Name: evaluation of cancer stem cell migration using compartmentalizing microfluidic devices and live cell imaging
Uploaded: 2011-12-23T12:00:00
Duration: 9 min 36 s
Description: Watch this Scientific Journal Video about Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging at JoVE.com

PAC is partially supported by a NIH T32 grant to University of Wisconsin Stem Cell Training Program (P. A. Clark). JSK was partially supported by the HEADRUSH Brain Tumor Research Professorship, Roger Loff Memorial GBM Research Fund, and the UW Foundation/ Neurosurgery Brain Tumor Research and Education Fund. JCW and YH are partially supported by NIH grant NIBIB 1R01EB009103-01.

1. BTSC's cell dissociation
BTSCs are derived from pre-existing cultures grown in serum-free stem cell medium as neurospheres. culture of which is previously described10, 11.
<ol>
 <li>Prepare cell suspension from BTSC-derived neurospheres. BTSC-derived neurospheres are collected in a 15 ml conical tube and centrifuged at 900 rpm for 5 minutes. Faster centrifugation can shear and/or damage the neurospheres. Supernatant is aspirated and neurosphere culture is resuspended in 0.5 ml of pre-warmed Accutase. This solution is incubated for 5-10 minutes at 37&deg;C allowing the neurospheres to loosen. Cells are mechanically disrupted with 10-20 gentle strokes of a P100 pipette and then 1.5 ml of stem cell medium is added to the cells to neutralize the Accutase.</li>
 <li>The BTSCs are then centrifuged at 1300 rpm for 5 minutes, and resuspended in 1 ml of stem cell medium. </li>
 <li>A 50 &mu;l aliquot is removed from the suspension and placed into a microcentrifuge tube for cell counting. 50 &mu;l of trypan blue is added to the Eppendorf tube and the suspension is placed in a hemocytometer for cell counting.</li>
 <li>If required after cell counting, additional stem cell medium is added to the BTSC suspension to give a cell density of 20,000 live cells/&mu;l media. </li>
</ol>
2. Fabrication of bi-layered su-8 master and molding of PDMS stamp (see figure 1)
We use optical lithography and soft lithography to fabricate su-8 master and PDMS stamp, which are essential for assembly of the microfluidic devices. Slightly different than the standard procedure12, our su-8 master is composed of two layers. The 3-&mu;m-tall microchannels are casted in the first/bottom layer earlier in the process, while the 250-&mu;m-tall seeding and receiving chambers are in the second/upper layer. In order for correct connection between two culture compartments (microchannels and chambers), the two layers have to be accurately aligned in position. However, thickness and opacity of the second layer are large enough to block the fiducial/optical aligner from accessing the bottom features. Here we design the masks with fiducial markers being remotely positioned. Thus, these markers in the first layer can be selectively shielded while spinning the second layer. As a result, both layers of features are made in one master and ready for molding in bas-relief of the PDMS stamp.
<ol>
 <li>Design and prepare two photo-masks. One mask consists of two fiducial markers and an array of microchannels (400 &mu;m long and 10-50 &mu;m wide) for the first layer. Another mask consists of seeding and receiving chambers (2 mm long and 600 &mu;m wide) for the second layer. Both masks are designed in Illustrator (Adobe, California), laser-printed onto transparency films (Thin Metal Parts, Colorado), cleaned with isopropanol and air-dried.</li>
 <li>Make the first layer with su-8 photoresist (MicroChem, Massachusetts) according to product manual from supplier. First, spin-coat the handle wafer (WRS Materials, California) with 3-&mu;m-thick photoresist of su-8 (5), followed by soft-baking. The photoresist is then ultraviolet-exposed and cured with the corresponding mask in close-contact, followed by post-baking and developing.</li>
 <li>Spin-coat the second layer with 250 &mu;m thick su-8 (2100). In order to prevent thick photoresist from blocking the fiducial markers from the first layer, we cover these areas with scotch tape in prior to spin-coating of the second layer. The tape is then peeled off to reveal the markers for alignment purpose.</li>
 <li>UV-expose the second layer with the second mask. With the alignment markers revealed, it is straightforward to align them to the ones in the second mask using optical mask aligner. The second layer of photoresist is then ultraviolet-exposed and cured in close-contact, followed by post-baking and developing.</li>
 <li>Anti-stick coat the resulted master. To assist in removal of cured PDMS, the su-8 master can be silanized via vapor deposition to make the surface a nonstick-type coating. Simply place it into a desiccator for at least one hour, with several drops of trichloro(1H,1H,2H,2H-perfluorooctyl)silane solution under vacuum.</li>
 <li>Mold PDMS with the master. Mix prepolymer base and curer of PDMS Sylgard 184 (Dow Corning, Michigan) in 10:1 ratio thoroughly. Place it in a desiccator for vacuum degassing until no air bubble is seen. Pour the mixture on the mask and degas again if new air bubble is introduced. Cure the mold on a level hotplate for 2 hours at 90 C. After it cools to room temperature, gently release the PDMS stamp. Thus, culturing channels are engraved in PDMS and ready for assembly of device. The master is reusable for molding until it is cracked or worn. The anti-stick coating needs to be performed again when the stickiness regains.</li>
</ol>
3. Assembly of microfluidic devices and cell culturing (figure 2)
In order to culture cells, feature-engraved PDMS stamp is attached to a glass coverslip to form enclosed channels. Inlets and outlets are created for loading the culture/media. Meanwhile, cleanup and other procedures to the glass substrate and PDMS stamp are necessary to ensure the cell-compatibility.
<ol>
 <li>Make reservoirs through the PDMS stamp using biopsy hole puncher. We choose their diameter to be 6 mm. These reservoirs serve for two purposes. One is to hold extra nutrient for cell growth. The other is for the access of pipette loading and vacuum aspiration.</li>
 <li>Soak the PDMS stamp in 70% of ethanol for 30 min and then rinse it with de-ionized water for another 10 min. The purpose is to clear potential organic residuals introduced during fabrication process, and uncrosslinked PDMS contamination. More intense and thorough cleaning processes such as organic extraction13 and Soxhlet extraction14 were used elsewhere. However, we found it unnecessary in BTSC culture.</li>
 <li>Sterilize the PDMS stamp using autoclave (121&deg;C, 35 min). This step further completes the crosslink reaction of PDMS. Starting from this step and until step 3.6, all procedures are taken in sterile manner.</li>
 <li>The PDMS stamp is then laid on a glass coverslip, which is previously coated with poly-L-lysine.15-17 The device is then coated with laminin by filling the channel with laminin solution overnight at 37&deg;C. Laminin is aspirated from the device and the device is washed with stem cell medium. </li>
 <li>Load cell suspension from step 1 into reservoirs and channels. 11 &mu;l of dissociated cells are placed in one seeding reservoir. Vacuum aspiration is used to force cells into the seeding chamber, if necessary. An additional 7 &mu;l of cells are placed the adjoining seeding reservoir. Note: Average cell density is 20,000 cells/&mu;l media. After five minutes, the seeding and receiving reservoirs are flooded with media.</li>
 <li>Place a 0.5-1 mm pre-autoclaved PDMS sheet on top. The naturally-occurred surface adhesion between two PDMS pieces will make the cell culture sealed and ready for transport, incubation and microscopic imaging.</li>
</ol>
4. Long-term time-lapse imaging of BTSC migration with BioStation IM (Nikon Instruments Inc, Melville, USA).
Combining camera, software and incubator all in one box, this microscopic system makes it possible to image cell culture with no disturbance for days. In addition, its unique motor design moves the objective lens and keeps sample stage stationary in the point-visiting feature. This makes it practical to image parallel experiments and track cell locomotion in high-throughput assay.
<ol>
 <li>Pre-equilibrate the Biostation IM for 45 minutes to stabilize the temperature, moisture and air supply. Addition of several water-contained dishes in sample chamber is used to maintain proper humidity.</li>
 <li>Load the cell-contained device in the sample chamber of microscope, and center it using micro tweezers.</li>
 <li>Set the focus, position-points and time-points in Biostation software and start the time-lapse.</li>
</ol>

5. Representative results:
Examples of visual inspection and characterization of cancer cell migration by using a compartmentalizing microfluidic device are illustrated in Figure 3 and Figure 4. The cell-line is BTSC. With our current setup, phase images of cell culture can be continuously recorded for as long as 5 days (Figure 3) and as frequent as every 2 seconds (Figure 4). Beyond 5 days, culture media need to be replaced for nutrient replenishment and waste removed. Our long-term time-lapse identifies a revolving sequence of six cell stages during its migration based on the morphological changes. As illustrated in Figure 3, we describe them as (i) pre-migration, (ii) initiation, (iii) path-exploration, (iv) cruising, (v) destination-exloration and (vi) post-migration. In the receiving chamber, spindle-shaped cells stay in stage (i) as in bulk tumors that are able to grow, divide and gradually migrate. As they approach the microchannel entrance, a few cells proceed to stage (ii) when they expand and generate adhesive protrusion. Only one of them is able to occupy the entrance and proceed to stage (iii), which can explore the migration direction. Once the cell determines the migration direction, it proceeds to stage (iv) to cruise through the microchannel in a steady and high speed, which is carried through the entire microchannel. At the end of microchannel, cell proceeds to stage (v) to explore the open space of receiving chamber, and then to stage (vi). In microchannel, migrating power is mostly generated by blebbing activity as illustrated in figure 4, similar to that of amoeboid cell. Cell blebbing and membrane deformation are fully recorded here.
<img src="/files/ftp_upload/3297/3297fig1.jpg" alt="Figure 1" /> 
Figure 1. Schematics of fabrication of microfluidic device. The entire process is carried on a 3-inch silicon handle wafer through a modified technique of soft lithography. 3-&mu;m-tall microchannels and alignment markers are made as in the first layer. Then 250-&mu;m-thick su-8 is spin-coated, while the alignment markers are covered with scotch tape, such that they can later be accessible to mask aligner without sight-blocking by thick photoresist. Thus, chamber features can be made as the second layer and accurately aligned to the first layer. PDMS stamp is eventually molded off the resulting master.
<img src="/files/ftp_upload/3297/3297fig2.jpg" alt="Figure 2" /> 
Figure 2. Schematics of device assembly and cell seeding process. Enclosed by PDMS and glass coverslip, the 3D culturing space is composed of chambers, reservoirs and microchannels. The seeding chamber is filled with cell culture, while the receiving chamber is initially filled with cell-free media. The microchannels that connect between them provide cell migrating trail from seeding side to receiving side.
<img src="/files/ftp_upload/3297/3297fig3.jpg" alt="Figure 3" /> 
Figure 3. BTSC migration through microchannel. Upper: snapshots of time-lapse show a single cell (highlighted in green) migrates over 400 &mu;m from the seeding side to the receiving side. The entire journey takes about 2 days, involving a sequence of cell morphological changes. The scale bar is 40 &mu;m. Lower: a cartoon representation of six migration stages. Pre-migration (i): In seeding chamber, spindle-shaped and dividable cells gradually migrate along each other. The cells near microchannel entrance race with each other by polarizing cell body and exerting lamellipodia protrusion. Initiation (ii): The migratory cell with the highest capacity occupies the channel entrance while its competitors will retreat. Path-exploration (iii): The migratory cell changes to an amoeboid mode with little polarity. This migration mode is extremely motile and able to explore path in all directions by blebbing small membrane protrusions. Cruising (iv): Once the migration path is determined, the cell transforms into an adapted amoeboid mode by maintaining an additional large protrusion heading forward. This way, cell upholds a high motility as well as a steady direction and speed. Destination-exploration (v): Upon path-end, the cell slows down and develops filopodias to explore the receiving chamber for any invasion target. Post-migration (vi): After entering the receiving chamber, cell turns into star-shaped and retains high motility but no determined direction.
<img src="/files/ftp_upload/3297/3297fig4.jpg" alt="Figure 4" /> 
Figure 4. Snapshots of time-lapse show the cycle of blebbing activity and membrane deformation of a BTSC: (A-B). initiation; (B-D). Expansion; (D-F). Retraction. The arrows and curve-lines represent the direction and location of membrane deformation, respectively. The snapshots are collected every 8 seconds.

<ol>
	<li>Carke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Stem+Cells-Perspectives+on+Current+Status+and+Future+Directions.">Cancer Stem Cells-Perspectives on Current Status and Future Directions.</a> AACR Workshop on Cancer Stem Cells. Cancer Research. 66, 9339-9344 (2006).</li><li>Stupp, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiotherapy+plus+concomitant+and+adjuvant+temozolomide+for+glioblastoma.">Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.</a> New England Journal of Medicine. 352, 987-996 (2005).</li><li>Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+cancer+cell+invasion.">Mechanisms of cancer cell invasion.</a> Current Opinion in Genetics &#38; Development. 15, 87-96 (2005).</li><li>Shackleton, M., Quintana, E., Fearon, E. R., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+Cancer:+Cancer+Stem+Cells+versus+Clonal+Evolution.">Heterogeneity in Cancer: Cancer Stem Cells versus Clonal Evolution.</a> Cell. 138, 822-829 (2009).</li><li>Karnoub, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+within+tumour+stroma+promote+breast+cancer+metastasis.">Mesenchymal stem cells within tumour stroma promote breast cancer metastasis.</a> Nature. 449, 557-557 (2007).</li><li>Huang, Y., Agrawal, B., Sun, D., Kuo, J. S., Williams, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics-based+Devices:+New+Tools+for+Studying+Cancer+and+Cancer+Stem+Cell+Migration.">Microfluidics-based Devices: New Tools for Studying Cancer and Cancer Stem Cell Migration.</a> Biomicrofluidics. 5, (2011).</li><li>Rolli, C. G., Seufferlein, T., Kemkemer, R., Spatz, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Tumor+Cell+Cytoskeleton+Organization+on+Invasiveness+and+Migration:+A+Microchannel-Based+Approach.">Impact of Tumor Cell Cytoskeleton Organization on Invasiveness and Migration: A Microchannel-Based Approach.</a> Plos One. 5, (2010).</li><li>Chung, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+into+scaffolds+under+co-culture+conditions+in+a+microfluidic+platform.">Cell migration into scaffolds under co-culture conditions in a microfluidic platform.</a> Lab on a Chip. 9, 269-275 (2009).</li><li>Irimia, D., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+migration+of+cancer+cells+under+conditions+of+mechanical+confinement.">Spontaneous migration of cancer cells under conditions of mechanical confinement.</a> Integrative Biology. 1, 506-512 (2009).</li><li>Svendsen, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+rapid+and+long+term+growth+of+human+neural+precursor+cells.">A new method for the rapid and long term growth of human neural precursor cells.</a> Journal of Neuroscience Methods. 85, 141-152 (1998).</li><li>Clark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma+cancer+stem+cells+exhibit+decreased+dependence+on+exogenous+growth+factors+for+proliferation+and+survival.">Glioblastoma cancer stem cells exhibit decreased dependence on exogenous growth factors for proliferation and survival.</a> , .</li><li>Xia, Y. N., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography.">Soft lithography.</a> Angewandte Chemie-International Edition. 37, 551-575 (1998).</li><li>Millet, L. J., Stewart, M. E., Sweedler, J. V., Nuzzo, R. G., Gillette, M. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+culturing+primary+mammalian+neurons+at+low+densities.">Microfluidic devices for culturing primary mammalian neurons at low densities.</a> Lab on a Chip. 7, 987-994 (2007).</li><li>Regehr, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+implications+of+polydimethylsiloxane-based+microfluidic+cell+culture.">Biological implications of polydimethylsiloxane-based microfluidic cell culture.</a> Lab on a Chip. 9, 2132-2139 (2009).</li><li>Dent, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filopodia+are+required+for+cortical+neurite+initiation.">Filopodia are required for cortical neurite initiation.</a> Nature Cell Biology. 9, 1347-1347 (2007).</li><li>Hu, X. D., Viesselmann, C., Nam, S., Merriam, E., Dent, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-Dependent+Dynamic+Microtubule+Invasion+of+Dendritic+Spines.">Activity-Dependent Dynamic Microtubule Invasion of Dendritic Spines.</a> Journal of Neuroscience. 28, 13094-13105 (2008).</li><li>Vitzthum, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+Na(+)/H(+)+exchange-mediated+pH(i)+regulations+in+neuronal+soma+and+neurites+in+compartmentalized+microfluidic+devices.">Study of Na(+)/H(+) exchange-mediated pH(i) regulations in neuronal soma and neurites in compartmentalized microfluidic devices.</a> Integrative Biology. 2, 58-64 (2010).</li><li>Clark, P. A., Treisman, D. M., Ebben, J., Kuo, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+signaling+pathways+in+brain+tumor-derived+stem-like+cells.">Developmental signaling pathways in brain tumor-derived stem-like cells.</a> Developmental Dynamics. 236, 3297-3308 (2007).</li><li>Chen, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Hierarchy+of+Self-Renewing+Tumor-Initiating+Cell+Types+in+Glioblastoma.">A Hierarchy of Self-Renewing Tumor-Initiating Cell Types in Glioblastoma.</a> Cancer Cell. 17, 362-375 (2010).</li><li>Pollard, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioma+Stem+Cell+Lines+Expanded+in+Adherent+Culture+Have+Tumor-Specific+Phenotypes+and+Are+Suitable+for+Chemical+and+Genetic+Screens.">Glioma Stem Cell Lines Expanded in Adherent Culture Have Tumor-Specific Phenotypes and Are Suitable for Chemical and Genetic Screens.</a> Cell Stem Cell. 4, 568-580 (2009).</li><li>Lobo, N. A., Shimono, Y., Qian, D., Clarke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+cancer+stem+cells.">The biology of cancer stem cells.</a> Annual Review of Cell and Developmental Biology. 23, 675-699 (2007).</li><li>Singh, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+human+brain+tumour+initiating+cells.">Identification of human brain tumour initiating cells.</a> Nature. 432, 396-401 (2004).</li><li>Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+stem+cells+derived+from+glioblastomas+cultured+in+bFGF+and+EGF+more+closely+mirror+the+phenotype+and+genotype+of+primary+tumors+than+do+serum-cultured+cell+lines.">Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines.</a> Cancer Cell. 9, 391-403 (2006).</li><li>Galli, R. <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+tumorigenic,+stem-like+neural+precursors+from+human+glioblastoma.">Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma.</a> Cancer Research. 64, 7011-7021 (2004).</li></ol>

No conflicts of interest declared.

The microfluidic device and image-recording technique presented here enables a visual characterization of cellular morphology during migration. Compared to existing conventional methods, the microfluidic platform features advantages of cost-effectiveness, high throughput and design flexibility. The presented microscopic visualization system permits study and recording of long-term live cell migration. The lens-motorized feature makes it possible to track multiple migration paths in a high image-resolution without disturbing the sample.
During assembly of the device, the PDMS stamp is non-permanently bonded to the glass coverslip for the convenience of PDL coating (step 3.4). It is critical to achieve a good seal for the success of this protocol. Contamination introduced from the device fabrication, such as air bubble in the stamp or dust/debris on the substrate, can jeopardize the bonding and result in fluid leaking. Therefore, treating the device in a dust-free manner is important to prevent device failure. Prior to the PDL-coating, the glass coverslips are nitric acid treated and the PDL solution is centrifuged to eliminate dust/debris. We find Scotch tape, alcohol rinse, and water bath very helpful in removing dust/debris from the PDMS stamp, if a clean room is not accessible. After the PDMS stamp making contact with the glass coverslip, tapping the stamp gently with tweezers facilitates the seal.
BTSCs can be enriched from human operating room specimens via sphere culture in stem cell medium, similar to culture of normal neural stem cells10. BTSCs enriched in this manner demonstrate stem cell-like properties, such as expression of stem cell markers (CD133, nestin), differentiation to multiple neural lineages (glial and neuronal), and tumor initiation in an orthotopic immunodeficient mouse model with as few as 100 cells18. Although some debate exists about purification and maintenance of BTSCs1, 19-21, sphere-grown BTSCs better maintain the phenotype and genotype of the parental tumor22-24.
The compartmentalized space mimics the physiological environment for cell migration. In our device, BTSC exhibit a strong capacity of migration through a size-constrained space by regulating cellular morphology. Our characterization of the cell morphological changes indicates mode-transformations in a six stage sequence that may model a possible new detailed description of BTSC invasion of adjacent brain. In early stages, the cells gain a significant amount of polarity and generate adhesive protrusions to effectively anchor themselves inside the microchannel. Once the cell occupancy in the microchannel is established, it converts to a cruising mode and maintains this mode for the entire journey through a microchannel. At this stage, BTSC maintain high motility and consistent direction but conserve energy. Interestingly, it appears that one microchannel is restricted to one cruising cell at a time, such that when a single cell proceeds to this stage, other cells retreat from the microchannel. This mechanism likely ensures effectiveness of tumor spreading and prevents overconsumption of nutrients in microchannel. After completing migration across the microchannel, a cell regains its propensity for multidirectional protrusions and further exploration for invasion targets or new migration paths. The compartmentalizing microfluidic device offers a novel in vitro means of creating a microenvironment for studying BTSC infiltration of brain parenchyma. 
This method can easily be adapted to the study of cancer stem cell (CSC) and migratory cell lines derived from other tumor types. Culturing cells in the microfluidic device can be performed in any modern biology laboratory that is equipped with an incubator and tissue culture expertise. Equipped with a su-8 master, PDMS casting and device assembly are feasible with some fundamental training in soft lithography. In addition, this platform may also be extended for other applications with integrating other functional modules such as gradient mixer, surface patterning, fluid control and microelectrode.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Dulbecco's modified eagle's medium (DMEM), high glucose</td>
			<td>Gibco / Invitrogen</td>
			<td>11965</td>
			<td>Brain tumor stem cell (BTSC) culture supplies</td>
		</tr>
		<tr>
			<td>Ham&#x2019;s F12</td>
			<td>Gibco / Invitrogen</td>
			<td>31765</td>
