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

This work was supported by Hong Kong Research Grant Council (grants 17122014, C1013-15G, 719813E, and 17304514). A. S. T. Wong is a recipient of the Croucher Senior Research Fellowship.

1. Microfluidic Device Design and Fabrication
<ol>
	<li>Microfluidic master design
	<ol>
		<li>Design and draw the microfluidic channel pattern with any computer-aided design (CAD) software. 
		NOTE: Normally, the CAD drawing can be sent to a photomask company to produce the photomask. The microfluidic design consists of three identical parallel channels, each with the following dimensions: 4 mm &#215; 25 mm &#215; 250 &#181;m (width &#215; length &#215; height) and set 2 mm apart. Both channel ends were designed ...

<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>

This assay offers a flexible and physiologically relevant model that can be incorporated with various biochemical and cell-based assays, including, but not limited to, adhesion assays, mesothelial clearance assays, and drug screening. It can be applied to the evaluation of the effect of the intraperitoneal microenvironment on cancer progression. However, several experimental conditions might need to be optimized, depending on the aims of the project (e.g., the number of HPMCs and cancer spheroids seeded per chan...

Ovarian cancer is the most lethal gynecological cancer and is characterized by widespread peritoneal dissemination and the formation of malignant ascites1. This extensive peritoneal metastasis represents a major clinical challenge and is associated with poor clinical outcomes. Unlike most solid carcinomas that metastasize via the blood, ovarian cancer primarily disseminates within the peritoneal cavity. Tumor cells exist as multicellular aggregates/spheroids during the process of metastasis2. The fact that suspension culture can enrich ovarian cancer stem/tumor-initiating cells further suggests that these spheroids may be associated with both tumor aggressiveness and enhanced chemoresistance3,4. There are differences in drug response between 2D and 3D cultures, which presumably have different molecular mechanisms5.
The essential interaction with the mesothelium constructs the primary microenvironment for ovarian tumor progression. These mesothelial cells lie on an extracellular matrix (ECM), where fibronectin is a ubiquitous constituent. A link between the increased expression of mesothelial cell-derived fibronectin and tumor progression has been shown. Fibronectin is abundantly present in malignant ascites6,7. Ovarian cancer cells are also able to induce the secretion of fibronectin from mesothelial cells in order to promote early ovarian cancer metastasis8.
Emerging evidence shows that mechanical stimuli, including shear stress, can modulate cell morphology, gene expression, and, thus, the phenotypes of tumor cells9,10,11. As malignant ascites develop and accumulate during tumor progression, ovarian tumor cells are exposed to fluid flow and the resulting shear stress. A number of groups, ours included, have shown the impact of shear stress on ovarian cancer progression, including cytoskeleton modifications, epithelial-to-mesenchymal transitions, and cancer stemness12,13,14,15. Thus, a physiologically relevant microenvironment is important for the investigation of tumor peritoneal metastasis. However, current in vitro hydrodynamic culture systems have limitations on mimicking and controlling a constant, low, physiologically relevant shear stress16,17,18,19. Conventional in vitro approaches focusing on either the cellular or mechanical environment are still limited in mimicking the complexity of the intraperitoneal microenvironment with proper physiological relevance.
Here, in order to engineer a new model of the peritoneum to overcome the limitations of conventional strategies and to advance the study of the intraperitoneal compartment in cancer metastasis, a 3D microfluidic-based platform with controlled fluid flow was designed. In this model, ovarian cancer spheroids were co-cultured with primary human peritoneal mesothelial cells in microfluidic chips under continuous fluidic flow (Figure1A). The mesothelial cells were plated on fibronectin. Non-adherent ovarian cancer spheroids were seeded into microfluidic channels with continuous flow medium perfused by a syringe pump. Both the 3D environment and the dynamic mechanical forces are very important factors of the metastatic cascade. This platform can be used for investigating the intraperitoneal microenvironment in terms of complex cellular and co-culture interactions, as well as with regard to dynamic mechanical cues.

<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

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with poor clinical outcomes and currently lacks effective treatment. Both the three-dimensional (3D) environment and the dynamic mechanical forces are very important factors in this metastatic cascade. However, traditional cell cultures fail to recapitulate this natural tumor microenvironment. Thus, in vivo-like models that can emulate the intraperitoneal environment are of obvious importance. In this study, a new microfluidic platform of the peritoneum was set up to mimic the situation of ovarian cancer spheroids in the peritoneal cavity during metastasis. Ovarian cancer spheroids generated under a non-adherent condition were cultured in microfluidic channels coated with peritoneal mesothelial cells subjected to physiologically relevant shear stress. In summary, this dynamic 3D ovarian cancer-mesothelium microfluidic platform can provide new knowledge on basic cancer biology and serve as a platform for potential drug screening and development.

Using this protocol, a microfluidic platform was set up to model ovarian cancer spheroids with mesothelial cells under hydrodynamic conditions. Primary human peritoneal mesothelial cells were cultured in the microdevice for 16 h and observed under a bright-field microscope. As shown in Figure 2A, the channel bottom was successfully covered with a monolayer of HPMCs. It is important to note that bubble formation during fibronectin or HPMC patterning will lead to channel co...

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

To study ovarian tumor progression in a physiologically relevant model, multicellular spheroids were cultured in a microdevice under simulated fluid flow. This dynamic 3D model emulates the intraperitoneal environment with the cellular and mechanical components where ovarian cancer metastasis occurs.

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

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

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

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

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

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

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

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

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

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

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