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 tissue culture hood for further processing within 45 min. Keep the vial on an ice block inside the hood.</li>
	<li>Pour the supplied tissue into a 6 well plate containing 6-7 ml of HBSS solution using a pipette. 
	Note: Amount of tissue per well is not critical in this rinsing step.</li>
	<li>Keep the 6 well plate on an ice block. Rinse the tissue in HBSS solution twice.</li>
	<li>Cut tissue cleanly into separated sections with sterile scissor. Mince tissue into smaller sections no greater than 1-2 mm3 in size with a sterile scalpel blade.</li>
	<li>Weigh a labelled 0.1% trypsin vial (containing 1 ml of 0.1% trypsin solution) before transferring the tissue. Transfer ~20 mg tissue into each of the 0.1% trypsin vials with a pipette.</li>
	<li>Try to minimize the transfer of HBSS into the trypsin vial to avoid further dilution of trypsin.</li>
	<li>Weigh the trypsin vials after tissue transfer; document the difference as the tissue mass.</li>
	<li>Pour ~20 mg tissue and 1 ml trypsin into each well of a 24 well plate.</li>
	<li>Put the 24 well plate on shaker in fridge at 4 &#176;C for 16-20 hr. During this wait period or at an earlier time, prepare polyacrylamide gel substrates and functionalize them with ECM proteins as described in section 3.</li>
</ol>
2. Enzymatic Dissociation of Tissue Sample into Single Cell Suspension and Culturing Isolated Cells on ECM Functionalized Elastic Substrates and Rigid Polystyrene Dishes
<ol>
	<li>After 16-20 hr of incubation in trypsin well at 4 &#176;C on shaker, transfer tissue from 4 &#176;C trypsin to 4 &#176;C complete growth media vial (containing 2-3 ml of complete growth media) using a pipette. Ensure minimal transfer of trypsin into growth media. 
	Note: Complete growth media formulation is as follows: RPMI 1640 base medium supplemented with horse serum to a final concentration of 10% and penicillin-streptomycin to 1% of total solution.</li>
	<li>Place the tissue containing media vial in a warm water bath at 37 &#176;C for 12-15 min.</li>
	<li>Transfer the tissue to a 15 ml vial containing HBSS solution using a pipette, shake gently.</li>
	<li>Repeat 2.3.</li>
	<li>Remove tissue from HBSS solution and transfer to 0.1% collagenase solution (in HBSS) vial containing 1 ml solution. Incubate for 45 min at 37 &#176;C and 5% CO2 environment in a humidified cell culture incubator. During the wait period, warm growth media in a 15 ml vial at 37 &#176;C in water bath.</li>
	<li>Pull out all tissue fragments into pipette minimizing collagenase carryover. Eject just the tissue fragments into pre-warmed culture media vials.</li>
	<li>Place a 40 &#181;m well-insert filter into each well of a 6 well plate. Moisten the filter with 1 ml cell culture media.</li>
	<li>Triturate tissue in media with a 10 ml glass pipette several times until tissue is completely dissociated.</li>
	<li>Maintain the pipette tip as close as possible to the base of the vial.</li>
	<li>Deposit the solution onto the filter to remove debris and undissociated parts.</li>
	<li>Centrifuge filtered cell suspension in media at 150 x g for 5 min at RT.</li>
	<li>Remove the supernatant afterwards and resuspend cell pellet with 2 ml fresh culture media.</li>
	<li>Count the cells using a hemocytometer (if required). Seed isolated primary cells on extracellular matrix (ECM) functionalized gels and polystyrene dishes at desired concentration.</li>
</ol>
3. Preparation and Functionalization of Different Stiffness Polyacrylamide (PA) Gels and Polystyrene Substrates
Note: For visual demonstration of Section 3, the viewers/readers are referred to a recent Journal of Visualized Experiments (JoVE) article17.
<ol>
	<li>Adopting published protocols18, 19, activate 12 mm2 glass cover slips chemically to ensure covalent binding of the hydrogel.
	<ol>
		<li>First, treat glass cover slips with 3- Aminopropyltrymethoxysilane (ATS) for 7 min at RT.</li>
		<li>Remove the ATS completely with DI water rinse and treat cover slips with 0.5% Glutaraldehyde (diluted in PBS from 70% Glutaraldehyde stock solution) solution for 30 min.</li>
	</ol>
	</li>
	<li>Obtain 2 kPa gel solutions by mixing 5% w/v acrylamide solution and 0.05% N,N&#39;-methylenebisacrylamide (bis) solution in 10 mM HEPES-buffered saline20. Use 8% w/v acrylamide and 0.13% N,N&#39;-methylenebisacrylamide (bis) solution in 10 mM HEPES-buffered saline for 10 kPa gel20.
	<ol>
		<li>In both cases, use 1:200 ammonium persulfate (10% w/v) and 1:2,000 N,N,N&#39;,N&#39;-tetramethylethylenediamine (TEMED) as the initiator and catalyst for the polymerization process, respectively.</li>
		<li>Also, add 100 &#181;l fluorescent beads to 2 kPa gel solution as fiduciary markers 21, 22.</li>
	</ol>
	</li>
	<li>Deposit a drop of 20 &#956;l pre-polymer PA gel solution on the 12 mm2 activated glass cover slip. Place another 12 mm2 regular glass cover slip on the drop. Ensure that the drop spreads between the cover slips due to capillarity. Invert the sandwich for 2 kPa gels to ensure that most of the beads come near the cell culture surface.</li>
	<li>Cure the PA gel for 45 min at RT.</li>
	<li>Peel off the top cover slip using a single edge razor. 
	Note: During peeling, detachment proceeds from one edge of the sandwich. The gel remains adherent to the activated glass slide.</li>
	<li>Store the gels in PBS solution before use.</li>
