This project was funded by the National Science Foundation ECCS grant 10-02165, and the Interdisciplinary Innovation Initiative Program, University of Illinois grant 12035. M.Y. A. was funded at UIUC from NIH National Cancer Institute Alliance for Nanotechnology in Cancer &#8216;Midwest Cancer Nanotechnology Training Center&#8217; Grant R25 CA154015A. Immnunostaining and confocal microscopy imaging were carried out at the Institute for Genomic Biology (IGB), UIUC. M.Y.A. acknowledges the discussions with B. J. Williams of UIUC regarding the isolation experiments. M.Y.A. acknowledges C. Nemeh and Abdul Bhuiya of UIUC for assistance in schematic and materials list preparation.

The protocol described below follows the guidelines of UIUC human research ethics committee.
1. Collection and Digestion of Surgical Tissue Sample
<ol>
	<li>Collect the tumor tissue sample right after colon resection (Figure 1A and 1B). Collect tissue from an adjacent healthy site as well.</li>
	<li>Transfer the tissue immediately to a 15 ml vial containing 12 ml HBSS solution. Keep the vial on ice inside an insulated foam box.</li>
	<li>Transport the tissue containing vial to a...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+hydrogel+substrates+with+tunable+mechanical+properties.">Preparation of hydrogel substrates with tunable mechanical properties.</a> Current Protocols in Cell Biology. , 10-16 (2010).</li><li>Tseng, Q., 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=Spatial+organization+of+the+extracellular+matrix+regulates+cell&#8211;cell+junction+positioning.">Spatial organization of the extracellular matrix regulates cell&#8211;cell junction positioning.</a> Proc. Natl. Acad. Sci. USA. 109 (5), 1506-1511 (2012).</li><li>Chopra, 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=Augmentation+of+integrin-mediated+mechanotransduction+by+hyaluronic+acid.">Augmentation of integrin-mediated mechanotransduction by hyaluronic acid.</a> Biomaterials. 35 (1), 71-82 (2014).</li><li>Damljanovic, V., Lagerholm, B. C., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+and+micropatterned+conjugation+of+extracellular+matrix+proteins+to+characterized+polyacrylamide+substrates+for+cell+mechanotransduction+assays.">Bulk and micropatterned conjugation of extracellular matrix proteins to characterized polyacrylamide substrates for cell mechanotransduction assays.</a> Biotechniques. 39 (6), 847-851 (2005).</li><li>Tilghman, R. 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Cellular traction stress has recently emerged as a potential biophysical indicator of metastatic state13. However, no experimental traction data with primary tumor cells exists in literature to date. Also, directly culturing isolated primary colon cells on different stiffness polyacrylamide gels is not reported yet. Hence, we establish an optimized primary colon cell culture conditions on gels and polystyrene (Figure 2). Fluorescent microbeads encapsulation near cell culture surface during sof...

