The authors thank Prof. M. Dietel and Dirk Schumacher for supporting this work and critically reading the manuscript.
The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement n&deg; 115234, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies' in kind contribution. This work was also supported by the Berliner Krebsgesellschaft (BKG).

The overall scheme of the experiment including the preparation of primary single-cells from tumor tissue, characterization of the primary cell culture, fibroblast depletion and magnetic cell sorting of CD133+ and CD133- melanoma cells is shown in Figure 1.
1. Sample Acquisition
<ol>
	<li>Check for ethical approval before considering the next steps.</li>
	<li>Prepare a 50 ml tube with sterile PBS supplemented with 1% Penicillin-Streptomycin final concentration and hand over to surgery OR team.</li>
	<li>Organize fast transport of tumor tissue from surgery to cell culture lab at 4 &deg;C.</li>
</ol>
2. Preparing Primary Melanoma Single-cells from Tumor Tissue
<ol>
	<li>Perform all steps under sterile conditions.</li>
	<li>Before resecting the tumor, one needs to prepare the dissociation mix. 10 ml dissociation mix per 2-4 g tissue is required. The mix can be used immediately or stored in 10 ml aliquots at -20 &deg;C for later use.
	<ol>
		<li>Prepare a solution containing 150 mM sodium chloride. Mix using a vortex until the solution is clear. If necessary sterile-filtrate sodium chloride solution using a 0.22 &mu;m filter.</li>
		<li>Weigh out DNase I and prepare a solution containing 10 mg/ml DNase I in 150mM sodium chloride. Mix by inverting until the solution is clear.</li>
		<li>Prepare a solution containing 100mg/ml Collagenase IV in PBS. Mix by inverting until the solution is clear. Aliquots of collagenase solution can be stored at -20 &deg;C for several months.</li>
		<li>Prepare the dissociation mix with a final concentration of 1% Penicillin-Streptomycin, 1 mg/ml Collagenase IV and 0.1 mg/ml DNase I in Quantum 263 medium. Mix using a vortex. Optional: when using melanoma tissue derived from skin add Amphotericin B solution to the dissociation mix to a final concentration of 1.5 &mu;g/ml Amphotericin B.</li>
	</ol>
	</li>
	<li>Pre-warm the required amounts of dissociation mix, PBS and Quantum 263 medium to 37 &deg;C.</li>
	<li>Place tumor tissue on a sterile Petri dish. Aspirate excessive solution from the tissue and add 500 &mu;l dissociation mix. Mince tumor into small pieces of 2-4 mm using fresh, sterile scalpels.</li>
	<li>Transfer 2-4 g minced tumor tissue into a gentleMACS C Tube containing 5 ml dissociation mix. Rinse Petri dish with additional 4.5 ml dissociation mix and add remaining tissue fragments to the gentleMACS C Tube.</li>
	<li>Close gentleMACS C tube and attach it upside down onto the sleeve of the gentleMACS Dissociator. Run the gentleMACS program "h_tumor_01".</li>
	<li>Detach the gentleMACS C tube from the dissociator and incubate the sample for 30 min at 37 &deg;C under continuous rotation using the MACSmix Tube Rotator on the highest run speed (12 rpm) or by turning the tube manually every 5 min to resuspend settled tissue fragments.</li>
	<li>Attach gentleMACS C tube upside down onto the sleeve of the gentleMACS Dissociator and run the gentleMACS program "h_tumor_02".</li>
	<li>Detach the gentleMACS C tube from the dissociator and incubate the sample for 30 min at 37 &deg;C under continuous rotation using the MACSmix Tube Rotator on the highest run speed (12 rpm) or by turning the tube manually every 5 min to resuspend settled tissue fragments.</li>
	<li>Attach gentleMACS C tube upside down onto the sleeve of the gentleMACS Dissociator and run the gentleMACS program "h_tumor_03".</li>
	<li>Detach the gentleMACS C tube from the dissociator, resuspend sample and apply the cell suspension to a 70 &mu;m cell strainer placed on a 50 ml tube. If clogging of the filter occurs, stir cell suspension with a sterile filter tip until the complete suspension ran through.</li>
	<li>Rinse cell strainer with 5 ml Quantum 263 medium.</li>
	<li>Optional: if you still have tissue fragments left in the filter, resuspend fragments in Quantum 263 medium and seed them in an appropriate cell culture dish. Incubate fragments at 37 &deg;C and 5% CO2.</li>
	<li>Discard cell strainer and close the 50 ml tube with the digested cell suspension. Centrifuge cell suspension for 5 min at 300 x g and discard supernatant.</li>
	<li>Optional: if cell pellet appears red due to a high amount of erythrocytes prepare 1x Red Blood Cell Lysis Solution by diluting 10x solution 1:10 in double-distilled water, resuspend cell pellet in 500 &mu;l PBS and add 5 ml 1x Red Blood Cell Lysis Solution. Vortex 5 sec and incubate at room temperature for 10 min. Centrifuge for 5 min at 300 x g and discard supernatant.</li>
	<li>Resuspend tumor cells with Quantum 263 and seed the cells in appropriate cell culture flask. Culture tumor cells at 37 &deg;C and 5% CO2 and routinely passage cells when they reach 80% confluence.</li>
</ol>
3. Characterization of Primary Cell Culture and Fibroblast Depletion
<ol>
	<li>Analyze primary cell culture phenotypically using a microscope.</li>
	<li>Harvest a small amount (0.5x 106 cells) of the primary cell culture and perform RT-PCR with primers for neoplastic-, melanoma-, stem cell- and stroma cell marker genes. The most important genes to analyze are CD90 (fibroblast marker), TYR (melanoma/melanocyte marker), CD133 (cancer stem cell marker), CD83 (marker for mature dendritic cells) and a housekeeping gene (e.g. HPRT or GAPDH).</li>
	<li>If microscopic analysis and high expression of CD90 during RT-PCR revealed a high contamination of the primary melanoma culture with fibroblasts, you need to deplete fibroblasts using the Anti-Fibroblasts MicroBeads and D Columns from Miltenyi.</li>
</ol>
4. Magnetic Cell Sorting of CD133+ and CD133- Melanoma Cells Using LS Columns
<ol>
	<li>Prepare MACS buffer containing 2 mM EDTA and 5% FCS in PBS. Mix by inverting and sterile-filtrate buffer using a 0.22 &mu;m Steritop-GP Filter Unit. Chill buffer to 4-8 &deg;C and degas before use.</li>
	<li>Pre-warm Accutase, PBS and Quantum 263 medium to 37 &deg;C.</li>
	<li>Wash 80% confluent cells with PBS. Add appropriate amount of Accutase and incubate until cells detach from the culture surface. Collect cells in a 50 ml tube using Quantum 263 medium.</li>
	<li>Determine the cell number and centrifuge the required number of cells (According to Miltenyi's protocol a maximum number of 2x 109 cells can be loaded per LS Column. Cell line specific optimal numbers should be determined and can be considerably lower.) for 5 min at 300 x g and 4-8 &deg;C. Aspirate supernatant completely.</li>
	<li>Resuspend a maximum of 1x 108 cells with 350 &mu;l MACS buffer (for higher cell numbers scale up all following MACS buffer, FcR Blocking Reagent, CD133/1-Biotin and anti-Biotin MicroBeads volumes accordingly).</li>
	<li>Add 100 &mu;l FcR Blocking Reagent.</li>
	<li>Add 50 &mu;l CD133/1-Biotin. Mix well using a vortex and incubate at 4-8 &deg;C for 10 min.</li>
	<li>Wash cells by adding 10 ml MACS buffer and centrifuge for 5 min at 300 x g and 4-8 &deg;C. Aspirate supernatant completely.</li>
	<li>Repeat washing step (4.8)</li>
	<li>Resuspend cell pellet with 400 &mu;l MACS buffer.</li>
	<li>Add 100 &mu;l anti-Biotin MicroBeads. Mix well using a vortex and incubate at 4-8 &deg;C for 15 min.</li>
	<li>Meanwhile, prepare the MACS separator for the magnetic separation. Attach QuadroMACS to MACS Separator Multi Stand. Insert the required number of LS columns with the column wings to the front in the magnetic field of the QuadroMACS. Place a Pre-Separation Filter (with 30 &mu;m nylon mesh) into each LS column and an appropriate collection tube (15 ml or 50 ml) under each column. Prepare filter and column by rinsing with 3 ml MACS buffer. Discard flow-through.</li>
	<li>Wash cells by adding 10 ml MACS buffer and centrifuge for 5 min at 300 x g and 4-8 &deg;C. Aspirate supernatant completely.</li>
	<li>Resuspend cells with 500 &mu;l MACS buffer and apply cell suspension onto the LS column with the Pre-Separation Filter.</li>
	<li>Wash three times with 3 ml MACS buffer per column. Only add new buffer when the column reservoir is empty.</li>
	<li>The collected total effluent contains the unlabelled CD133- cell fraction.</li>
	<li>Discard the Pre-Separation Filter, remove column from the separator and place it on a suitable collection tube.</li>
	<li>Apply 5 ml MACS buffer. Immediately flush out the labelled CD133+ cells by firmly pushing the plunger into the column.</li>
	<li>To increase the purity of the negative fraction, apply cell suspension onto a new equilibrated LS column inserted in the QuadroMACS. The effluent contains the CD133- fraction.</li>
	<li>Discard columns.</li>
</ol>

