Name: an efficient and flexible cell aggregation method for 3d spheroid production
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Description: Watch this Scientific Journal Video about An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production at JoVE.com

The authors thank M. Gordon of the Queen's University Biomedical Imaging Centre for assistance. This work was supported by operating grants from the Cancer Research Society of Canada (19439) and the Canadian Institutes for Health Research (MOP-142303) (LMM), and by Ontario Graduate Scholarships and studentships from the Terry Fox Research Institute Training Program in Transdisciplinary Cancer Research (SMM, EYL), and by a Craig Jury Summer Studentship (SMM).

NOTE: All reagents and consumables are listed in the Materials List.
1. Spheroid Production by Cell Aggregation
<ol>
	<li>Methyl cellulose solution: Prepare 100 mL of 100 mg/mL methyl cellulose.
	<ol>
		<li>Heat 50 mL ultrapure H2O to 80 &#176;C. Add 10 g methyl cellulose powder and agitate until particles are evenly dispersed.</li>
		<li>Bring to final volume with cold ultrapure H2O and stir at 4 &#176;C until the solution becomes clea...

<ol>
	<li>Zimmermann, M., Box, C., Eccles, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+vs.+three-dimensional+in+vitro+tumor+migration+and+invasion+assays.">Two-dimensional vs. three-dimensional in vitro tumor migration and invasion assays.</a> Methods Mol Biol. 986 (986), 227-252 (2013).</li><li>Albini, A., Noonan, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+'chemoinvasion'+assay,+25+years+and+still+going+strong:+the+use+of+reconstituted+basement+membranes+to+study+cell+invasion+and+angiogenesis.">The 'chemoinvasion' assay, 25 years and still going strong: the use of reconstituted basement membranes to study cell invasion and angiogenesis.</a> Curr Opin Cell Biol. 22 (5), 677-689 (2010).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. 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=The+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nat. Rev. Mol. Cell Biol. 8 (10), 839-845 (2007).</li><li>Fennema, E., Rivron, N., Rouwkema, J., van Blitterswijk, C., de Boer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+culture+as+a+tool+for+creating+3D+complex+tissues.">Spheroid culture as a tool for creating 3D complex tissues.</a> Trends Biotechnol. 31 (2), 108-115 (2013).</li><li>Carletti, E., Motta, A., Migliaresi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolds+for+tissue+engineering+and+3D+cell+culture.">Scaffolds for tissue engineering and 3D cell culture.</a> Methods Mol Biol. 695, 17-39 (2011).</li><li>Foty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+hanging+drop+cell+culture+protocol+for+generation+of+3D+spheroids.">A simple hanging drop cell culture protocol for generation of 3D spheroids.</a> J Vis Exp. (51), (2011).</li><li>Handschel, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospects+of+micromass+culture+technology+in+tissue+engineering.">Prospects of micromass culture technology in tissue engineering.</a> Head Face Med. 3, 4 (2007).</li><li>Stuckhoff, A. P., DelValle, L., Amini, S., White, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuronal Cell Culture Methods and Protocols Vol. 1078 Methods in Molecular Biology. 1078, 119-132 (2013).</li><li>Stewart, G. J., Wang, Y., Niewiarowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylcellulose+protects+the+ability+of+anchorage-dependent+cells+to+adhere+following+isolation+and+holding+in+suspension.">Methylcellulose protects the ability of anchorage-dependent cells to adhere following isolation and holding in suspension.</a> Biotechniques. 19 (4), 598-604 (1995).</li><li>Vinci, 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=Advances+in+establishment+and+analysis+of+three-dimensional+tumor+spheroid-based+functional+assays+for+target+validation+and+drug+evaluation.">Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation.</a> BMC Biol. 10, 29 (2012).</li><li>Nagelkerke, A., Bussink, J., Sweep, F. C. G. J., Span, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+multicellular+tumor+spheroids+of+breast+cancer+cells:+How+to+go+three-dimensional.">Generation of multicellular tumor spheroids of breast cancer cells: How to go three-dimensional.</a> Anal. Biochem. 437 (1), 17-19 (2013).</li></ol>

We present a rapid and flexible method for producing 3D cell spheroids to model the architecture of in vivo tissues using inexpensive and widely available reagents. Our protocol exploits the non-cytotoxic and adhesion-promoting properties of MC8,9 to mediate cell aggregation and minimize cell monolayer formation. Unlike protein-based matrices isolated from animal sources, MC is inert, contains no growth factors, and is easily removed by washing, allowing...