In recent years it has become increasingly evident that mechanical micro-environment, in addition to bio-chemical factors, plays an important role in regulating cell functionalities. Cells can sense and respond to the substrate stiffness on which they are adhered to (as in 2D culture) or surrounded by (as in 3D culture) 3-7. By doing so, cells can modulate their differentiation3, morphology4, migration/motility5, bio-physical properties6, growth7, and other processes.
Cancer cells also respond to the 2D and 3D matrix stiffness using a coordinated, hierarchical mechano-chemical combination of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. For example, mammary epithelial cells (MECs) form normal acinar parenchyma when cultured on 150 Pa substrates that is similar to the stiffness of healthy mammary tissue. Interestingly, they exhibit the hallmarks of a developing tumor, both structural and transcriptional, when cultured on stiffer substrates (&#62; 5,000 Pa) that mimic the stiffness of a tumor stroma8. In addition, another experiment shows that breast tumorigenesis is accompanied by collagen crosslinking and ECM stiffening9. Recent experiments show that human colon carcinoma (HCT-8) cells display metastasis like phenotype (MLP) when they are cultured on 2D substrates having physiologically relevant stiffness (20&#8211;47 kPa), but not on very stiff (3.6 GPa) substrates10-12.These cells first form tumor-like cell clusters and then dissociate from one another, starting from the periphery. As this epithelial to rounded morphological (E to R transition) change occurs, they proliferate, reduce cell-cell and cell-ECM adhesion, and become migratory. HCT-8 cells cultured on very hard polystyrene substrates do not exhibit these malignant traits. Thus it has been hypothesized that HCT-8 cells become metastatic due to their exposure to appropriate micro-environment. It is worth noting that these experiments are carried out with immortalized cancer cell lines or murine derived primary cells, not with primary human cancer cells.
A recent study proposes that augmented cellular traction stress may be used as a potential biophysical signature for metastatic cells13.The study involves measuring traction force for different human cancer cell lines on polyacrylamide gels. It is found that metastatic cancer cells can exert significantly higher traction stress compared to non-metastatic cells in all cases13. However, these results directly contradict the earlier published findings on murine derived breast cancer cell lines14. Also, a recent study highlights remarkable differences between immortalized and primary human cells in their cytoskeletal remodeling protein profiling and cell survival protein expression15. Hence, it is important to revisit many of the biophysical assays including traction for primary human cancer cells. This will address the question whether the primary cells recapitulate immortalized cancer cell lines traction trend.
The protocol described here is optimized for isolation of primary human colon cells (both healthy and cancerous), and for culturing them on soft substrates (polyacrylamide hydrogels) as well as on Petri dishes. The protocol is based on digestion and consequent enzymatic dissociation of surgical tissue sample into single cell suspension16. To our knowledge, this is the first demonstration of culturing isolated primary colon tumor and normal cells directly on soft hydrogel substrates with embedded fluorescent microbeads for traction cytometry. Transparent gel substrates also allow immunostaining. This assay revealed differences in F-actin organization and focal adhesions in primary human colon cells as substrate stiffness changes. This cell culture platform opens up the possibility of exploring various biophysical properties of primary human cells such as cell stiffness and traction as parameters for cancer prognostics.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td ></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr >
			<td >HBSS</td>
			<td>Life technologies</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr >
			<td >PBS</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin</td>
			<td>Worthington</td>
			<td>LS003736</td>
			<td></td>
		</tr>
		<tr >
			<td >Collagenese</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td></td>
		</tr>
		<tr >
			<td >3- Aminopropyltrymethoxysilane (ATS)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td >281778</td>
			<td></td>
		</tr>
		<tr >
			<td >Glutaraldehyde</td>
			<td>Polysciences, Inc.</td>
			<td>01201-5</td>
			<td></td>
		</tr>
		<tr >
			<td >Acrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>A4058</td>
			<td></td>
		</tr>
		<tr >
			<td >N- methylenebisacrylamide (bis)</td>
			<td>Sigma-Aldrich</td>
			<td>M1533</td>
			<td></td>
		</tr>
		<tr >
			<td >HEPES buffer solution</td>
			<td>Sigma-Aldrich</td>
			<td >83264</td>
			<td></td>
		</tr>
		<tr >
			<td >Ammonium persulfate</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td></td>
		</tr>
		<tr >
			<td >N&#714;-tetramethylethylenediamine (TEMED)</td>
			<td>Bio-Rad</td>
			<td>161-0801</td>
			<td></td>
		</tr>
		<tr >
			<td >Human fibronectin</td>
			<td>BD biosciences</td>
			<td >354008</td>
			<td></td>
		</tr>
		<tr >
			<td >Hydrazine hydrate</td>
			<td>Sigma-Aldrich</td>
			<td >18412</td>
			<td>Hazardous</td>
		</tr>
		<tr >
			<td >Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr >
			<td >Paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>RT15710</td>
			<td></td>
		</tr>
		<tr >
			<td >Signal enhancer</td>
			<td>Life technologies</td>
			<td>I36933</td>
			<td></td>
		</tr>
		<tr >
			<td >Monoclonal anti vinculin antibody&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>V9131</td>
			<td></td>
		</tr>
		<tr >
			<td >Alexa fluor 488 goat anti-mouse IgG</td>
			<td>Life technologies</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr >
			<td >TRITC phalloidin conjugates&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1951</td>
			<td></td>
		</tr>
		<tr >
			<td >0.1 &#181;m fluroscent beads</td>
			<td>Life technologies</td>
			<td>F8801</td>
			<td></td>
		</tr>
		<tr >
			<td >0.25% Trypsin-EDTA</td>
			<td>Life technologies</td>
			<td>25200-056</td>
			<td></td>
		</tr>
	
		<tr >
			<td ></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		</tr>
		<tr >
			<td >12 mm2 glass cover slips</td>
			<td>Corning</td>
			<td>2865-12</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of primary human colon tumor cells from surgical tissues and culturing them directly on soft elastic substrates for traction cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

The protocol described above is successfully employed for multiple tissue samples (n=12) from four different patients under guidelines of the institutional review board. Figure 1A illustrates a representative colorectal tumor right after surgery from which the tissue sections for cell cultures are obtained. A typical tissue section in HBSS solution after transfer to the laminar hood for further processing is shown in Figure 1B. A schematic of digestion and enzymatic dissociation of surgi...

Watch this Scientific Journal Video about Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry at JoVE.com

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

isolation-primary-human-colon-tumor-cells-from-surgical-tissues

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15.4K visualizações.  University of Illinois at Urbana-Champaign. A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses. 

Vídeo: Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry