1. Assay Set-up
<ol>
	<li>Thaw a small aliquot (&lt;500 &mu;l) of Matrigel on ice at 4 &deg;C (approximately 2 hr).</li>
	<li>Prepare gel recipe (see example volumes in table below): 10x PBS (1x in total volume), 1N sodium hydroxide (equivalent to 0.023 volumes of added collagen, or per the collagen manufacturer's recommendations, as appropriate), Matrigel and collagen type I to final concentrations of 1 mg/ml and 1.3 mg/ml respectively (other matrix formulations may be used depending on cell type and experiment).</li>
</ol>
Example gel recipe
<table border="1">
	<tbody>
		<tr>
			<td>Components</td>
			<td>Stock Concentration</td>
			<td>Final Concentration</td>
			<td>Add volume</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>10X</td>
			<td>1X</td>
			<td>0.090 ml</td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>0.346 ml</td>
		</tr>
		<tr>
			<td>1N NaOH</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>0.008 ml</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>9.90 mg/ml</td>
			<td>1 mg/ml</td>
			<td>0.101 ml</td>
		</tr>
		<tr>
			<td>Rat tail type I collagen</td>
			<td>3.66 mg/ml</td>
			<td>1.3 mg/ml</td>
			<td>0.355 ml</td>
		</tr>
	</tbody>
</table>
<ol start="3">
	<li>Mix components of the gel on ice in the same sequence as above and incubate final solution for 1 hr at 4 &deg;C. In our experience, a 1 hr incubation prior to cell seeding results in a more uniform gelation of the collagen. Make sure to keep Matrigel and collagen on ice at all times and to work as fast as possible to prevent gelation.</li>
	<li>Place 12 mm diameter 8 &mu;m pore cell culture inserts into a 12-well plate using sterilized forceps.</li>
	<li>Count cells and resuspend in serum-free media at 5 x 106 cells/ml (total volume should be 10% of the gel solution).</li>
	<li>Add 100 &mu;l of cell suspension to 900 &mu;l of gel solution (final cell concentration 5 x 105 cells/ml) and mix thoroughly by pipetting gently up and down.</li>
	<li>Add 150 &mu;l of the final mixture to each insert and transfer to a 37 &deg;C, 5% CO2 incubator for 30 minutes until gel polymerizes.</li>
	<li>Using serum-free media,</li>
</ol>
<ul>
	<li>Add 100 &mu;l on top of the gel and 1200 &mu;l under the insert for the STATIC condition. The fluid levels inside the insert and outside in the well should be approximately equal, resulting in minimal pressure difference across the gel and no interstitial flow.</li>
	<li>Add 100 &mu;l under the insert and 650 &mu;l above gel for the FLOW condition. Be careful to avoid any air bubbles beneath the insert, as they will prevent cells from migrating through the membrane at these locations. The pressure difference generated by these volumes is approximately 1.3 cm H2O (or 1 mm Hg).</li>
</ul>
<ol start="9">
	<li>Place plate in a 37 &deg;C, 5% CO2 incubator for 24 hr.</li>
</ol>
2. Cell Staining and Counting
<ol>
	<li>Add 500 &mu;l of 1X PBS per well into new 24-well plate; this will be used to wash the inserts.</li>
	<li>Remove the medium remaining in the upper portion of the flow transwells and determine the volume. Calculate total eluted volume by subtracting the remaining volume from the total volume originally added, 650 &mu;l (this will be used for flow rate calculations, see below). Use cotton swabs to remove the gel from the inserts and to wipe the top surface of the membrane to remove non-invaded cells. Place the inserts into the 24-well plate containing 1X PBS for 15 s to wash inserts.</li>
	<li>Remove PBS and add 500 &mu;l of 4% paraformaldehyde (PFA) underneath each insert and incubate for 30 minutes at room temperature to fix the transmigrated cells.</li>
	<li>Remove the PFA and rinse once with 500 &mu;l 1X PBS to remove residual fixative.</li>
	<li>Add 500 &mu;l of 0.5% Triton X-100 solution beneath the inserts and incubate for 10 minutes at room temperature to permeabilize the cells.</li>
