Name: Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay (Video) | JoVE Journal
Uploaded: 2011-10-30T14:00:00.000+00:00
Duration: 9 min 24 s
Description: Learn to isolate and expand stem-like cells from human glioblastoma multiforme tumor tissue using the neurosphere assay culture method. This protocol enables researchers to study tumor biology and identify therapeutic targets.

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This work was supported by Grants from the Florida Centre for Brain Tumor Research; Preston A. Wells Jr. Center for Brain Tumor Therapy.

1. Collection of Primary GBM Tissue
<ol>
	<li>A glioblastoma mutliforme (GBM) tumor is obtained from a patient diagnosed with the cancer and undergoing surgery. </li>
	<li>Arrangements must be made with neurosurgery team. The neurosurgeon will place the resected GBM tumor in an appropriate size tube containing neural stem cell (NSC) basal medium supplemented with 10-15% antibiotics (Peniciline/Streptomycine). Cold medium must be provided to operating room by lab. Alternatively, cold HEPES-buffered minimum essential medium (HEM) or phosphate buffered saline (PBS) with high concentration of antibiotics can also be used for this purpose.</li>
	<li>The resected tumor tissue is delivered to the lab on ice and placed under the hood.</li>
</ol>
2. Dissociation of Primary Tumor into Single-Cell Suspension
<ol>
	<li>Excess of the original medium/PBS is removed from the falcon tube and the sample is washed 2-3 times with 5-10ml of PBS/NSC basal medium to remove blood and debris. The PBS/Medium is removed and the GBM tumor tissue is placed in a Petri dish. </li>
	<li>The tissue is cut into small pieces and minced with a No. 10 scalpel blade into tiny pieces to increase the surface area for trypsinization process. Mincing can take 1-3 minutes depending on the size of the tumor. </li>
	<li>The minced tissue is trypsinized in 3-5ml of pre-warmed %0.05 trypsin-EDTA for 10-15 minutes at a 37°C water bath. An electronic pipette is used to transfer the tiny tumor pieces and trypsin into a 15ml Falcon tube. </li>
	<li>An equal volume of soybean trypsin inhibitor is added to stop the enzymatic trypsin reaction after the incubation period.</li>
	<li>Trypsin inactivation is ensured by pipetting the suspension up and down several times. Then, the suspension is pelleted down by centrifuging at 800rpm (110g) for 5min.</li>
	<li>The supernatant is discarded and the tissue pieces are resuspended in 1ml of sterile NSC basal medium. The clumps are dissociated by gently pipetting up and down (3-7 times) until a smooth milky single cell suspension is achieved. The number of pipetting steps directly depends on the size of particles in the minced tissue. Lengthy and vigorous mechanical dissociation should be avoided as it might result in cell death and a reduction in sphere formation.</li>
	<li>To remove un-dissociated pieces and debris, 10-15 ml of basal medium is added to the tube and the cell suspension is filtered through a 40 micron cell strainer into a 50ml tube.</li>
	<li>The filtered suspension is centrifuged at 800rpm (110g) for 5min. The supernatant is discarded afterwards.</li>
	<li>Pelletted cells are then resuspended in 1-2ml of complete NSC medium for cell counting. </li>
</ol>
3. Cell Count and Plating
<ol>
	<li>10 μL of the cell suspension is added to 90 μL of 0.04% Trypan blue in a 1ml eppendorf tube. 
	Note: other appropriate cell dilutions may also be used.</li>
	<li>Pipette up and down to mix the suspension. 10 μL of the cells/trypan blue mixture is transferred to hemocytometer in order to count cell density.</li>
	<li>Cells are plated in complete NSC medium (a mixture of NSC basal medium and NSC proliferation supplement at a 9:1 ratio) supplemented with 20ng/ml EGF, 10ng/ml bFGF and 1μl/ml of 0.2% heparin (2μg/ml) in appropriate tissue culture vessels. 5, 20 and 40 ml of medium is used for T25, T80 and T175 flasks, respectively. Antibiotics may be added to the medium at a concentration of 1:100 to decrease chance of contamination.</li>
	<li>The flask is placed in an incubator set at 37°C and 5% CO2.</li>
</ol>
4. Passaging and expansion of GBM derived spheres:
<ol>
	<li>When the neurospheres reached an average size of 150-200 μm in diameter, the culture is ready for subculture. The content of each flask is removed and placed in an appropriate size sterile tissue culture tube, and centrifuged at 800 rpm (110 g) for 5 min at room temperature.</li>
	<li>The supernatant is removed and the pellet is resuspended in 1 ml of %0.05 trypsin-EDTA.</li>
	<li>To achieve an optimal trypsinization, the cell suspension is incubated at 37°C in a water bath for 2-3 min. To stop the trypsin activity an equal volume of soybean trypsin inhibitor is added to the cell suspension and the cell suspension is gently pipetted up and down.</li>
	<li>The cell suspension is centrifuged at 800 rpm (110g) for 5 min. Then, the supernatant is removed and the cells are resuspended in 1 ml of NSC medium. 	</li>
	<li>A cell count is performed as described earlier.</li>
	<li>The cells are plated at a concentration of 5x104 cells/ml in complete NSC medium supplemented with growth factors and plated in appropriate size tissue culture vessels as described in previous part.</li>
	<li>Secondary neurospheres are formed in 7-10 days when incubated at 37° C in a humidified incubator with 5% CO2.</li>
</ol>
5. Representative Results:
After plating the single cells harvested from GBM tumor tissue, tumor stem-like cells proliferate and generate small clusters of cells composed of few cells in 3-4 days (see the video and Figure 1). As these clusters grow, they acquire a more spherical shape so that by 7-8 days, proper phase bright spheres with an average diameter of 150-200 microns form (See the video and Figure 2). At higher magnification, healthy spheres usually demonstrate microspikes at their periphery. Having large sphere like clusters in 1-2 days after culture initiation is due to existence of non-dissociated clumps at the binging of the culture and should not be mistaken as true spheres. The amount of debris in primary tumor sphere culture varies depending on the initial source of tissue and whether or not it includes any surrounding brain tissue. Proper tissue preparation techniques including enzymatic and mechanical dissociation, and subsequent filtration of the sample with sufficient amount of medium can result in less debris in culture.
<img src="/files/ftp_upload/3633/3633fig1.jpg" alt="Cell division process in microscope images, showing various stages of cellular mitosis." data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Primary GBM sphere culture 4 days after plating. Tumor stem-like cells proliferate and generate small clusters of cells in 3-4 days. Original Magnification; 20x.
<img src="/files/ftp_upload/3633/3633fig2.jpg" alt="Spheroid formation, microscopy image, cell culture technique, in vitro 3D model, cancer research." data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Passage one GBM sphere culture 8 days after plating. Original Magnification; 20x.

