Name: patient derived cell culture and isolation of cd133+ putative cancer stem cells from melanoma
Uploaded: 2013-03-13T12:00:00
Description: Watch this Scientific Journal Video about Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma at JoVE.com

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 concent...

<ol>
	<li>Garibyan, L., Fisher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+sunlight+causes+melanoma.">How sunlight causes melanoma.</a> Curr. Oncol. Rep. 12, 319-326 (2010).</li><li>Radovic-Kovacevic, 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=Survival+analysis+in+patients+with+cutaneous+malignant+melanoma.">Survival analysis in patients with cutaneous malignant melanoma.</a> Srp. Arh. Celok. Lek. 125, 132-137 (1997).</li><li>Lens, M. B., Eisen, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+chemotherapy+in+the+treatment+of+malignant+melanoma.">Systemic chemotherapy in the treatment of malignant melanoma.</a> Expert Opin. Pharmacother. 4, 2205-2211 (2003).</li><li>Nashan, D., Muller, M. L., Grabbe, S., Wustlich, S., Enk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+therapy+of+disseminated+malignant+melanoma:+an+evidence-based+overview+of+the+state-of-the-art+in+daily+routine.">Systemic therapy of disseminated malignant melanoma: an evidence-based overview of the state-of-the-art in daily routine.</a> J. Eur. Acad. Dermatol. Venereol. 21, 1305-1318 (2007).</li><li>Eigentler, T. 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. B., 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=Improved+survival+with+vemurafenib+in+melanoma+with+BRAF+V600E+mutation.">Improved survival with vemurafenib in melanoma with BRAF V600E mutation.</a> N. Engl. J. Med. 364, 2507-2516 (2011).</li><li>Burdall, S. E., Hanby, A. M., Lansdown, M. R., Speirs, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+cell+lines:+friend+or+foe.">Breast cancer cell lines: friend or foe.</a> Breast Cancer Research : BCR. 5, 89-95 (2003).</li><li>Osborne, C. K., Hobbs, K., Trent, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+differences+among+MCF-7+human+breast+cancer+cell+lines+from+different+laboratories.">Biological differences among MCF-7 human breast cancer cell lines from different laboratories.</a> Breast Cancer Research and Treatment. 9, 111-121 (1987).</li><li>Hiorns, L. R., 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=Variation+in+RNA+expression+and+genomic+DNA+content+acquired+during+cell+culture.">Variation in RNA expression and genomic DNA content acquired during cell culture.</a> Br. J. Cancer. 90, 476-482 (2004).</li><li>Nelson-Rees, W. A., Daniels, D. W., Flandermeyer, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-contamination+of+cells+in+culture.">Cross-contamination of cells in culture.</a> Science. 212, 446-452 (1981).</li><li>MacLeod, R. A., 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=Widespread+intraspecies+cross-contamination+of+human+tumor+cell+lines+arising+at+source.">Widespread intraspecies cross-contamination of human tumor cell lines arising at source.</a> Int. J. Cancer. 83, 555-563 (1999).</li><li>Masters, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cancer+cell+lines:+fact+and+fantasy.">Human cancer cell lines: fact and fantasy.</a> Nature reviews. Molecular Cell Biology. 1, 233-236 (2000).</li><li>Pennartz, S., Reiss, S., Biloune, R., Hasselmann, D., Bosio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Single-Cell+Suspensions+from+Mouse+Neural+Tissue.">Generation of Single-Cell Suspensions from Mouse Neural Tissue.</a> J. Vis. Exp. (29), e1267 (2009).</li><li>Zabierowski, S. E., Fukunaga-Kalabis, M., Li, L., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dermis-derived+stem+cells:+a+source+of+epidermal+melanocytes+and+melanoma.">Dermis-derived stem cells: a source of epidermal melanocytes and melanoma.</a> Pigment Cell Melanoma Res. 24, 422-429 (2011).</li><li>Singh, S. K., 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=Identification+of+human+brain+tumour+initiating+cells.">Identification of human brain tumour initiating cells.</a> Nature. 432, 396-401 (2004).</li><li>Ieta, K., 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=Biological+and+genetic+characteristics+of+tumor-initiating+cells+in+colon+cancer.">Biological and genetic characteristics of tumor-initiating cells in colon cancer.</a> Ann. Surg. 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. P., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+CD4++T+cells+from+Mouse+Lymph+Nodes+Using+Miltenyi+MACS+Purification.">Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification.</a> J. Vis. Exp. (9), e409 (2007).</li><li>Watkins, S. K., Zhu, Z., Watkins, K. E., Hurwitz, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Immune+Cells+from+Primary+Tumors.">Isolation of Immune Cells from Primary Tumors.</a> J. Vis. Exp. (64), e3952 (2012).</li><li>Mollamohammadi, S., 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+simple+and+efficient+cryopreservation+method+for+feeder-free+dissociated+human+induced+pluripotent+stem+cells+and+human+embryonic+stem+cells.">A simple and efficient cryopreservation method for feeder-free dissociated human induced pluripotent stem cells and human embryonic stem cells.</a> Hum. Reprod. 24, 2468-2476 (2009).</li></ol>

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

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

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 ...

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 ...