This work was funded by the Ontario Institute of Cancer Research (OICR), the Terry Fox Foundation and the American Association of Neurological Surgeons.

1. Culture of Brain Tumor Tissue
<ol>
 <li>Add 200 &mu;l thawed Liberase (Roche Applied Science) to 15 ml of artificial CSF (aCSF- see Table 1) and place into 37 &deg;C water bath. Liberase TM is a mix of proteolytic enzymes used to dissociate primary tissue samples, as well as cultured tumorspheres. Unlike Trypsin-EDTA, the Liberase method preserves the surface antigen CD133. For a tissue sample of about 0.5 cm3, we use 200 &mu;l of Liberase. If the tissue is smaller, we use 100 ul.</li>
 <li>Bring ammonium chloride solution (Stem cell technologies) to room temperature. Ammonium Chloride solution gently lyses red blood cells with minimal effect on other cells. It does not contain a fixative.</li>
 <li>In sterile biological safety cabinet, add 5 ml of aCSF to specimen container, swirl to rinse tissue, then pipette off. This step helps to remove red blood cells (RBC).</li>
 <li>Transfer brain tumor tissue to a sterile 100 mm Petri dish.</li>
 <li>Using fine scissors or scalpels and forceps, disaggregate tissue to slurry consistency.</li>
 <li>Collect sample using a 10 ml regular pipette or forceps and transfer fragments into the tube containing pre-warmed aCSF with Liberase.</li>
 <li>Place on incubator-shaker (30 rpm) and set to 37 &deg;C, for 15 min.</li>
 <li>Filter the tissue lysate through 70 &mu;m cell strainer into a 50 ml Falcon tube.</li>
 <li>Spin the filtrate down at 280 x g for 5 min.</li>
 <li>Remove supernatant carefully and evaluate size and colour of the resulting cell pellet: pellets which are pink or red indicate increasing numbers of red blood cells.</li>
 <li>Resuspend pellet in 1 ml PBS.</li>
 <li>Add an appropriate amount of ammonium chloride solution (4-12 ml) based on pellet size and red cell contamination (the ammonium chloride solution is very gentle and increased amounts are not harmful to cells other than red cells).</li>
 <li>Incubate at room temperature for 5 min.</li>
 <li>Spin cells down at 280 x g for 5 min.</li>
 <li>Wash once with 10 ml of sterile PBS.</li>
 <li>Resuspend in 5 ml NSC complete medium (Table 2) and transfer to an ultra-low binding 60 mm tissue culture plate (Corning). We use ultra-low binding culture plates with covalently bonded hydrogel surfaces that are hydrophilic and neutrally charged, to minimize cell attachment-mediated differentiation.</li>
</ol>
For the initial days in culture, do not change the media: top-up only with 1-2 ml media as needed, then continue to observe culture and change media when the color of the media becomes slightly yellow.
2. Tumorsphere Dissociation for Flow Cytometry
<ol>
 <li>Evaluate tumorspheres under microscope: if the sphere size is &gt;100 &mu;m, dissociation is recommended as larger spheres may become necrotic in the center.</li>
 <li>Transfer culture to 15 ml conical tube.</li>
 <li>Add 2-3 ml sterile PBS to rinse plate thoroughly and add to conical tube.</li>
 <li>Centrifuge at 280 x g for 5 min.</li>
 <li>Remove supernatant and resuspend in 1- 2 ml sterile PBS.</li>
 <li>Add 10 &mu;l Liberase.</li>
 <li>Incubate in 37 &deg;C water bath for 3 min. Remove and visually evaluate suspension, if multiple clumps seen, triturate gently using a 1,000 &mu;l pipette tip.</li>
 <li>If clumping persists, continue incubation for an additional 1-2 min.</li>
 <li>Wash the cells by adding 5 ml sterile PBS and centrifuging at 280 x g for 5 min.</li>
 <li>Remove supernatant and resuspend in 500-1,000 &mu;l sterile PBS+2 mM EDTA.</li>
 <li>Assess cell number and viability using trypan blue.</li>
 <li>Adjust cell count to 1 million/ml in PBS+2 mM EDTA.</li>
</ol>
3. Surface Staining
