This study was supported by a grant from the Baden-W&#252;rttemberg Stiftung (Adult Stem Cells Program II) awarded to C.L. We thank Dr. Martina Konantz for critical input and review of the manuscript. We thank Emmanuel Traunecker and Toni Krebs from the DBM FACS Facility (University Hospital Basel) for assistance with FACS sorting.

1. דור של תאי השחלה אדם OVCAR-3 Carcinoma transduced ביציבות עם Lentiviruses מכיל לבנות כתב Sox2 התקינה האזור <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ליצור חלקיקי lentiviral על ידי transfecting קו תא 293T-אריזת HEK עם לבנות כתב הכרת אזור רגולציה Sox2 כ10,21 תיארו. הערה: הכתב לבנות עוד מכיל תחום יציבות של מערכת חומת ProteoTuner לפני חלבון פלואורסצנטי tdTomato. Shield1 נקשר לתחום היציבות ובכך למנוע פרוטאזום להשפיל חלבון פלואורסצנטי 22. </li><li style=";text-align:right;direction:rtl"> Transduce OVCAR-3 תאים עם חלקיקי lentiviral על פני תקופה של 24 שעות זמן. לאחר מכן, להסיר את supernatant ויראלי ולשטוף את התאים עם ופר פוספט (PBS) ותרבית בינונית שלם (RPMI בתוספת 10% FBS, 100 U / ml פניצילין, 100 מיקרוגרם / מיליליטר סטרפטומיצין). </li><li style=";text-align:right;direction:rtl"> 48 שעות מאוחר יותר, 10 מיקרוגרם / מיליליטר פורmycin נוספו לתרבויות ונשמר במשך 5 ימים, כדי לאפשר בחירה של תאי transduced כראוי. </li></ol> 2. הכנת Cell המיון והציפוי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להוסיף Shield1 ב 1: 1000 שעות דילול 24 לפני מיון תא. השתמש OVCAR-3 תאי transduced ביציבות ללא טיפול Shield1 כפקדים שליליים (איור 1). תקשורת לשאוב מבקבוק, לשטוף תאים עם 1x PBS וtrypsinize תאים עם 0.05% טריפסין- EDTA במשך 3 דקות. </li><li style=";text-align:right;direction:rtl"> להפסיק טריפסין באמצעות מדיום מלא (ראה לעיל), לספור מספרי תא, תאי צנטריפוגות ב 300 XG ב RT (15 - 25 מעלות צלזיוס) במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> למזוג supernatant ותאי resuspend בזהירות ב 0.5 - PBS סטרילי 1 מיליליטר. </li><li style=";text-align:right;direction:rtl"> השתמש במסנן כובע מסננת תא 40 מיקרומטר להשיג השעיה תא בודד. </li><li style=";text-align:right;direction:rtl"> התאם ספירת תאים 5 מיליון תאים לכל מיליליטר. </li><li style=";text-align:right;direction:rtl"> הכן 96-גם צלחות נמוכות מצורפים אולטרה עם 100 μl הישות הזכרית והנקביות בינוניים (MEGM בתוספת גורמי גדילה, ציטוקינים, ותוספי מזון,B-27, heparine-נתרן; או DMEM / F12 בתוספת גורמי גדילה, ציטוקינים, ותוספי מזון, B-27, heparine-נתרן, עם או בלי תוספת של 1% methylcellulose, ראה גם לוח 1). לחלופין להוסיף אנטיביוטיקה למדיום בריכוז של 100 U פניצילין מיליליטר / 100 מיקרוגרם / מיליליטר סטרפטומיצין כדי למזער את הסיכון לזיהום אפשרי. </li><li style=";text-align:right;direction:rtl"> מיין + RFP ותאי RFP- ל96-גם צלחות שהוכנו מלמעלה, תא 1 בכל טוב (assay מבוסס תאים בודדים תחומים) וכל גם 100 תאים (assay תחומים מבוססי תאים רב), בהתאמה. בצע סדרן תא מין על זמין מסחרי (ראה חומרים) שימוש במצב תא בודד, מיין התקנה: 100 μ זרבובית, לחץ נדן 20 psi, ומסכת תשואת 0, מסכת טוהר 32, מסכת שלב 16. </li><li style=";text-align:right;direction:rtl"> להעריך את היעילות על ידי ציפוי מיקרוסקופי הבקיע בארות המכילות תאים (עבור assay מבוסס התאים הבודד) ועל ידי ספירת מספר תאים בבארות בודדות (לassay מבוסס תאים הרב; איור 2 </stronז&gt;). </li><li style=";text-align:right;direction:rtl"> דגירה תאים בתנאים סטנדרטיים בתחומים בינוניים (להרכב ראה שלב 2.6) על 37 מעלות צלזיוס, 5% CO 