</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA solution&#160;</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>0.05% Trypsin -0.01% EDTA, phenol red</td>
		</tr>
		<tr>
			<td>Fibronectin human</td>
			<td>BD</td>
			<td>354008</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>Invitrogen</td>
			<td>15510-027</td>
			<td></td>
		</tr>
		<tr>
			<td>5-chloromethylfluorescein diacetate</td>
			<td>Life technologies</td>
			<td>C7025</td>
			<td>Green CMFDA</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>SANYO</td>
			<td>MCO-18AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Hitachi</td>
			<td>CT15RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Model: 80i or ECLIPSE Ti; software: SPOT</td>
		</tr>
		<tr>
			<td>SKOV-3&#160;</td>
			<td></td>
			<td></td>
			<td>Gift from Dr. N Auersperg (University of British Columbia)</td>
		</tr>
	</tbody>
</table>

modeling ovarian cancer multicellular spheroid behavior in a dynamic 3d peritoneal microdevice

난소 암은 일반적으로 악성 복수에서 발견 된 종양 분야와 광범위한 복막 전이 특징으로한다. 이것은 가난한 임상 결과와 관련, 현재 효과적인 치료가 부족합니다. 모두 3 차원 환경과 동적 기계적 힘이 전이성 캐스케이드에서 매우 중요한 요소이다. 그러나, 기존의 세포 배양이 자연 종양 미세 환경 요점을 되풀이하지 못한다. 따라서, 복강 환경을 에뮬레이션 할 수 있습니다 생체 -like 모델은 분명 중요하다. 이 연구에서, 복막의 새로운 미세 전이 플랫폼 동안 복강 난소 암 타원체의 상황을 모방하기 위해 설립되었다. 비 - 부착 성 조건하에 생성 된 난소 암 타원체는 생리 학적으로 관련이 전단 응력을 실시 복막 중피 세포로 코팅 된 미세 채널에서 배양 하였다. 요약하면,이 역동적 인 3D 난소 암 mesothelium 마이크rofluidic 플랫폼은 기본 암 생물학에 새로운 지식을 제공하고 잠재적 인 약물 검사 및 개발을위한 플랫폼 역할을 할 수 있습니다.

이 프로토콜을 이용하여 미세 유체 플랫폼은 유체 역학적 조건 중피 세포 난소 암 타원체를 모델링하기 위해 설정되었다. 차 인간 복막 중피 세포는 16 시간을위한 마이크로 디바이스에서 배양하고, 명 시야 현미경으로 관찰 하였다. 도 2a에 도시 된 바와 같이, 채널 하부 성공적 HPMCs의 단층으로 덮었다. 이는 채널 코팅 실패로 이어질 피브로넥틴 또는 HPMC 패터...

Watch this Scientific Journal Video about Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice at JoVE.com

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

생리 관련 모델에서 난소 종양의 진행을 연구하기 위해, 다세포 회전 타원체는 시뮬레이션 된 유체의 흐름에 따라 마이크로 디바이스에서 배양 하였다. 이 동적 3D 모델은 난소 암 전이가 발생 셀룰러 및 기계 부품과 복강 환경을 에뮬레이트합니다.

동적 3D 복막의 마이크로 디바이스의 모델링 난소 암 다세포 회전 타원체 동작

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

The overall goal of this procedure is to create a dynamic microfluidic-based co-culture platform to emulate the intraperitoneal environment with the cellular and mechanical components where ovarian cancer metastasis occurs. By using this micro for this platform, it helps to answer the key questions in the cancer biology field, such as the microenvironment of peritoneal dissemination. The main advantage of this technique is that a 3D co-culture under precise-controlled free-flow better mimics the in vivo communication between ovarian cancer cells and methothedia cells that occurs in a peritoneal cavity.After designing and drawing the microfluidic channel pattern and having it produced according to the text protocol, spin coat a layer of SU82075 Photoresist onto the silicone wafer with a spinning speed of 1800 RPM. Bake the Photoresist-coated wafer directly onto a hot plate at 65 degrees Celsius for five minutes. And then at 95 degrees Celsius for 25 minutes to remove excess solvent.Spin coat a second layer of SU82075 Photoresist onto the wafer for a final thickness of 250 micrometers. Place the Photoresist-coated wafer directly on a hot plate at 65 degrees Celsius for seven minutes and then at 95 degrees Celsius for 30 minutes before slowly cooling the wafer to room temperature. Next, align and put the photo mask directly on top of the wafer.Then expose it to UV light for 20 seconds to cross length the pattern. On a hot plate, bake the coated wafer at 65 degrees Celsius for five minutes and 95 degrees Celsius for 12 minutes to remove the solvent for prelimerization. Remove the noncross-lengthed Photoresist by immersing the wafer in an SU8 developer for 25 minutes.Then with isopropanal, rinse the wafer and use pressurized air to dry it. Combine the Polydimethylsiloxane or PDMS base and curing agent at a 10-to-one mass ratio and use the centrifugal mixer under the mixing and degas function to mix the solution. Slowly pour the PDMS mixture onto the wafer to a height of approximately five millimeters.Degas the PDMS in a vacuum desiccator for 40 minutes. Then cure the PDMS in the oven at 65 degrees Celsius for two hours. Carefully peel the PDMS slab off the master and trim the PDMS along the channels until it is the size of a glass slide.Then with a one-millimeter biopsy punch, punch the PDMS to create the inlets and outlets of the device. Use pressurized air to clean the PDMS surface and then with adhesive tape, remove the dust and PDMS debris. Now place the PDMS slab with the channel side facing upwards into a plasma cleaner.Then position a clean glass slide into the plasma cleaner alongside the PDMS slab. Treat the PDMS and glass slide under air plasma at a high-radio frequency or RF level for one minute. Then after removing the samples from the plasma cleaner, bind the PDMS to the glass slide to create an irreversible covalent bond.Place the PDMS chip on a hot plate at 150 degrees Celsius for one hour to enhance the bonding strength and to return the hydrophobicity of the PDMS before use. To coat microfluidic channels with fibronectin, begin by using 30 micro-liters of 75%ethanol to sterilize the channels. Remove the ethanol and use PBS to rinse the channels twice.Then thoroughly aspirate the PBS from the channels. Prepare 10 micrograms per milliliter of fibronectin solution in serum-free M199MCDB105 medium with 30 microliters of the solution, slowly fill up the channels and use tape to seal them. Then incubate the chip at four degrees Celsius overnight.To seed primary peritoneal mesothelia cells or HPMCs into the microfluidic channels, after rinsing cultured cells according to the text protocol, use two milliliters of 10%FBS culture medium to re-suspend the HPMCs. Using a hemocytometer, count the cells and adjust the cell concentration to 3.5 times 10 to the sixth cells per milliliter. Warm the fibronectin-coated microfluidic chip in a 37 degrees Celsius incubator for five minutes before use.Then completely aspirate the fibronectin solution and slowly pipet 30 microliters of HPMC suspension into each channel. Use tape to seal the channels and place the device in the CO2 incubator overnight. To induce ovarian cancer spheroid formation, dissolve 0.5%agarose in sterilized water.Dispense 50 microliters of the agarose solution into the wells of 96 well plates. After counting previously-cultured human epithelial cancer cells, re-suspend the cells in 5%FBS culture medium at a density of 5, 000 cells per milliliter. Transfer 200 microliters of cell suspension into each agarose-coated well.And culture the cells at 37 degrees Celsius and five percent CO2 for 48 hours. Transfer the cancer spheroids to a 50-milliliter centrifuge tube prewetted with one milliliter of serum-free medium and spin down the spheroids at 750 times G for five minutes. Following the preparation of 2.5 micrograms per milliliter of CMFDA solution in serum-free M199MCDB105 medium.Re-suspend the cancer spheroids in one milliliter of the solution and incubate the spheroids at 37 degrees Celsius for 30 minutes. Then count the spheroid number and use serum-free medium to adjust the concentration to 2, 000 spheroids per milliliter. Prior to spheroid loading, slowly inject 100 microliters of serum-free M199MCDB105 medium to wash away the non-adhered mesothelio cells inside the channels.Load 30 microliters of fluorescent-labeled spheroid suspension into each channel. Then place the microfluidic chip into the CO2 incubator. Assemble a profusion platform by attaching a syringe loaded with fresh serum-free M199MCDB105 medium to a one-meter long tube.Place the syringe on the syringe pump and secure the plunger and the body of the syringe. Then install the syringe pump in a horizontal position at the same height as the microfluidic chip inside the incubator. Quickly infuse the whole tube with medium by pressing and holding the fast forward button until the medium overflows in the tubing.Then run the injection at the desired flow rate. Connect the tubing to the inlet of the channel according to the text protocol. Then use a short-output tube to connect the outlet of the channel to a 50-milliliter conical tube to collect the outflow medium.Finally, use a bright field or fluorescence microscope to observe the HPMCs and cancer spheroids. Using the microfluidic platform described in this video, ovarian cancer spheroids were modeled with mesothelial cells under hydrodynamic conditions. As shown in this figure, mesothelial cells that were cultured in the microdevice for 16 hours successfully formed a monolayer of HPMCs.In this experiment, multi-cellular spheroids with diameters of approximately 100 micrometers which are similar in size to those found in patient societies were labeled with a green fluorescent dye CMFDA. The spheroids were then transferred to HPMC-coated microfluidic channels and remained in suspension and the cell morphologies were captured. Once mastered, this technique can be done in several hours over three days if it is performed properly.One in 10 in this procedure, it is important to remember to avoid introducing air bubbles into the microfluidic channels. Following this procedure, other methods, like drug treatment, adhesion assays, mesothelia-clearance assays can be performed to answer additional questions, like the effect of the introperitoneal microenvironment on cancer progression. After its development, this technique paved a way for researchers in the field of cancer biology to explore into peritoneal mestastasis in humans.After watching this video, you should have a good understanding of how to generate a peritoneal microdevice with fluidic flow to starting ovarian cancer behavior in peritoneal cavity. Don't forget that working with organic sulfurs such as acetone, as a developer and cyline can be extremely hazardous. MP cautions such as wearing gloves and a lab coat should always be taken when you first perform this procedure.All of the procedures dealing with this office should be performed under extreme cover.

Research

JoVE Journal

Bioengineering

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

Proteolytically 분해 3 차원 시스템에서 난소 대과의 캡슐화 및 문화에 대한 방법

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

연구

생체공학

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

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Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

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3D 상호 작용의 연구에 대한 3D 콜라겐 젤과 마이크로 채널 준비<em&gt; 생체</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

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The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

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Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

신경 임 플 란 트의 연 화 행동을 예측 하기 위해 환경 동적 기계적인 분석

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

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Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

미생물 행동을 연구하기 위한 제어된 동적 화학 적 경관 생성

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...