<ol>
 <li>Transfer 100 &mu;l of 1x106/ml cell suspension per test to 12x75 mm flow tubes.</li>
 <li>Add appropriate amount of antibodies. Matched Isotype controls should be used for each antibody (see Table 3).</li>
 <li>Incubate for 30 min on ice.</li>
 <li>Add 2 ml PBS+2 mM EDTA to each flow tube.</li>
 <li>Centrifuge at 200 x g for 4 min, decant and blot.</li>
 <li>Resuspend pellet in 300 &mu;l PBS+2 mM EDTA.</li>
 <li>Add 10 &mu;l 7AAD viability dye to each tube and incubate for at least 15 min on ice.</li>
 <li>Analyze by flow cytometry.</li>
</ol>
4. Flow Cytometry Acquisition and Analysis
The specifics of acquisition and analysis of flow data are instrument-dependent. Representative negative samples are run and instrument settings established for Forward (size) and Side (granularity) scatter. This scatter pattern allows the end-user to view all cells in the sample, including debris. A region is usually drawn around the cells of interest. (Figure 4a) Settings are then established for all fluorescence detectors required to position the negative cells within the first decade of a fluorescence intensity plot. When more than one dye or fluorochrome is used, single stained controls must be run to establish color compensation values (subtraction of interfering fluorescence emissions). When running live cells, a viability dye such as 7-AAD (7-Amino-actinomycin D - excitation 488/emission 655) is used and a second region drawn to exclude dead cells (Figure 4b). Both of these regions are applied to any further analysis of the samples.
5. Self-renewal Assay
The key assay that is greatly facilitated by clonal sphere growth is the in vitro test of self-renewal capacity, the limiting dilution assay. Tumorspheres are dissociated and distributed at dilutions down to a single cell per well, and the rate of subsequent sphere formation over 7 days in culture is calculated. On day 7, the percentage of wells not containing spheres for each cell plating density (F0) is calculated and plotted against the number of cells per well (x). The number of cells required to form at least one tumorsphere in every well is determined from the point at which the line crosses the 0.37 level (F0 = e -x). In a Poisson distribution of cells, F0 = 0.37 corresponds to the dilution of one cell per well, so that this calculation (the 37% intersect) reflects the frequency of clonogenic stem cells in the whole cell population (Figure 5).
6. Single Cell RT-PCR
Single cells from different populations are sorted by Beckman Coulter MoFlo XDP on reaction sites on an AmpliGrid slide (Beckman Coulter cat#AG480F). Single cell RT-PCR is performed using AmpliGrid Single One Step RT-PCR system (Beckman Coulter cat#OAX04515) according to manufacturer's specifications. Briefly, a single cell is deposited into each reaction site of an AmpliGrid slide; the presence of a single cell in each reaction site confirmed by microscopy. Reverse transcription (RT) is performed immediately after sorting to prevent the sample from being compromised. The RT master mix is prepared fresh, according to kit instructions (Table 4). We used 0.03 &mu;l of 25 &mu;M random primers (Promega) per reaction; 1 &mu;l of master mix is added to the centre of each reaction site. This is immediately coated with 5 &mu;l of sealing solution (Beckman Coulter cat#OAX04503). Using an AmpliSpeed slide cycler (Beckman Coulter; cat#OAX04101), slides are incubated at 25 &deg;C for 10 min, 42 &deg;C for 10 min, and 55 &deg;C for 45 min. After reverse transcription, 3 &mu;l of DNase/RNase-free water is added to each reaction site, and the entire mixture transferred to a PCR tube (1 tube/site). In a PCR plate, a reaction volume of 10 &mu;l is used for quantitative PCR: 8 &mu;l qPCR master mix (5 &mu;l Sybr Green (Quanta), 1 &mu;l water, 1 &mu;l of 10 &mu;M forward and reverse primer mix) plus 2 &mu;l of the RT mixture. For real-time quantification, samples are run on a BioRad cycler using the following program: 95 &deg;C for 10 min, 45 cycles of 95 &deg;C for 30 sec, 60 &deg;C for 60 sec, 72 &deg;C for 60 sec, followed by 10 min at 72 &deg;C and melting curve analysis. Ct values were determined using Opticon Monitor 3 (BioRad). Three genes can be assessed per cell, including GAPDH as a housekeeping gene. Gene expression is normalized to GAPDH expression, according to 2-&Delta;Ct. At least a dozen single cells of the same population from the same sort can be used as biological replicates to compensate for lack of technical replicates.
7. Representative Results
Figure 1 shows a Magnetic Resonance Imaging (MRI) scan of a representative patient with a GBM. Brain tumor samples are obtained from consenting patients immediately after surgery, as approved by the Hamilton Health Sciences/McMaster Health Sciences Research Ethics Board. A part of each specimen is given to the neuropathologist for routine clinical diagnosis. The remaining sample is processed as shown in Figure 2. The tumor cells, once plated in complete neural stem cell media with growth factors, form tumorspheres as shown in Figure 3.
The tumor cells are labeled with neural surface stem cell markers CD133 and CD15 and analyzed by flow cytometry as shown in Figure 4.
Single cells from different populations for e.g., CD15+/CD133+, CD15+/CD133-, CD15-/CD133+, CD15-/CD133- are then sorted into 96 well plates (Figure 5a) or into AmpliGrid slides (Figure 6a).
In the 96 well plate, single cells which possess self-renewal capacity (e.g. CD133+ cells), (Figure 5b), can form tumorspheres (Figure 5c) following incubation. A self-renewal graph can be plotted (Figure 5d) by counting the number of spheres formed per well. Single cells sorted into the reaction site of the Ampligrid slide (Figure 6a). The RNA extracted from single cells can be reverse transcribed using the Advalytix AmpliSpeed (Figure 6b). The cDNA obtained is used to perform Real time RT-PCR (Figure 6c).
<img alt="Figure 1" src="/files/ftp_upload/4111/4111fig1.jpg" /> 
Figure 1. Magnetic resonance image (MRI) of a patient GBM. MRI scan of the brain of a 16-year old girl with a month-long history of headache and a one week history of vomiting, lethargy and visual blurring. Axial FLAIR sequence shows a large bi-hemispheric ("butterfly") GBM.
<img alt="Figure 2" src="/files/ftp_upload/4111/4111fig2.jpg" /> 
Figure 2. Brain tumor processing. Brain tumor samples are obtained from consenting patients immediately after surgery. They are mechanically dissociated as shown (a) and are then enzymatically dissociated in aCSF with Liberase by incubating in a 30 rpm rocking incubator at 37 &deg;C for 15 min (b).
<img alt="Figure 3" src="/files/ftp_upload/4111/4111fig3.jpg" /> 
Figure 3. BTIC populations form tumorspheres in culture. Dissociated brain tumor cells plated in complete neural stem cell media with growth factors form tumorspheres.
<img alt="Figure 4" src="/files/ftp_upload/4111/4111fig4.jpg" /> 
Figure 4. Flow cytometric analysis of BTIC populations. (a) Forward versus side scatter properties provide an image of all cells, including debris (b) Cells which stain with the viability dye 7-AAD are excluded from analysis. (c) The position of the statistical quadrants is determined using appropriate Isotype controls (d) Tumor cells stained with surface markers CD15-PE and CD133-APC. (d) Moflo XDP cell sorter. <a href="https://www.jove.com/files/ftp_upload/4111/4111fig4large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 5" src="/files/ftp_upload/4111/4111fig5.jpg" /> 
Figure 5. Limiting dilution analysis of BTICs reveals their self-renewal capacity. (a) Cells of varying dilutions are sorted into 96 wells containing 200 &mu;l of NSC to (b) a single cell per well. (c) Following a 7 day incubation period, a tumorsphere is formed. The morphology of secondary tumorspheres is identical to that of primary tumorspheres. (d) The mean x-intercept values can be calculated from limiting dilution analysis for each tumor subtype to reveal the number of cells required to form at least one tumorsphere per well.
<img alt="Figure 6" src="/files/ftp_upload/4111/4111fig6.jpg" /> 
Figure 6. Gene expression analysis of a single BTIC cell is possible using single cell RT-PCR technology. (a) Single cells from a BTIC population can be sorted onto AmpliGrid slides. (b) cDNA synthesis is performed on single cells on AmpliGrid slides using the Advalytix AmpliSpeed. (c) Quantitative Realtime PCR is performed on the cDNA synthesized from single BTIC cells.
<table border="1">
 <tbody>
 <tr>
 <td>2M NaCl</td>
 <td>62 ml</td>
 </tr>
 <tr>
 <td>1M KCl</td>
 <td>5 ml</td>
 </tr>
 <tr>
 <td>1M MgCl2</td>
 <td>3.2 ml</td>
 </tr>
 <tr>
 <td>155 mM NaHCO3</td>
 <td>169 ml</td>
 </tr>
 <tr>
 <td>1M Glucose</td>
 <td>10 ml</td>
 </tr>
 <tr>
 <td>108 mM CaCl2</td>
 <td>0.9256 ml</td>
 </tr>
 <tr>
 <td>Purelab Ultra H20</td>
 <td>749.84 ml</td>
 </tr>
 </tbody>
</table>
Table 1. Artificial Cerebrospinal Fluid (aCSF) - 1,000 ml.
<table border="1">
 <tbody>
 <tr>
 <td>Stock Basal media</td>
 <td>500 ml </td>
 </tr>
 <tr>
 <td>1:1 DMEM:F12</td>
 <td>480 ml</td>
 </tr>
 <tr>
 <td>N2 supplement</td>
 <td>5 ml</td>
 </tr>
 <tr>
 <td>1M HEPES</td>
 <td>5 ml</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>3.0 g</td>
 </tr>
 <tr>
 <td>N-acetylcysteine (60 &mu;g/ml)</td>
 <td>1 ml</td>
 </tr>
 <tr>
 <td>Neural survival factor-1 (NSF-1)</td>
 <td>10 ml</td>
 </tr>
 </tbody>
</table>
Table 2. Neural stem cell media.
NSC complete media (made fresh prior to use): 
NSC basal media + 20 ng/ml EGF (epidermal growth factor) + 20 ng/ml bFGF (basic fibroblast growth factor) + 10 ng/ml LIF (leukemia inhibitory factor)* + 10 &mu;l/ml antibiotic-antimycotic
*Leukemia inhibitory factor (LIF): This reagent from Millipore contains an interleukin 6 class cytokine, a protein which suppresses spontaneous differentiation of stem cells promoting longer term maintenance of stem cell cultures.
<table border="1">
 <tbody>
 <tr>
 <td>Antibodies</td>
 <td>Supplier/CAT#</td>
 <td>&mu;l/test (in 100 &mu;l)</td>
 </tr>
 <tr>
 <td>CD133/2 APC (293C3)</td>
 <td>Miltenyi Biotec/130-090-854</td>
 <td>5</td>
 </tr>
 <tr>
 <td>IgG2b APC (Isotype control)</td>
 <td>Miltenyi Biotec/130-092-217</td>
 <td>5</td>
 </tr>
 <tr>
 <td>CD15PE</td>
 <td>Beckman Coulter/IM1954U</td>
 <td>5</td>
 </tr>
 <tr>
 <td>IgG2a PE (Isotype control)</td>
 <td>Beckman Coulter/A09141</td>
 <td>5</td>
 </tr>
 <tr>
 <td>7AAD</td>
 <td>Beckman Coulter/A07704</td>
 <td>10</td>
 </tr>
 </tbody>
</table>
Table 3. Flow antibodies.
<table border="1">
 <tbody>
 <tr>
 <td>Component</td>
 <td>Volume (1 reaction)</td>
 <td>Volume ( 48 reaction)</td>
 </tr>
 <tr>
 <td>2x Single Cell RT reaction Buffer</td>
 <td>0.50 &mu;l</td>
 <td>30.0 &mu;l</td>
 </tr>
 <tr>
 <td>RNase Inhibitor (10 U/&mu;l)</td>
 <td>0.02 &mu;l</td>
 <td>1.2 &mu;l</td>
 </tr>
 <tr>
 <td>5X Single Cell RT enhancer</td>
 <td>0.15 &mu;l</td>
 <td>9.00 &mu;l</td>
 </tr>
 <tr>
 <td>Single Cell RT Enzyme Mix</td>
 <td>0.04 &mu;l</td>
 <td>2.4 &mu;l</td>
 </tr>
 <tr>
 <td>Advablue (OAX04227)</td>
 <td>0.1 &mu;l</td>
 <td>6.00 &mu;l</td>
 </tr>
 <tr>
 <td>Nuclease free water</td>
 <td>make up to 1 &mu;l</td>
 <td>make up to 60 &mu;l</td>
 </tr>
 </tbody>
</table>
Table 4. Composition of RT master mix for Single Cell RTPCR (cat# OAX04515).