2. להשלים bFGF היומי (20 ng / ml) וEGF (מיליליטר / 20 ng). </li><li style=";text-align:right;direction:rtl"> אחרי שבוע אחד, לספור מספרים של מתעוררים תחומי גידול באמצעות מיקרוסקופ רגיל עם הגדלה 4X או 10X ומיקרוסקופ פלואורסצנטי כדי לזהות אותות הקרינה ממערכת הכתב המשולבת. רוזן התחומים בקוטר העולה על כתחומים &quot;גדולים&quot; 100 מיקרומטר, ותחומים בקוטר 100 - 50 כתחומים &quot;קטנים&quot; מיקרומטר (איור 3). להיות בטוח שאתה לספור תחומים אמיתיים ולא תא אשכולות. הערה: במבחנים מבוססי תאים בודדים היווצרות כדור קלה יותר להבקיע מיקרוסקופי לאחר 10 (לעומת 7) ימים של התרבות. </li><li style=";text-align:right;direction:rtl"> חשב את חלקם של תאי יוצרי כדור ב+ RFP ותאי בהתאמה RFP- במבחנים מבוססי תאים בודדים (96-גם צלחת אחת לכל ניסוי בודד) או תא-ba הרבמבחני SED תחומים (טוב אחד עבור כל ניסוי בודד) כפי שהוצג על ידי et al שו. 16 הערה: חלקם של תאי תחום יצירה (%) = (מספר תחומים) / (מספר תאי זרע) x 100 </li></ol> 3. Passaging הסידורי של Spheres <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הנח את התוכן של כל אחד גם בצינור סטרילי מתאים וצנטריפוגות ב XG 300 עבור 10 דקות ב RT. עבור מבחני מבוססי תאים רב התחומים, לאסוף יחד את התחומים מאחד גם. לשטוף היטב 3-5 פעמים עם PBS ו צנטריפוגות 2 דקות יותר. עבור מבחני מבוססי תאים בודדים תחומים, לאסוף כדורים בודדים. לאור המספרים הנמוכים של תאים, להשתמש צינורות 1.5 מיליליטר לצעדי צנטריפוגה וכביסה. </li><li style=";text-align:right;direction:rtl"> הסר supernatant וגלול גלול ב200 μl של 0.05% טריפסין- EDTA. </li><li style=";text-align:right;direction:rtl"> על מנת להשיג הפרדת תא אופטימלית, דגירה ההשעיה התא ב 37 מעלות צלזיוס למשך 5 דקות בייקרו רך. לייעל trypsinization זמן לקו הסלולרי שלך לשיעור מוות של תאים תחתון: אם לא גדולהתחומים הוא triturate הגלוי בעדינות באמצעות קצה פיפטה 100 μl. במקרה תחומים גדולים עדיין קיימים, דגירה עם טריפסין עוד 3 דקות ולאחר מכן להמשיך לשלב טחינה דקה. במקרה של יחיד מבחני מבוססי תאים לוודא כדי לייעל את הזמן לתשואת תא אופטימלי של תאי חיים במהלך שלב trypsinization. </li><li style=";text-align:right;direction:rtl"> כדי להשבית טריפסין, להוסיף 500 μl בינוני ו צנטריפוגות מלאים ב XG 300 במשך 10 דקות, במקרה של מבחני תא בודדים, צנטריפוגות במשך 2 דקות נוספות. </li><li style=";text-align:right;direction:rtl"> הסרת תאי supernatant ו resuspend בזהירות במדיום תחומים. </li><li style=";text-align:right;direction:rtl"> שימוש במסנן כובע מסננת תא 40 מיקרומטר לקבל השעיה תא בודד. </li><li style=";text-align:right;direction:rtl"> במקרה של שימוש ב+ RFP ותאי RFP- במבחנים-bassed תא רב, להעריך את אחוז תאי ניאון בכל אחד גם לאחר resuspension באמצעות cytometer זרימת הניתוח. </li><li style=";text-align:right;direction:rtl"> עבור מבחני סידוריים replating של תאים בודדים, זרע תא 1 לכל גם לתוך צלחת אולטרה חדשה נמוכה מצורף 96-גם הכינו כמפורט לעיל. מכדור אחד בודד, זרע כ 20 בארות בודדות. עבור מבחני replating תחומים עיקריים המבוסס על תאים רב, תאי זרע המתקבלים מכן אחד מתחומים עיקריים לצלחת 96-היטב חדשה ולספור את מספר התאים למחרת על ידי מיקרוסקופ. </li><li style=";text-align:right;direction:rtl"> להעריך את חלקם של תאי יוצרי כדור במבחני תחומים משניים באמצעות הנוסחא המתוארת בשלב 2.11. </li></ol> ניתוח 4. תוצאה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לנתח את התוצאות מניסויים שבוצעו בtriplicates העצמאי ולהשתמש במבחן t דו-צדדי לנתח ערכים מתפלגים נורמלי ואחר Mann-Whitney-בדיקות לניתוח סטטיסטי. </li></ol>