<ol>
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K., Meier, F., Keilholz, U., Hauschild, A., Garbe, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Der Onkologe. 16, 1160-1166 (2010).</li><li>Keilholz, U., Tilgen, W., Hohenberger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemische+Therapie+des+metastasierten+Melanoms:+Ergebnisse+randomisierter+Studien+der+letzten+zehn+Jahre.">Systemische Therapie des metastasierten Melanoms: Ergebnisse randomisierter Studien der letzten zehn Jahre.</a> Deutsches &#196;rzteblatt. 100, (2003).</li><li>Chapman, P. 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Oncol. 15, 638-648 (2008).</li><li>Bertolini, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+tumorigenic+lung+cancer+CD133++cells+display+stem-like+features+and+are+spared+by+cisplatin+treatment.">Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 16281-16286 (2009).</li><li>Monzani, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanoma+contains+CD133+and+ABCG2+positive+cells+with+enhanced+tumourigenic+potential.">Melanoma contains CD133 and ABCG2 positive cells with enhanced tumourigenic potential.</a> Eur. J. Cancer. 43, 935-946 (2007).</li><li>Gedye, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer/testis+antigens+can+be+immunological+targets+in+clonogenic+CD133++melanoma+cells.">Cancer/testis antigens can be immunological targets in clonogenic CD133+ melanoma cells.</a> Cancer Immunology, Immunotherapy : CII. 58, 1635-1646 (2009).</li><li>Kobari, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133++cell+selection+is+an+alternative+to+CD34++cell+selection+for+ex+vivo+expansion+of+hematopoietic+stem+cells.">CD133+ cell selection is an alternative to CD34+ cell selection for ex vivo expansion of hematopoietic stem cells.</a> J. Hematother. Stem Cell Res. 10, 273-281 (2001).</li><li>Quirici, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+expansion+of+endothelial+cells+from+human+bone+marrow+CD133(+)+cells.">Differentiation and expansion of endothelial cells from human bone marrow CD133(+) cells.</a> Br. J. Haematol. 115, 186-194 (2001).</li><li>Pfenninger, C. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133+is+not+present+on+neurogenic+astrocytes+in+the+adult+subventricular+zone,+but+on+embryonic+neural+stem+cells,+ependymal+cells,+and+glioblastoma+cells.">CD133 is not present on neurogenic astrocytes in the adult subventricular zone, but on embryonic neural stem cells, ependymal cells, and glioblastoma cells.</a> Cancer Res. 67, 5727-5736 (2007).</li><li>Dubreuil, V., Marzesco, A. M., Corbeil, D., Huttner, W. B., Wilsch-Brauninger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midbody+and+primary+cilium+of+neural+progenitors+release+extracellular+membrane+particles+enriched+in+the+stem+cell+marker+prominin-1.">Midbody and primary cilium of neural progenitors release extracellular membrane particles enriched in the stem cell marker prominin-1.</a> J. Cell Biol. 176, 483-495 (2007).</li><li>Florek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+neural+and+hematopoietic+stem+cell+marker,+is+expressed+in+adult+human+differentiated+cells+and+certain+types+of+kidney+cancer.">a neural and hematopoietic stem cell marker, is expressed in adult human differentiated cells and certain types of kidney cancer.</a> Cell Tissue Res. 319, 15-26 (2005).</li><li>Matheu, M. 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The authors declare that they have no competing financial interests.