			<td>Brain tumor stem cell (BTSC) culture supplies</td>
		</tr>
		<tr>
			<td>B27 supplement minus vitamin A</td>
			<td>Gibco / Invitrogen</td>
			<td>12587-010</td>
			<td>Brain tumor stem cell (BTSC) culture supplies</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (PSA)</td>
			<td>Gibco / Invitrogen</td>
			<td>15240</td>
			<td>Brain tumor stem cell (BTSC) culture supplies</td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF), human recombinant</td>
			<td>Gibco / Invitrogen</td>
			<td>PHG0313</td>
			<td>Brain tumor stem cell (BTSC) culture supplies</td>
		</tr>
		<tr>
			<td>basic Fibroblast Growth Factor (bFGF) , human recombinant</td>
			<td>Gibco / Invitrogen</td>
			<td>PHG0021</td>
			<td>Brain tumor stem cell (BTSC) culture supplies</td>
		</tr>
		<tr>
			<td>Heparin sodium salt, from porcine intestinal mucosa</td>
			<td>Sigma</td>
			<td>H1027-250KU</td>
			<td>Brain tumor stem cell (BTSC) culture supplies</td>
		</tr>
		<tr>
			<td>Laminin (natural mouse)</td>
			<td>Gibco / Invitrogen</td>
			<td>23017-015</td>
			<td>Brain tumor stem cell (BTSC) culture supplies</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Millipore</td>
			<td>SCR005</td>
			<td>Brain tumor stem cell (BTSC) culture supplies</td>
		</tr>
		<tr>
			<td>Stem cell medium</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td><ul>
				<li>30 % Hams F12 </li>
				<li>70% DMEM</li>
				<li>1% antibiotic-antimycotic</li>
				<li>2% B27 without vitamin A</li>
				<li>EGF (20 ng/mL) and bFGF/heparin (20 ng/mL bFGF, 5 mg/ml heparin) Mix ingredients and sterile filter before use</li>
			</ul></td>
		</tr>
		<tr>
			<td>Basic Fibroblast Growth Factor (bFGF)/ Heparin</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td><ul>
				<li>Aliquot concentration 20 &mu;g/ml</li>
				<li>For 25 &mu;g: Add 1.25 mL of 5mg/mL Heparin to 25 &mu;g bFGF.</li>
				<li>For 250 &mu;g: Add 1 mL 5mg/mL Heparin to 250 &mu;g bFGF. Transfer to 15 mL conical and add 11.5 mL 5 mg/mL Heparin to conical. </li>
				<li>Store as 50 &mu;l or 250 &mu;l aliquots at -80&deg;C.</li>
			</ul></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td><ul>
				<li>Aliquot concentration 20 &mu;g/ml</li>
				<li>For 200 &mu;g: Add 1 mL sterile DMEM (Gibco 11965-118) to 200 &mu;g EGF. Transfer to 15 mL conical and add 9 mL DMEM.</li>
				<li>For 500 &mu;g: Add 1 mL sterile DMEM (Gibco 11965-118) to 200 &mu;g EGF. Transfer to 50 mL conical and add 24 mL DMEM.
				<li>Store as 50 &mu;l or 250 &mu;l aliquots at -80&deg;C. </li>
			</ul></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td><ul>
				<li>Aliquot concentration 5 mg/ml</li>
				<li>Weigh out 100 mg heparin.</li>
				<li>Add 100 mg heparin to 20 mL (70%)DMEM-(30%)F12-(1%)PSA (14 mL DMEM, 6 mL F12, 200 &mu;L PSA)</li>
				<li>Sterile filter</li>
				<li>Store as 50 &mu;l or 250 &mu;l aliquots at -80&deg;C</li>
			</ul></td>
		</tr>
		<tr>
			<td>su-8 photoresist</td>
			<td>MicroChem</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Silicon handle wafer</td>
			<td>WRS Materials</td>
			<td>3P01-5SSP-INV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>trichloro(1H,1H,2H,2H-perfluorooctyl)silane</td>
			<td>Sigma-Aldrich</td>
			<td>448931</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PDMS Sylgard 184</td>
			<td>Dow Corning</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>laminin</td>
			<td>BD Bioscience</td>
			<td>&nbsp;</td>
			<td>50 &mu;g/ml in PBS buffer for the final concentration</td>
		</tr>
		<tr>
			<td>Biostation IM</td>
			<td>Nikon instruments</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

evaluation of cancer stem cell migration using compartmentalizing microfluidic devices and live cell imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

Watch this Scientific Journal Video about Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging at JoVE.com

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

evaluation-cancer-stem-cell-migration-using-compartmentalizing

Research

JoVE Journal

Medicine

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.
A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure1,2, alterations in brain vasculature of a Alzheimer's disease mouse model3, portal vein abnormalities in portal hypertensive and cirrhotic mice4, developmental steps in rat lymphatic maturation between immature and adult lungs5, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury6.
Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...