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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=Non-small+cell+lung+cancer+cells+expressing+CD44+are+enriched+for+stem+cell-like+properties.">Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties.</a> PLoS One. 5 (11), e14062 (2010).</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 (38), 16281-16286 (2009).</li><li>Ma, L., Lai, D., Liu, T., Cheng, W., Guo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem-like+cells+can+be+isolated+with+drug+selection+in+human+ovarian+cancer+cell+line+SKOV3.">Cancer stem-like cells can be isolated with drug selection in human ovarian cancer cell line SKOV3.</a> Acta Biochim Biophys Sin (Shanghai). 42 (9), 593-602 (2010).</li><li>Wang, H., Zhang, Y., Du, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+and+breast+cancer+spheres+are+similar+in+transcriptomic+features+and+sensitive+to+fenretinide).">Ovarian and breast cancer spheres are similar in transcriptomic features and sensitive to fenretinide).</a> Biomed Res Int. 2013, 510905 (2013).</li><li>Du, Y. 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=Effects+and+mechanisms+of+anti-CD44+monoclonal+antibody+A3D8+on+proliferation+and+apoptosis+of+sphere-forming+cells+with+stemness+from+human+ovarian+cancer.">Effects and mechanisms of anti-CD44 monoclonal antibody A3D8 on proliferation and apoptosis of sphere-forming cells with stemness from human ovarian cancer.</a> Int J Gynecol Cancer. 23 (8), 1367-1375 (2013).</li><li>Xiang, T., 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=Interleukin-17+produced+by+tumor+microenvironment+promotes+self-renewal+of+CD133+cancer+stem-like+cells+in+ovarian+cancer.">Interleukin-17 produced by tumor microenvironment promotes self-renewal of CD133 cancer stem-like cells in ovarian cancer.</a> Oncogene. , (2013).</li><li>Liu, T., Cheng, W., Lai, D., Huang, Y., Guo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+primary+ovarian+cancer+cells+in+different+culture+systems.">Characterization of primary ovarian cancer cells in different culture systems.</a> Oncol Rep. 23 (5), 1277-1284 (2010).</li><li>Soritau, O., 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=Enhanced+chemoresistance+and+tumor+sphere+formation+as+a+laboratory+model+for+peritoneal+micrometastasis+in+epithelial+ovarian+cancer.">Enhanced chemoresistance and tumor sphere formation as a laboratory model for peritoneal micrometastasis in epithelial ovarian cancer.</a> Rom J Morphol Embryol. 51 (2), 259-264 (2010).</li><li>Shi, M. F., 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+cancer+stem+cell-like+cells+from+human+epithelial+ovarian+carcinoma+cell+line.">Identification of cancer stem cell-like cells from human epithelial ovarian carcinoma cell line.</a> Cell Mol Life Sci. 67 (22), 3915-3925 (2010).</li><li>Zhang, 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=Identification+and+characterization+of+ovarian+cancer-initiating+cells+from+primary+human+tumors.">Identification and characterization of ovarian cancer-initiating cells from primary human tumors.</a> Cancer Res. 68 (11), 4311-4320 (2008).</li><li>Liang, D., 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=The+hypoxic+microenvironment+upgrades+stem-like+properties+of+ovarian+cancer+cells.">The hypoxic microenvironment upgrades stem-like properties of ovarian cancer cells.</a> BMC Cancer. 12, 201 (2012).</li></ol>

The authors have nothing to disclose.

תרבויות תחומים הן שיטה נפוצה לassay תא גזע סרטני פוטנציאלי ולהעשיר לתאי גזע כמו-במגוון רחב של תאים סרטניים אנושיים 15,25,26. בתנאים אלה התרבות, תאי סרטן שאין יכולת התחדשות עצמית צפויים לבדל וסופו של דבר לעבור מוות של תאים. למרות שהם עשויים בתחילה יוצרים צבירי תאים או ?...