In order to remove erythrocyte contaminations from the tumor cell pellet we highly recommend using the red blood cell lysis solution from Miltenyi. The advantages of lysing the erythrocytes over the traditional Ficoll density gradient centrifugation are that it is faster and simpler. Furthermore, contaminations with Ficoll or red blood cells and the loss of tumor cells are avoided. When you observe the growth of fibroblasts in the primary cell culture they should be removed immediately since they normally overgrow the tu...

Cutaneous malignant melanomas are the least common, yet most deadly type of skin cancer. Due to an increasing incidence, a high grade of malignancy and a rapid dissemination, melanomas now account for 75% of malignant skin tumors related deaths1,2. Besides surgical excision of the primary tumor, chemotherapy, radiotherapy, immunotherapy and combined chemo- and immunotherapy of metastasized melanoma are the state-of-the-art strategies for melanoma treatment3,4. However, malignant melanoma is characterized by a poor response to chemotherapeutics (5-12%)5,6, and only those 50-70% of melanoma patients carrying the BRAF V600E gene mutation benefit from promising new targeted therapies as treatment with vemurafenib7 To improve overall survival, a much better understanding of the mechanisms of melanoma tumorigenesis is needed.
To study those mechanisms in the past, well-established commercially available cell lines were used. More recent approaches to study the molecular events of cancer initiation and progression utilize primary cultures derived directly from tumor tissue - a strategy bearing manifold benefits: The researcher has full control of patient and tissue selection. The sampled cells gained from expertly selected tumor tissue, resemble the tumor with all its heterogeneity and follow-up data of the patient and a detailed pathology is available to allow the characteristics of the culture to be compared with those of the original tumor8.
In contrast, the use of established cell lines as relevant model systems in cancer research is discussed controversially. Already in 1987 Osborne and colleagues described significant biological differences among MCF-7 human breast cancer cell lines from different laboratories9. This extensive genomic instability and variation in RNA expression during subculture was confirmed by Hiorns et al. and provided supportive data for evidence that cell lines do evolve in culture, thereby weakening the direct relevance of such established cultures as models of human cancer10.
Another important consideration, which has largely been ignored over the years, is the risk of contamination or overgrowth of cultures with unrelated 'false' cells, which was first highlighted over twenty years ago by demonstrating that a large number of cell lines were contaminated by HeLa cells8,11. The misinterpretation of data from 'false' cell lines has recently come to light in the literature again. Using a combination of DNA profiling and molecular cytogenetics, MacLeod et al. revealed that of 252 new tumor-derived human cell lines deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ), nearly 18% were found to be intraspecies or interspecies cross-contaminants12,13.
To avoid these problems and to make use of the advantages mentioned above, we decided to establish low-passage melanoma cell lines from freshly excised metastases.