	<li>Cut membranes out of the insert using a razor blade and place into 100 &mu;l of 2 &mu;g/ml DAPI in 1X PBS solution, being careful to place the underside of the membrane face down (transmigrated cells face down).</li>
	<li>Wrap plate in aluminum foil and place on a shaker at 150 rpm for 30 minutes at room temperature.</li>
	<li>Wash membranes in 500 &mu;l 1X PBS on shaker (repeat 3 times for 10 minutes each) to remove free DAPI.</li>
	<li>Place membranes on glass slides with transmigrated cells facing up, add mounting solution and cover slip.</li>
</ol>
3. Data Analysis
<ol>
	<li>Count DAPI-stained nuclei on 5 randomly selected locations of each membrane (stay away from the edges and use a 10X or 20X objective lens).</li>
	<li>Calculate the average cell count and obtain the percent invasion by using the following formula: 
	Percent invasion = 100% x (average cell count x membrane surface area) / (surface area of the microscope image x number of cells seeded) 
	The percent invasion can be normalized to some control condition (typically the static condition) to allow for comparison between independent experiments.</li>
	<li>Calculate the average flow rate: divide the total eluted volume in the flow condition by the incubation time (e.g., 24 hr).</li>
	<li>Calculate the average flow velocity: divide the average flow rate by the cross-sectional area of the gel/membrane (in this case 60 mm2).</li>
</ol>
4. Representative Results
To measure tumor cell invasion under interstitial flow, we performed our 3-D flow invasion assay using MDA-MB-435S metastatic melanoma cells. These cells have previously been shown to invade in response to interstitial fluid flow 13-14. The cells were embedded in a matrix composed of 1.3 mg/ml rat tail tendon collagen type I and 1 mg/ml Matrigel basement membrane matrix at a final cell concentration of 5 x 105 cells/ml. Two different conditions were compared: (1) average interstitial flow = 0.1 &mu;m s-1 and (2) static condition = no measurable flow rate (Figure 1).
After 24 hr, the cells that invaded through the pores of the membrane were stained with DAPI to facilitate cell counting. Figure 2 shows a representative image of the invaded cells. Under brightfield, only the pores of the membrane are visible. Using fluorescence, the DAPI-stained nuclei were used for cell counting and the phalloidin stained F-actin structures were used to visualize the cell body (optional). Using a Student's t-test assuming equal variances, we showed that interstitial flow significantly increases MDA-MB-435S cell invasion by 2.3-fold over static conditions (p = 0.003) (Figure 3). This corroborates similar findings (but with different matrix conditions and therefore flow velocities) using this cell line 13-14.
<img alt="Figure 1" src="/files/ftp_upload/4159/4159fig1.jpg" /> 
Figure 1. Schematic of the 3-D interstitial fluid flow invasion assay. First prepare gel solution using appropriate concentrations and volumes. Then add cells to gel solution and transfer to cell culture inserts. Finally add appropriate volume of media to each condition and incubate. Interstitial fluid flow is driven by a fluid pressure head.
<img alt="Figure 2" src="/files/ftp_upload/4159/4159fig2.jpg" /> 
Figure 2. Transmigrated MDA-MB-435S cells on membrane. Invaded cells were fixed after our interstitial fluid flow invasion assay and stained with DAPI and Alexa Fluor 488-conjugated phalloidin to facilitate counting of invaded cells; A) Picture of the membrane under bright field; B) DAPI stained nuclei (in blue); C) Alexa Fluor 488-phalloidin stained F-actin (in green). Scale bar represents 50 &mu;m.
<img alt="Figure 3" src="/files/ftp_upload/4159/4159fig3.jpg" /> 
Figure 3. Increased invasion of MDA-MB-435S cells under flow. Cell invasion after 24 hr significantly enhanced by interstitial flow (p = 0.003). The results are normalized to the average static condition and values represent mean &plusmn; SEM of 6 cell culture inserts.