<ol><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li>
<li>Siebzehnrubl, F. A., Vedam-Mai, V., Azari, H., Reynolds, B. A., Deleyrolle, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+Characterization+of+Adult+Neural+Stem+Cells%5BTitle%5D)+AND+%22+Methods+Mol+Biol%22%5BJournal%5D)">Isolation and Characterization of Adult Neural Stem Cells.</a> Methods Mol Biol. 750, 61-77 (2011).</li>
<li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J. Vis. Exp. (45), e2393-e2393 (2010).</li>
<li>Azari, H., Sharififar, S., Rahman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> J. Vis. Exp. (47), e2457-e2457 (2011).</li>
<li>Eramo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+expansion+of+the+tumorigenic+lung+cancer+stem+cell+population%5BTitle%5D)+AND+%22Cell+Death+Differ%22%5BJournal%5D)">Identification and expansion of the tumorigenic lung cancer stem cell population.</a> Cell Death Differ. 15, 504-514 (2008).</li>
<li>Patrawala, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+purified+CD44%2B+prostate+cancer+cells+from+xenograft+human+tumors+are+enriched+in+tumorigenic+and+metastatic+progenitor+cells%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells.</a> Oncogene. 25, 1696-1708 (2006).</li>
<li>Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intrinsic+resistance+of+tumorigenic+breast+cancer+cells+to+chemotherapy%5BTitle%5D)+AND+%22J+Natl.+Cancer.+Inst%22%5BJournal%5D)">Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy.</a> J Natl. Cancer. Inst. 100, 672-679 (2008).</li>
<li>Galli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+characterization+of+tumorigenic%2C+stem-like+neural+precursors+from+human+glioblastoma%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma.</a> Cancer Res. 64, 7011-7021 (2004).</li>
<li>Deleyrolle, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+for+label-retaining+tumour-initiating+cells+in+human+glioblastoma%5BTitle%5D)+AND+%22Brain%22%5BJournal%5D)">Evidence for label-retaining tumour-initiating cells in human glioblastoma.</a> Brain. 134, 1331-1343 (2011).</li>
</ol>

No conflicts of interest declared.

To isolate neural stem and progenitor cells from normal adult and fetal brains1, 2, 3, 4 and also tumor stem-like cells from cancer tissues such as lung5, prostate6, breast7 and brain8, 9 the neurosphere assay has been frequently used as the method of choice. Using this simple and reproducible assay, one can generate an indefinite number of cells from resected tumor tissue that show similar characteristics as somatic stem cells; ex vivo multipotency, the ability to create new tumors upon implantation, and self-renewal. These cells could be used to study the basic cancer cell biology including cell-to-cell interactions, and differentiation, migration, invasion and cell death. In addition, isolated tumor stem-like cells provide an invaluable tool to study how tumors form, progress, and relapse and also to unravel the underlying deriving cellular mechanisms that eventually could provide insights to therapeutic options.

<table><tbody><tr><td>NeuroCult NSC Basal Medium (Human)</td><td>Medium</td><td>Stem Cell Technologies</td><td>05750</td><td></td></tr><tr><td>NeuroCult NSC Proliferation Supplements (Human)</td><td>Medium supplement</td><td>Stem Cell Technologies</td><td>05753</td><td></td></tr><tr><td>%0.05 trypsin-EDTA</td><td>Reagent</td><td>GIBCO, by Life Technologies</td><td>25300-062</td><td></td></tr><tr><td>*MEM</td><td>Reagent</td><td>GIBCO, by Life Technologies</td><td>41500-018</td><td>HEM component</td></tr><tr><td>*HEPES</td><td>Reagent</td><td>Sigma-Aldrich</td><td>H4034</td><td>HEM component</td></tr><tr><td>*Distilled water</td><td>Reagent </td><td>GIBCO, by Life Technologies</td><td>15230-147</td><td></td></tr><tr><td>**DNase I</td><td>Reagent</td><td>Roche Group</td><td>104159</td><td></td></tr><tr><td>**Soybean trypsin inhibitor </td><td>Reagent</td><td>Sigma-Aldrich</td><td>T6522</td><td></td></tr><tr><td>Pen/Strep </td><td>Reagent</td><td>GIBCO, by Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>No. 10 scalpel blade</td><td>Surgical tool</td><td>BD Biosciences</td><td>371610</td><td></td></tr><tr><td>Petri Dish</td><td>Culture ware</td><td>BD Biosciences</td><td>353003</td><td></td></tr><tr><td>Small forceps</td><td>Surgical tools</td><td>Fine Science Tools</td><td>11050-10</td><td></td></tr><tr><td>Cell strainer </td><td>Sieve</td><td>BD Biosciences</td><td>352340</td><td></td></tr><tr><td>T25 flask</td><td>Culture ware</td><td>Nalge Nunc international</td><td>136196</td><td></td></tr><tr><td>T80 flask</td><td>Culture ware</td><td>Nalge Nunc international</td><td>178905</td><td></td></tr><tr><td>15 ml tubes</td><td>Culture ware</td><td>BD Biosciences</td><td>352096</td><td></td></tr><tr><td>50 ml tubes</td><td>Culture ware</td><td>BD Biosciences</td><td>352070</td><td></td></tr><tr><td>EGF</td><td>Growth factor</td><td>R&amp;amp;D Systems</td><td>2028-EG</td><td></td></tr><tr><td>b-FGF</td><td>Growth factor</td><td>R&amp;amp;D Systems</td><td>3139-FB</td><td></td></tr><tr><td>Heparin</td><td>Growth factor</td><td>Sigma-Aldrich</td><td>H4784</td><td>Reconstituted in PBS</td></tr><tr><td colspan="5">* To make HEM, mix 1x10L packet of MEM and160ml of 1M HEPES and bring the volume to 8.75 L using distilled water. Set the final PH to 7.4 and store it at 4&amp;#x00B0;C.</td></tr><tr><td colspan="5">** To prepare trypsin inhibitor solution, first make 10 ml of DNase I solution (100 mg DNase dissolved in 100 ml of HEM) and then add 0.14 g of trypsin inhibitor to DNase solution and finally make the volume up to 1 Liter using HEM. Keep aliquots of the final products in -20&amp;#x00B0;C freezer.</td></tr></tbody></table>

isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

The goal of this protocol is the isolation and expansion of human glioblastoma multiform tumor cells using the neurospheres asset. This is achieved by first mincing the harvested tumor tissue, followed by ization mechanical dissociation, and finally plating the resulting cells To grow in neuros sphere culture. In this Video, we are going to show how to isolate and expand stem-like cells from human glioblastoma tumor tissue using the neuros sphere culture method.This method can also be used to culture cells from other brain tumors as well as solid tumors such as breast, prostate, liver, and lung. Okay, let's get started. After receiving the resected GBM tumor Tissue from surgery, the medium is removed.The tumor is washed two or three times using sterile PBS to remove contaminants Such as blood and debris. After washing, place the resected tumor into sterile petri Dish. First, cut the tumor into smaller portions, then transfer a portion of tumor tissue into a separate Petri dish, mince the tissue using a number 10 scalpel into tiny pieces.This will increase surface area available for enzymatic dissociation. Mincing can take one to three minutes depending on the size of the tumor. Now add three to five ml of prewarm trypsin to the mince tissue and transfer it to a 15 ml sterile falcon tube.Place the 15 Ml tube in 37 degree water bath for 10 to 15 minutes. Shake the tube every five minutes. To ensure better tissue digestion, add an equal volume of trypsin inhibitor to stop the reaction.Trypsin in activation is insured by pipetting up and down several times. Centrifuge the suspension at 800 RRP M or 110 G for five Minutes. Remove the supernatant, then resuspend the pallet in One ml of neuros stem cell basal media, placing the tip against the bottom of the tube.Pipette up and down three to seven times to break up clumps until you achieve a milky suspension over pipetting might result in cell death. Place a 40 micron filter on a 50 ml tube. Now add 10 to 15 ml of basal medium to cell suspension and mix it gently pass the cell suspension through the 40 micron filter so as to remove debris in unassociated clumps.PT the cells And vacuum off the supernat. Resuspend the Cells in one to two mls of complete medium. Then gently pipette up and down to homogenize the suspension.Mix 10 microliter of cell suspension in 90 microliter of Trian blue. Mix the suspension well and transfer 10 microliter to hemo.Cytometer. Perform a cell count as shown here.A single cell suspension is required with no clumps to have an accurate cell count. Do not include trian blue positive cells. When counting to plate the cells in a T 80 flask.Add 20 ml of complete neuro stem cell media to a 50 ml tube. Add appropriate amount of cell suspension to reach a density of 200, 000 cells per ml. Now add EGF to reach a final concentration Of 20 nanogram per ml.Add 0.2%heparin at concentration of one microliter per ml. Finally, add BFGF to reach a Final concentration of 10 nanogram per ml. Mix the Cells in complete media supplemented with growth factors.Well then transfer the suspension to a T 80 label. The flask then transfer to incubator at 37 degrees Celsius and 5%CO2 for seven to 10 days. After eight days in culture, the spheres are fully grown and ready to be passaged.Collect the contents of the flask and transfer to a 50 ml two centrifuge. The cells at 800 RPM for five minutes. Vacuum off the supernat, then resuspend the pallet in one ml of prewarm trypsin.Incubate the cells in water bath at 37 degrees Celsius for two minutes. Add an equal amount of trypsin inhibitor and mix thoroughly to ensure an activation. Then pellet the cells.Remove the supernat and resuspend the cells in one ml of complete medium pipet up and down gently to homogenize the cell suspension. Perform a cell count as described earlier to plate the cells in a T 80 flask at 20 ml of complete medium in a 50 ml tube. Add an appropriate amount of cell suspension to reach a density of 50, 000 cells per ml.Now add Growth factors including E-G-F-B-F-G-F, and heparin at the concentration described earlier. Mix the resulting cell suspension gently, then transfer the suspension to a T 80 and place in the incubator for seven to 10 days. Here's an example of primary GBM Culture.After four days at this stage, you can see the cells are proliferating and beginning to form spheres. You should not observe large spherical clusters of cells at this stage. This would've resulted from inappropriate tissue preparation.After being in culture of seven to 10 days, spheres of varying size have formed when the spheres have reached an average diameter of 150 to 200 microns, They're ready to be passaged. This is an example of passage GBM culture after eight days. Again, you can see the spheres of varying sizes, healthy spheres, exhibit micro spikes on their periphery.Preparing a single cell Suspension is a very important issue in any sphere cultures. Otherwise, you might end up observing cell aggregates even on the day after plating cells. These aggregates be assumed as spheres, but it should be noted that it takes at least a week for perforating cells to form true spheres.The amount of debris in primary tumor sphere culture varies depending on many factors such as source of tissue and that how precise the tissue has been removed and also the tissue processing technique. We hope you find this video useful and wish you good luck with your experiments.

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Learn to isolate and expand stem-like cells from human glioblastoma multiforme tumor tissue using the neurosphere assay culture method. This protocol enables researchers to study tumor biology and identify therapeutic targets.

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

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