Biologically relevant assessment of tumor cell behavior is challenging using traditional two-dimensional (2D) cell culture methodologies, in part because these do not adequately reflect the cell microenvironment found in vivo. Alternative approaches incorporating extracellular matrix components into the culture (e.g., Boyden chamber assays) are more physiologically representative of the in vivo tissue environment. However, they can be limited to assessment of individual cell behavior, and do not recapitulate the complex in vivo combinations of cell-matrix and cell-cell interactions that contribute to tissue or tumor growth1,2,3.
The use of multicellular spheroids is a recent approach that more accurately reproduces the compact architecture of in vivo cell growth1,4. Spheroids can be used to investigate cell-matrix interactions of normal cells, but can also act as tumor analogues to model characteristics of tumor progression, such as metastatic growth or drug resistance4.
Spheroids may be formed by the proliferation of single cells embedded in a matrix5, or more rapidly, by promoting the aggregation of multiple cells to form a single cell cluster (e.g., hanging drop, centrifugation methods)6,7. Existing cell aggregation techniques may require costly materials or specialized equipment. In addition, these spheroids have a wide range of sizes and morphologies and may be difficult to produce in large quantities, making comparisons between growth conditions or treatments difficult. Finally, spheroids generated by these methods can be difficult to isolate from the proteinaceous extracellular matrix in which they are embedded for use in other applications.
Here, we describe a robust and easily modifiable cell aggregation methodology for the rapid formation of consistently sized cell spheroids using commercially available U-bottom cell-repellent plates and an inert adhesion-promoting matrix, methyl cellulose. Once formed, these multicellular spheroids are readily isolated for use in a wide range of applications. The protocol is also easily adapted to generate spheroids through cell proliferation, which may be used to assess other cell processes. Here, we show cell invasion assays, quantified by immunofluorescence staining, and an anoikis assay, as example applications of these two different spheroid formation protocols.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x Phosphate buffered saline</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Solution</td>
			<td>Sigma-Aldrich</td>
			<td>21114</td>
			<td>Used for PBS* wash buffer; Do not autoclave PBS* wash buffer upon addition of calcium chloride</td>
		</tr>
		<tr>
			<td>Magnesium Chloride Solution</td>
			<td>Sigma-Aldrich</td>
			<td>M1028</td>
			<td>Used for PBS* wash buffer; Do not autoclave PBS* wash buffer upon addition of magnesium chloride</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>For Spheroid Formation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well U-bottom Cell-Repellent Plate</td>
			<td>Greiner Bio-One</td>
			<td>650970</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Sigma-Aldrich</td>
			<td>D5546</td>
			<td>For culturing SH-SY5Y, PANC-1, TPC-1 cell lines</td>
		</tr>
		<tr>
			<td>F12K Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>2112722</td>
			<td>For culturing TT cell line</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F1051</td>
			<td>Filter prior to use to remove particulate contaminants</td>
		</tr>
		<tr>
			<td>Methyl cellulose</td>
			<td>Sigma-Aldrich</td>
			<td>M7027</td>
			<td>Prepare in water to 100 mg/mL</td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute Medium</td>
			<td>Sigma-Aldrich</td>
			<td>R8758</td>
			<td>For culturing HCT-116, BxPC-3 cell lines</td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Thermo Fisher Scientific</td>
			<td>12605028</td>
			<td>Dissociation buffer</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>For Invasion Assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Type I Collagen</td>
			<td>Corning Incorporated</td>
			<td>354231</td>
			<td>Stock 3.1mg/ml; Maintain on ice when in use</td>
		</tr>
		<tr>
			<td>DMEM Phenol Red Free Low Glucose&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>11054-20</td>
			<td>Less background fluorescence compared to Phenol Red supplemented medium</td>
		</tr>
		<tr>
			<td>Glial Cell Line Derived Neurotrophic Factor</td>
			<td>Peprotech</td>
			<td>450-10</td>
			<td>Chemoattractant</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>For Immunofluorescence Microscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>#1.5 Coverglass</td>
			<td>Electron Microscopy Sciences</td>
			<td>72225-01</td>
			<td>For mounting excised spheroids</td>
		</tr>
		<tr>
			<td>Alexa-Fluor 488 Phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12379</td>
			<td>Used to stain actin at 1:200</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Bioshop Canada Incorporated</td>
			<td>ALB001</td>
			<td>Used in BSA blocking buffer</td>
		</tr>
		<tr>
			<td>Dabco 33-LV</td>
			<td>Sigma-Aldrich</td>
			<td>290734</td>
			<td>Antifade</td>
		</tr>
		<tr>
			<td>Glycerol&#160;</td>
			<td>Bioshop Canada Incorporated</td>
			<td>GLY001</td>
			<td>Used in MOWIOL mounting medium</td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>Freeware, NIH</td>
			<td>-</td>
			<td>Used for image analysis&#160;</td>
		</tr>
		<tr>
			<td>Microslides</td>
			<td>VWR International</td>
			<td>48312-024</td>
			<td>For mounting excised spheroids</td>
		</tr>
		<tr>
			<td>MOWIOL 4-88</td>
			<td>EMD-Millipore</td>
			<td>475904</td>
			<td>Used in MOWIOL mounting medium</td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>EMD-Millipore</td>
			<td>PX0055-3</td>
			<td>Used in fixation buffer</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Bioshop Canada Incorporated</td>
			<td>TRX777</td>
			<td>Used in permeabilization buffer</td>
		</tr>
	</tbody>
</table>

an efficient and flexible cell aggregation method for 3d spheroid production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

We describe a flexible and efficient method to generate discrete spheroids using cell-repellent plates and spheroid formation media supplemented with MC. Under the appropriate conditions of MC and serum, individual cells settle and adhere together at the center of the well to form spheroids with minimal adherence to the well bottom. Using this protocol, spheroids were generated from a variety of cell lines (Figure 2B). Titration of MC and serum concentrations is required ...

Watch this Scientific Journal Video about An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production at JoVE.com

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

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