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Oncol. 103, 468-474 (2011).</li><li>Provenzano, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+density+promotes+mammary+tumor+initiation+and+progression.">Collagen density promotes mammary tumor initiation and progression.</a> BMC Med. 6, 11 (2008).</li><li>Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126, 677-689 (2006).</li><li>Paszek, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8, 241-254 (2005).</li><li>Levental, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139, 891-906 (2009).</li><li>Butler, T. P., Grantham, F. H., Gullino, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+transfer+of+fluid+in+the+interstitial+compartment+of+mammary+tumors.">Bulk transfer of fluid in the interstitial compartment of mammary tumors.</a> Cancer Res. 35, 3084-3088 (1975).</li><li>Dafni, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+vascular+endothelial+growth+factor+165+drives+peritumor+interstitial+convection+and+induces+lymphatic+drain:+magnetic+resonance+imaging,+confocal+microscopy,+and+histological+tracking+of+triple-labeled+albumin.">Overexpression of vascular endothelial growth factor 165 drives peritumor interstitial convection and induces lymphatic drain: magnetic resonance imaging, confocal microscopy, and histological tracking of triple-labeled albumin.</a> Cancer Res. 62, 6731-6739 (2002).</li><li>Chary, S. R., 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=Direct+measurement+of+interstitial+convection+and+diffusion+of+albumin+in+normal+and+neoplastic+tissues+by+fluorescence+photobleaching.">Direct measurement of interstitial convection and diffusion of albumin in normal and neoplastic tissues by fluorescence photobleaching.</a> Proc. Natl. Acad. Sci. U.S.A. 86, 5385-5389 (1989).</li><li>Heldin, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+interstitial+fluid+pressure+-+an+obstacle+in+cancer+therapy.">High interstitial fluid pressure - an obstacle in cancer therapy.</a> Nat. Rev. Cancer. 4, 806-813 (2004).</li><li>Fukumura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microvasculature+and+microenvironment:+novel+insights+through+intravital+imaging+in+pre-clinical+models.">Tumor microvasculature and microenvironment: novel insights through intravital imaging in pre-clinical models.</a> Microcirculation. 17, 206-225 (2010).</li><li>Shields, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+chemotaxis+as+a+mechanism+of+tumor+cell+homing+to+lymphatics+via+interstitial+flow+and+autocrine+CCR7+signaling.">Autologous chemotaxis as a mechanism of tumor cell homing to lymphatics via interstitial flow and autocrine CCR7 signaling.</a> Cancer Cell. 11, 526-538 (2007).</li><li>Shieh, A. C. <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+invasion+is+promoted+by+interstitial+flow-induced+matrix+priming+by+stromal+fibroblasts.">Tumor cell invasion is promoted by interstitial flow-induced matrix priming by stromal fibroblasts.</a> Cancer Res. 71, 790-800 (2011).</li><li>Shi, Z. D., Wang, H., Tarbell, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparan+sulfate+proteoglycans+mediate+interstitial+flow+mechanotransduction+regulating+MMP-13+expression+and+cell+motility+via+FAK-ERK+in+3D+collagen.">Heparan sulfate proteoglycans mediate interstitial flow mechanotransduction regulating MMP-13 expression and cell motility via FAK-ERK in 3D collagen.</a> PLoS One. 6, e15956 (2011).</li><li>Fleury, M. E., Boardman, K. C., 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=Autologous+morphogen+gradients+by+subtle+interstitial+flow+and+matrix+interactions.">Autologous morphogen gradients by subtle interstitial flow and matrix interactions.</a> Biophys J. 91, 113-121 (2006).</li><li>Miteva, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmural+flow+modulates+cell+and+fluid+transport+functions+of+lymphatic+endothelium.">Transmural flow modulates cell and fluid transport functions of lymphatic endothelium.</a> Circ. Res. 106, 920-931 (2010).</li><li>Ng, C. P., Hinz, B., 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=Interstitial+fluid+flow+induces+myofibroblast+differentiation+and+collagen+alignment+in+vitro.">Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro.</a> J. Cell. Sci. 118, 4731-4739 (2005).</li><li>Kunder, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell-derived+particles+deliver+peripheral+signals+to+remote+lymph+nodes.">Mast cell-derived particles deliver peripheral signals to remote lymph nodes.</a> J. Exp. Med. 206, 2455-2467 (2009).</li><li>Helm, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergy+between+interstitial+flow+and+VEGF+directs+capillary+morphogenesis+in+vitro+through+a+gradient+amplification+mechanism.">Synergy between interstitial flow and VEGF directs capillary morphogenesis in vitro through a gradient amplification mechanism.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 15779-15784 (2005).</li><li>McGuire, P. G., Seeds, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+plasminogen+activator+with+a+reconstituted+basement+membrane+matrix+and+extracellular+macromolecules+produced+by+cultured+epithelial+cells.">The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells.</a> J Cell Biochem. 40, 215-227 (1989).</li><li>Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+type+IV+procollagen,+laminin,+and+heparan+sulfate+proteoglycan+from+the+EHS+sarcoma.">Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma.</a> Biochemistry. 21, 6188-6193 (1982).</li><li>Haessler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+dynamics+of+breast+cancer+cells+in+a+tunable+3D+interstitial+flow+chamber.">Migration dynamics of breast cancer cells in a tunable 3D interstitial flow chamber.</a> Integr. Biol. (Camb). , (2011).</li><li>Polacheck, W. J., Charest, J. L., Kamm, R. 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No conflicts of interest declared.