There is increasing evidence that ovarian carcinomas are comprised of heterogeneous mixtures of cells and harbor so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for disease initiation, maintenance and relapse after conventional cytotoxic therapies1-3. Therefore, the development of molecular strategies targeting ovarian CSCs is an important goal and promises to improve the therapy of ovarian cancer patients.
A pre-requisite for the understanding of the molecular features of CSCs is their reliable isolation from the non-CSCs. However, identification of ovarian CSCs appears challenging. While CD133 expression and aldehyde dehydrogenase (ALDH) activity4,5 have been reported to mark ovarian CSCs, some data indicate that these markers are unstable6. Consistently, in ovarian cancer, other than for example in breast carcinoma7, expression of ALDH1 associates with favorable outcome8 and expression of the proposed stem cell marker CD44 variant has no prognostic value9. More recently, we have shown that expression of the embryonic stem cell protein SOX2 confers stemness to ovarian carcinoma cells10 and high SOX2 expression associates with clinically aggressive ovarian and breast carcinomas11,12. Therefore, in this report we use a lentiviral reporter construct containing a red fluorescence protein (RFP) whose expression is controlled by a SOX2 regulatory region, as a method to isolate putative ovarian CSCs.
By definition, CSCs can both self-renew and differentiate, giving rise to all tumor cell types. Putative CSC populations need to be analyzed in functional assays performed in vivo. For obvious reasons, in human cells such functional tests are confined to xenograft assays, comprising mostly transplantation of human tumor cells into immuno-compromised mice10,13.
An alternative in vitro method was offered by Brent Reynolds and Sam Weiss who firstly reported the so-called neurosphere assay as a surrogate assay evaluating stem potential in neural cells14. Dontu and colleagues later confirmed the use of this assay for evaluation of stem cell potential in breast cells15,16. Here, human mammary cells were plated in different numbers in serum-free medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), B-27 and heparin and cultured under non-adherent conditions for seven to ten days before sphere formation was scored by microscopy. Following this protocol with some adjustments in cell numbers, growth medium and supplements, several groups have explored in vitro stem cell potential from several cancer types such as breast17, brain18, pancreas19 and colon20 tumors. In ovarian carcinoma, we have recently reported feasibility of the spheres assay and compared its results to those collected in in vivo murine xenograft models10. We found that overexpression of the stem cell protein SOX2 enhanced both in vitro sphere formation as well as in vivo tumorigenicity of human ovarian carcinoma cells10. However, the frequency of sphere-initiating cells was higher than the frequency of tumor-initiating cells measured in vivo10 suggesting that either the sphere assay may lead to false positive results due to technical reasons or, alternatively, the in vivo assay may be inefficient and result in false negative results.
In this report, we analyze multi cell-based ovarian spheres assays in more detail, review the different protocols available in the literature and compare them to a single cell-based assay. We show that the single cell-based assay provides more accurate and reproducible results than multi cell-based assays, which can be highly influenced by the density of plated cells unless methylcellulose is added to the cultures to immobilize cells. However, also in single cell-based assays, in vitro sphere-initiating potential is observed at higher frequency than in vivo tumor-initiating potential.

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			<td>Lonza</td>
			<td>CC-3151</td>
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			<td>end concentration: 20 ng/ml</td>
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			<td height="21" style="height:21px;">Heparin-Natrium-25000 IE</td>
			<td>Ratiopharm</td>
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			<td>Gibco</td>
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			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>Gibco</td>
			<td style="width:119px;">21875-034</td>
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			<td height="21" style="height:21px;">Trypsin-EDTA&#160;</td>
			<td>Gibco</td>
			<td style="width:119px;">25300-054</td>
			<td></td>
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			<td height="21" style="height:21px;">Dulbecco&#8217;s PBS (1x)</td>
			<td>Gibco</td>
			<td style="width:119px;">14190-094</td>
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			<td height="21" style="height:21px;">Shield1&#160;</td>
			<td>Clontech</td>
			<td style="width:119px;">632189</td>
			<td>dilution 1:1000</td>
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			<td>Gibco</td>
			<td style="width:119px;">21041-025</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">DMEM/F12 (powder)</td>
			<td>Gibco</td>
			<td style="width:119px;">42400-010</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methyl cellulose</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">M0387</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Puromycin dihydrochloride</td>
			<td>applichem</td>
			<td style="width:119px;">A2856</td>
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			<td height="21" style="height:21px;">cell sorter&#160;</td>
			<td style="width:112px;">BD</td>
			<td style="width:119px;"></td>
			<td>Aria III cell sorter&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS analyser</td>
			<td style="width:112px;">BD</td>
			<td style="width:119px;"></td>
			<td>accuri c6 flow cytometer</td>
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		<tr height="21">
			<td height="21" style="height:21px;">microscope</td>
			<td style="width:112px;">Olympus&#160;</td>
			<td style="width:119px;"></td>
			<td>IX50 Osiris</td>
		</tr>
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evaluation of stem cell properties in human ovarian carcinoma cells using multi and single cell-based spheres assays

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

במבחני התחומים קונבנציונליים, כמעט 40% מRFP + OVCAR-3 תאים לעומת 20% מתאי RFP- הולידו תחום גידול בודד בassay תחומים העיקרי (איור 4 א). יתר על כן, תחומים שהוקמו על ידי RFP + תאים היו גדולים יותר בגודל יותר מאלה שהוקמו על ידי תאי RFP-. כאשר מצו?...

Watch this Scientific Journal Video about Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays at JoVE.com

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

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

הערכה של מאפייני תא גזע בקרצינומה של תאי השחלות אדם באמצעות Multi ומבחני Spheres מבוסס תא בודדים

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

evaluation-stem-cell-properties-human-ovarian-carcinoma-cells-using

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Medicine

9.5K Views.  University Hospital Basel. In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

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

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

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

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

הערכה של נדידת תאי גזע סרטני באמצעות מכשירי microfluidic מידור והדמית תא חייה

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

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

גזע השתלת תאים ב במבחנה דגם סימולציה איסכמיה / reperfusion

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

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

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

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

עיבוד של רקמת גידול במוח העיקרית למבחנים בתאי גזע וזרימת מיון

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

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.

בידוד, אפיון והשוואתי התמיינות של תאי גזע אנושיים שיניים זולות נגזרה שיניים קבועות על ידי שימוש בשתי שיטות שונות

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

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells&#39; responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

פרוטוקול assay המבוסס תא לחיזוי הפרוגנוזה של Idiopathic עקמת באמצעות סלולרית דיאלקטרי ספקטרוסקופיה

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current &#39;gold standard&#39; method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The presence of high-risk HPV in head and neck tumor tissue is associated with favorable outcomes. The recently developed RNA in situ hybridization technique called RNAscope allows direct visualization of HPV E6/E7 mRNA in FFPE tissue sections.

RNAscope עבור<em&gt; באתרו</em&gt; איתור של נגיף הפפילומה אנושי תעתיק פעיל בראש וצוואר קשקש קרצינומה של תאים

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection ...

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.

העשרה לתאי גזע Chemoresistant סרטן השחלות משורות תאים אנושיות

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

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

בידוד של תאי האנדותל אדם הלימפה על ידי מיון תא הקרינה המופעל Multi-פרמטר

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

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.

לומד סרטן לבלב גזע מאפייני תא לפיתוח טיפול חדש אסטרטגיות

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

Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local recurrence and distant metastasis. One hypothesis to explain this recurrence is the presence of cancer stem-like cells (CSCs) that present inherent chemo- and radio-resistance. In order to develop new therapeutic strategies, it is necessary to have experimental models that validate the effectiveness of targeted treatments and therefore to have reliable methods for the identification and isolation of CSCs. To this end, we present a protocol for the isolation of CSCs from human HNSCC cell lines that relies on the combination of two successive cell sortings performed by fluorescence activated cell sorting (FACS). The first one is based on the property of CSCs to overexpress ATP-Binding Cassette (ABC) transporter proteins and thus exclude, among others, vital DNA dyes such as Hoechst 33342. The cells sorted with this method are identified as a &#34;side population&#34; (SP). As the SP cells represent a low percentage (&#60;5%) of parental cells, a growing phase is necessary in order to increase their number before the second cell sorting. The next step allows for the selection of cells that possess two other HNSCC stem cell characteristics i.e. high expression level of the cell surface marker CD44 (CD44high) and the over-expression of aldehyde dehydrogenase (ALDHhigh). Since the use of a single marker has numerous limitations and pitfalls for the isolation of CSCs, the combination of SP, CD44 and ALDH markers will provide a useful tool to isolate CSCs for further analytical and functional assays requiring viable cells. The stem-like characteristics of CSCs was finally validated in vitro by the formation of tumorispheres and the expression of &#946;-catenin.

Understanding the role of cancer stem-like cells in tumor recurrence and resistance to therapies has become a topic of great interest in the last decade. This article describes the isolation and characterization of the sub-population of cancer stem-like cells from head and neck squamous carcinoma cell lines (HNSCC).

בידוד ואפיון של ראש וצוואר קשקש Cell Carcinoma-אוכלוסיית מאפייני תאי גזע לאחר

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local ...