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+does+not+predict+tumor+growth+potential+in+mouse+models+of+high-grade+glioma.">Self-renewal does not predict tumor growth potential in mouse models of high-grade glioma.</a> Cancer Cell. 21, 11 (2012).</li><li>Venugopal, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmi1+marks+intermediate+precursors+during+differentiation+of+human+brain+tumor+initiating+cells.">Bmi1 marks intermediate precursors during differentiation of human brain tumor initiating cells.</a> Stem Cell Res. 8, 141 (2012).</li><li>Gerlinger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumor+heterogeneity+and+branched+evolution+revealed+by+multiregion+sequencing.">Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.</a> N. 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J., KAErn, M., Cantor, C. R., Collins, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise+in+eukaryotic+gene+expression.">Noise in eukaryotic gene expression.</a> Nature. 422, 633 (2003).</li><li>Elowitz, M. B., Levine, A. J., Siggia, E. D., Swain, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+gene+expression+in+a+single+cell.">Stochastic gene expression in a single cell.</a> Science. 297, 1183 (2002).</li><li>Maheshri, N., O'Shea, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+with+noisy+genes:+how+cells+function+reliably+with+inherent+variability+in+gene+expression.">Living with noisy genes: how cells function reliably with inherent variability in gene expression.</a> Annu. Rev. Biophys. Biomol. Struct. 36, 413 (2007).</li><li>Raj, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+mRNA+synthesis+in+mammalian+cells.">Stochastic mRNA synthesis in mammalian cells.</a> PLoS Biol. 4, e309 (2006).</li><li>Ross, I. L., Browne, C. M., Hume, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+of+individual+genes+in+eukaryotic+cells+occurs+randomly+and+infrequently.">Transcription of individual genes in eukaryotic cells occurs randomly and infrequently.</a> Immunol. Cell Biol. 72, 177 (1994).</li><li>Kubista, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+real-time+polymerase+chain+reaction.">The real-time polymerase chain reaction.</a> Mol. 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No conflicts of interest declared.