Here we have described a methodology for quantifying the effect of interstitial flow on tumor cell invasion, using cells embedded in a 3-D matrix within a cell culture insert. This and similar methods have been used to study the effect of interstitial flow on a variety of cell types 13-15, 17. Our approach partially mimics the matrix microenvironment of the tumor by using type I collagen and Matrigel which contain proteins found in the basement membrane of epithelial tissue and the surrounding stroma 21-2...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Collagen (Rat Tail)</td>
			<td>BD</td>
			<td>354236</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Millicell cell culture insert</td>
			<td>Millipore</td>
			<td>PI8P01250</td>
			<td>8 &mu;m pore diameter, polycarbonate membrane</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD</td>
			<td>354234</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich</td>
			<td>100M-8202</td>
			<td>10x for preparing gel solution, 1x for washing steps</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide, 1.0N Solution</td>
			<td>Sigma Aldrich</td>
			<td>S2770</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>DMEM 1X</td>
			<td>CellGro</td>
			<td>10-013-CV</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>511150</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>CellGro</td>
			<td>30002CI</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100-500 ml</td>
			<td>0.5% in PBS</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>04042-500</td>
			<td>4% in PBS</td>
		</tr>
		<tr>
			<td>Deionized Water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>4',6-diaminido-2-phenylindole (DAPI)</td>
			<td>MP Biomedicals</td>
			<td>0215757401</td>
			<td>1 mg/ml stock solution</td>
		</tr>
		<tr>
			<td>Mounting Solution</td>
			<td>Thermo Scientific</td>
			<td>TA-030-FM</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>CellGro</td>
			<td>25-052-CI</td>
			<td>Keep sterile</td>
		</tr>
	</tbody>
</table>

three-dimensional cell culture model for measuring the effects of interstitial fluid flow on tumor cell invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Watch this Scientific Journal Video about Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion at JoVE.com

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

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Research

JoVE Journal

Bioengineering

16.5K visualizações.  Drexel University. Fluxo de fluido intersticial é elevada em tumores sólidos e pode modular a invasão de células tumorais. Descrevemos aqui uma técnica para aplicar o fluxo de fluido intersticial para as células embebidas numa matriz e, em seguida medir os seus efeitos sobre a invasão celular. Esta técnica pode ser facilmente adaptado para estudar outros sistemas.

Vídeo: Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...