	<li>Functionalize PA gels and glass with ECM molecules, human fibronectin at a concentration of 50 &#181;g/ml following published methods 23.
	<ol>
		<li>Briefly, incubate substrates with 2 ml pure hydrazine hydrate O/N.</li>
		<li>Remove hydrazine hydrate and rinse the substrates thoroughly with DI water.</li>
		<li>Wash the substrates with 5% acetic acid for 30 min.</li>
		<li>Remove acetic acid and rinse the substrates thoroughly with DI water.</li>
		<li>Keep the substrates immersed in DI water for 30 min.</li>
		<li>Incubate the substrates with oxidized fibronectin for 35 min at a concentration of 50 &#181;g/ml.</li>
		<li>Rinse the substrates with PBS on shaker at low rpm for 10 min.</li>
		<li>Incubate all substrates at 37 &#176;C in 2-3 ml culture media for 30 min before plating the cells. 
		Note: Plate cells in a sparsely populated manner (1,000-3,000 cells/cm2). Each gel-covered glass slip needs to be contained in a 35 mm Petri dish. Allow cells to adhere completely before any microscopy (at least O/N).</li>
	</ol>
	</li>
</ol>
4. Traction Force Microscopy and Immunofluorescence Microscopy Assays
<ol>
	<li>Traction Force Microscopy Assay
	<ol>
		<li>Warm 0.25% trypsin-EDTA / 10% SDS solution at 37 &#176;C in water bath for 10 minutes before traction experiments start.</li>
		<li>Remove one gel from the cell culture incubator at a time and place on the fluorescent inverted microscope stage (32X magnification).</li>
		<li>Find a single cell in field of view. Remove the Petri dish lid.</li>
		<li>Take a phase contrast image of cell (e.g., Figure 3).</li>
		<li>Switch the imaging mode to fluorescence and select appropriate filter. Don&#8217;t move the microscope stage or sample during this time.</li>
		<li>Take an image of the fluorescent beads displaced by cellular traction (Figure 4C).</li>
		<li>Add 1 ml trypsin/SDS solution to Petri dish to detach the cell from gel. Don&#8217;t move the microscope stage or sample during this time. Also, take a control image of the cell to ensure complete removal from gel.</li>
		<li>Take a reference (null force) image of the beads after the cell is removed.</li>
		<li>Repeat steps 4.1.2-4.1.8 for all other gels.</li>
		<li>Make an image stack using ImageJ from the two images acquired in step 4.1.6 and 4.1.8 for each case. To generate the image stack, use following sequence of commands in ImageJ after opening the images: Image &#8594; Stacks &#8594; Images to stack.</li>
		<li>To obtain the displacement field and traction, use ImageJ plugins following published methods 21, 22. Obtain codes and detailed tutorials for these plugins using the following link: <a href="https://sites.google.com/site/qingzongtseng/imagejplugins">https://sites.google.com/site/qingzongtseng/imagejplugins</a>.
		<ol>
			<li>Align the images in the stack using the &#8216;Template Matching&#8217; plugin. Use following sequence of commands in ImageJ after opening the image stack generated in step 4.1.10: Plugins &#8594; Template Matching &#8594; Align Slices in Stack &#8594; OK. Save the image stack consequently. Use this new image stack in the following steps.</li>
			<li>Obtain the displacement field using the PIV (particle image velocimetry) plugin (Figure 4D). Use following sequence of commands after opening the image stack saved in step 4.1.11.1: Plugins &#8594; PIV &#8594; Iterative PIV (Basic) &#8594; OK &#8594; Accept this PIV and output &#8594; Ok. Save the PIV output in the same directory as in the original image stack. Use this as input in next step.</li>
			<li>Finally use the FTTC (Fourier transform traction cytometry) plugin to obtain the traction map (Figure 4E). Use following sequence of commands: Plugins &#8594; FTTC &#8594; Insert material properties, i.e., Young&#8217;s modulus and Poisson&#8217;s ratio of PA gel &#8594; OK &#8594; Select the PIV output file saved in step 4.1.11.2 &#8594; OK. Save FTTC results in the same directory as in the image stack and PIV output.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Immunofluorescence Microscopy Assays
	<ol>
		<li>Take the substrates to be immunostained to the laminar hood and remove the culture media.</li>
		<li>Rinse the substrates with PBS and fix the cells with 4% paraformaldehyde in PBS for 20 min at RT.</li>
		<li>Wash the substrates with PBS for 3 times (5 min each time). Consequently, incubate the substrates with 500 &#181;l signal enhancer for 30 min and rinse with PBS.</li>
		<li>Incubate cells with monoclonal anti-vinculin antibody at a 1:250 dilution in PBS for 45 min at RT. Wash the substrates with PBS for 3 times (5 min each time).</li>
		<li>Incubate the samples with secondary antibody Alexa Fluor 488 goat anti-mouse IgG at a 1:200 dilution in PBS at RT for 30 min. Wash the substrates with PBS for 3 times (5 min each time).</li>
		<li>To visualize the F-actin structure, incubate cells with TRITC phalloidin conjugates at a concentration 50 &#181;g/ml for 45 min at RT. Wash the substrates with PBS for 3 times (5 min each time).</li>
		<li>Image the samples using confocal scanning laser microscope (Figure 5A-5D).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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

Research

JoVE Journal

Bioengineering

15.4K Views.  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. 

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

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.