The cancer stem cell hypothesis10, based on work in leukemia21, breast cancer11 and brain cancer 4,5, suggests that only a relatively small fraction of cells in the tumor, termed cancer stem cells, possess an ability to extensively proliferate and self-renew. Most of the tumor cells lose the ability to proliferate and self-renew as they differentiate into cells that become the phenotypic signature of the tumor. Finding the key cells in the brain tumor population that are able t...

Brain tumors are among the most aggressive and heterogeneous cancers known in humans. Although their earlier detection and diagnosis have been facilitated by modern neuro-imaging technology, we still lack curative therapies for many brain tumors, particularly for diffuse, invasive ones or those located deep in the brain.
Brain tumors represent the leading cause of cancer mortality in children due to their highly aggressive and often incurable nature. Glioblastoma (GBM), the most common primary brain tumor in adults, is one of the most aggressive human cancers, feared for its uniformly fatal prognosis1. This highly malignant astrocytic tumor (WHO Grade 4) usually occurs in the cerebral hemispheres of adults, and can also occur in young children and infants. Its growth is rapid and infiltrative, and diagnostic pathological features include nuclear pleomorphism, microvascular proliferation, and necrosis2,3. For adults with newly diagnosed GBM, median survival rarely extends beyond 12 months1, with generally poor responses to all therapeutic modalities. We noted that there are many functional and genetic similarities shared by somatic stem cells and cancer cells, and that the molecular pathways that regulate normal brain development are often dysregulated in cancer. In applying stem cell biology paradigms to the study of brain tumors, we were the first researchers to prospectively identify and purify a subpopulation of cells from human GBMs which exhibited the stem cell properties of proliferation, self-renewal, and differentiation in vitro4 and in vivo5. We applied culture conditions and assays originally used to characterize normal neural stem cells (NSCs) in vitro 6,7 to multiple pediatric and adult brain tumors, and enriched for these stem-like cells by cell sorting for the neural progenitor cell surface marker CD1338,9. The CD133+ brain tumor fraction contained cells that had a much higher frequency of tumor initiation than the CD133- fraction in NOD-SCID mouse brains5,10. This formally established that only a rare subset of brain tumor cells with stem cell properties are tumor-initiating, earning them the name "brain tumor initiating cells" or "BTICs". The novel identification of BTICs provides new insights into human brain tumorigenesis, giving strong support for the cancer stem cell hypothesis10-13 as the basis for many solid tumors, and establishes a novel cellular target for more effective cancer therapies14-20. Therapies that focus on killing the bulk of the tumor may miss the rare stem-like fraction, allowing the tumor to continue to grow. Therapies that focus on killing the cancer stem cell may provide better treatment and prognosis for patients with brain tumors.
In order to study BTIC populations, we have refined our culture protocols to specifically select for cell populations within human brain tumors that possess stem cell properties. Serum-free, neural stem cell (NSC) medium allows for the maintenance of an undifferentiated stem cell state, and the addition of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and leukemia inhibitory factor (LIF) allows for the proliferation of multi-potent, self-renewing, and expandable human tumorspheres. Here, we describe the methods involved in processing of primary brain tumors and culturing them in NSC medium to enrich for BTIC populations. We have called our experimental model system "BTIC patient isolates" to emphasize the fact that these cells are only minimally cultured under stem cell conditions to select for stem cell populations. Subsequent immunolabelling of BTIC populations for key stem cell markers such as CD133 and CD15 and flow cytometry analysis is also described. We then discuss the limiting dilution analysis, which aids in studying the self-renewal potential of BTICs. Finally, we explore the gene expression analysis of these rare cells by sorting single cells onto AmpliGrid slides and performing single cell RT-PCR. These techniques are also applicable to other brain tumors such as medulloblastoma, ependymoma and pediatric gliomas.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>1:1 DMEM:F12</td>
 <td>Invitrogen</td>
 <td>11320-082</td>
 </tr>
 <tr>
 <td>N2 supplement</td>
 <td>Invitrogen</td>
 <td>17502-048</td>
 </tr>
 <tr>
 <td>1M HEPES</td>
 <td>Wisent</td>
 <td>330-050-EL</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>Invitrogen</td>
 <td>15023-021</td>
 </tr>
 <tr>
 <td>N-acetylcysteine</td>
 <td>Sigma Aldrich</td>
 <td>A9165-25g</td>
 </tr>
 <tr>
 <td>Neural survival factor -1 (NSF-1)</td>
 <td>Lonza Clonetics</td>
 <td>CC-4323</td>
 </tr>
 <tr>
 <td>Epidermal growth factor (EGF)</td>
 <td>Sigma Aldrich</td>
 <td>E9644</td>
 </tr>
 <tr>
 <td>Basic fibroblast growth factor (bFGF)</td>
 <td>Invitrogen</td>
 <td>PHG0261</td>
 </tr>
 <tr>
 <td>Leukemia inhibitory factor (LIF)</td>
 <td>Millipore</td>
 <td>LIF1010</td>
 </tr>
 <tr>
 <td>Antibiotic/mycotic</td>
 <td>Wisent</td>
 <td>450-115-EL</td>
 </tr>
 <tr>
 <td>Liberase TM</td>
 <td>Roche</td>
 <td>05 401 119 001</td>
 </tr>
 <tr>
 <td>Ammonium chloride solution</td>
 <td>Stem Cell Technologies</td>
 <td>07850</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting at JoVE.com

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

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 ...

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17.9K Views.  McMaster University. 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.

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

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer1-7. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens7. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to na&iuml;ve counterparts7-10. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression11,12. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.
Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to na&iuml;ve MDSC 13. MDSC percentages are significantly less in lymphoid compartments of na&iuml;ve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1+ leukocytes from na&iuml;ve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1+ leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1+ leukocytes from spleens of na&iuml;ve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas na&iuml;ve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from na&iuml;ve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1+ leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1+ enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (na&iuml;ve vs. tumor- bearing) in in vivo and in vitro assays.

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

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 ...

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 ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...