Robust cell separation and analysis technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface proteins. A benchtop instrument for the automated dissociation of tissues into single-cell suspensions is the gentleMACS Dissociator manufactured by Miltenyi. When used in combination with gentleMACS C Tubes and optimized dissociation solutions, an effective and gentle dissociation of tumor tissue in a closed system is achieved while preserving antigen epitopes and reducing cell loss. The instrument offers optimized, pre-set programs for a variety of specific applications and guaranties standardized preparation of single-cell suspensions from melanoma tissue14.
Recent reports revealed that the majority of tumors harbor a small subpopulation of so called cancer stem cells (CSCs), which exclusively exhibit tumor-initiating and self-renewing capacity. The identification of melanocyte-producing stem cells in the dermis of the skin led to the hypothesis that these cells might be the origin of cancer stem cells (CSCs) in melanoma since exposure to UVA radiation can prime these cells for malignant transformation15. The result of such a genetic lesion would be cells that harbor a combination of tumor and stem cell characteristics.
One of the key markers proposed to represent the subpopulation of CSCs in melanoma is CD13316-20. CD133 (also known as Prominin 1), a member of pentaspan transmembrane glycoproteins, is expressed in hematopoietic stem cells, endothelial progenitor cells, neuronal and glial stem cells21-23. Expression of CD133 is correlated with asymmetric cell division24, and the glycosylated epitope of CD133 was shown to be downregulated upon cell differentiation25. Recently, CD133+ melanoma cells were shown to have self-renewal and tumor-initiation capacity19,20.
To study the function of CD133+ putative melanoma CSCs, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) system from Miltenyi Biotech to obtain highly enriched and viable populations of CD133+ and CD133- cells.
Magnetic bead-based cell separation allows for either negative selection as shown by Matheu et al.26 or positive selection as we show here. During positive selection the particular target cell type, e.g. CD133 expressing cells, is magnetically labeled with MicroBeads, 50-nm superparamagnetic particles that are conjugated to highly specific antibodies against a particular cell surface antigen. During separation, the magnetically labeled cells are retained within the column in the magnetic field of the separator, whereas unlabeled cells flow through. Following washing steps, the column is removed from the magnetic field of the separator, and the target cells are eluted from the column. The LS or MS columns used during positive selection result in fractions of labeled and unlabeled cells with high purity. On the other hand, LD columns, used for negative selection, result in a slightly lower purity of the labeled fraction. Due to the denser packing of their matrix the flow rate in these columns is lower resulting in a higher risk of unlabeled cells to be trapped in the column. LD columns should therefore be used only for stringent depletion of an unwanted cell subpopulation. Positive selection can be performed by direct (antibody coupled to MicroBeads) or indirect magnetic labeling (incubation with MicroBeads following the incubation with the primary antibody). Specific MACS MicroBeads are available for the positive selection of numerous cell types of primary tumor cells like prostate or melanoma27.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Accutase</td>
 <td>PAA Laboratories GmbH</td>
 <td>L11-007</td>
 <td></td>
 </tr>
 <tr>
 <td>Amphotericin B solution 250 &mu;g/mL in deionized water</td>
 <td>Sigma-Aldrich, Inc.</td>
 <td>A2942-50ml</td>
 <td></td>
 </tr>
 <tr>
 <td>anti-fibroblasts microbeads</td>
 <td>Miltenyi Biotec GmbH</td>
 <td>130-050-601</td>
 <td></td>
 </tr>
 <tr>
 <td>CD133/1 (W6B3C1)</td>
 <td>Miltenyi Biotec GmbH</td>
 <td>130-092-395</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase IV</td>
 <td>Sigma-Aldrich, Inc.</td>
 <td>C5138</td>
 <td></td>
 </tr>
 <tr>
 <td>DNase I</td>
 <td>Applichem GmbH</td>
 <td>A3778.0050</td>
 <td></td>
 </tr>
 <tr>
 <td>D-PBS without Ca, Mg </td>
 <td>PAA Laboratories GmbH</td>
 <td>H15-002</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA)</td>
 <td>Sigma-Aldrich, Inc.</td>
 <td>E-7889</td>
 <td></td>
 </tr>
 <tr>
 <td>FBS Superior</td>
 <td>Biochrom</td>
 <td>S0615</td>
 <td></td>
 </tr>
 <tr>
 <td>Indirect CD133 Micro-Bead Kit human</td>
 <td>Miltenyi Biotec GmbH</td>
 <td>130-091-895</td>
 <td>Includes: CD133/1(AC133)-Biotin, anti-Biotin MicroBeads and FcR Blocking Reagent</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin, liquid (100x)</td>
 <td>Invitrogen GmbH</td>
 <td>15140-122</td>
 <td></td>
 </tr>
 <tr>
 <td>Quantum 263 </td>
 <td>PAA Laboratories GmbH</td>
 <td>U15-815</td>
 <td></td>
 </tr>
 <tr>
 <td>Red Blood Cell Lysis Solution (10x)</td>
 <td>Miltenyi Biotec GmbH</td>
 <td>130-094-183</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Cell strainer (70 &mu;m)</td>
 <td>BD Biosciences</td>
 <td>352350</td>
 <td></td>
 </tr>
 <tr>
 <td>gentleMACS C Tubes</td>
 <td>Miltenyi Biotec GmbH</td>
 <td>130-093-237</td>
 <td></td>
 </tr>
 <tr>
 <td>gentleMACS dissociator</td>
 <td>Miltenyi Biotec GmbH</td>
 <td>130-093-235</td>
 <td></td>
 </tr>
 <tr>
 <td>MACS Separator Multi Stand</td>
 <td>Miltenyi Biotec GmbH</td>
 <td>130-042-303</td>
 <td></td>
 </tr>
 <tr>
 <td>MACS Seperation Columns 25 LS Columns </td>
 <td>Miltenyi Biotec GmbH</td>
 <td>130-042-401</td>
 <td></td>
 </tr>
 <tr>
 <td>MACSmix Tube Rotator</td>
 <td>Miltenyi Biotec GmbH</td>
 <td>130-090-753</td>
 <td></td>
 </tr>
 <tr>
 <td>Natriumchloride</td>
 <td>Merck KGaA</td>
 <td>567440-1KG</td>
 <td></td>
 </tr>
 <tr>
 <td>Pre-Separation Filters</td>
 <td>Miltenyi Biotec GmbH</td>
 <td>130-041-407</td>
 <td></td>
 </tr>
 <tr>
 <td> QuadroMACS Separator </td>
 <td>Miltenyi Biotec GmbH</td>
 <td>130-090-976</td>
 <td></td>
 </tr>
 <tr>
 <td>Rotilabo-syringe filters (0.22 &mu;m, PES)</td>
 <td>Carl Roth GmbH &amp; Co. KG </td>
 <td>P668.1</td>
 <td></td>
 </tr>
 <tr>
 <td>Steritop-GP Filter Unit 500 ml</td>
 <td>Miltenyi Biotec GmbH</td>
 <td>SCGPS05RE</td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

Preparation of single-cells from tumor tissue
Figure 2 exemplifies a resected lymph node metastasis of a late stage melanoma patient before (A) and after (B) mechanical dissociation into 2-4 mm pieces. Following enzymatic dissociation of the tissue and filtration through a 70 &mu;m nylon mesh, cells were pelleted and the supernatant discarded. At this step we observed a high contamination with erythrocytes as indicated by the reddish color of the cell pellet in Figur...

Watch this Scientific Journal Video about Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma at JoVE.com

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).

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 ...

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21.1K Views.  Charité - Universitätsmedizin Berlin. 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).

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

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 ...

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 ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

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, ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...