This publication is funded through Cure CMD, an Association Contre Les Myopathies (AFM) grant (project 16297), and by the National Institutes of Health (grant numbers K08 AR059750, L40 AR057721 and 2R01NS047727).

הערה: פרוטוקולים לאוסף של רקמות אדם צריכים להיות נבדקו ואושרו על ידי הוועדה המוסדית IRB. אוסף של רקמה אנושית שהושלכה, מזוהה de אושר על ידי בית החולים לילדים בבוסטון וועדות IRB Brigham and בית החולים לנשים. השיטות שתוארו להלן יושמו בבידוד של תאי myogenic מדה-מזוהה, שהושלך רקמה. השיטות שתוארו חלות על רקמה שנאספו מחומר מטופל הסכים. 1. בידוד תא <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ניתוק של ביופסיה של שריר וטיהור של תאי myogenic <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> טרום לשקול צלחת תרבית רקמת 10 סנטימטרים במנדף בטיחות ביולוגי בתרבית רקמה, ולאחר מכן לשקול מחדש את הצלחת המכילה את הביופסיה של השריר כדי לחשב את כמות הרקמה שניתק. </li><li style=";text-align:right;direction:rtl"> באמצעות אזמלים סטריליים, רקמת שריר בשר טחון דק ולהוסיף כמה טיפות של סטרילית 1x HBSS כדי למנוע רקמות מהתייבשות. </li><li style=";text-align:right;direction:rtl"> להוסיף 3.5 מיליליטר כל אחת מdispase השני וcollagenase D לגרם של רקמת שריר להתעכל. פיפטה רקמות טחונות ופתרון אנזים באמצעות פיפטה 25 מיליליטר סטרילי כמה פעמים. דגירה את הצלחת בתרבית רקמת חממה ב 37 מעלות צלזיוס עם 5% CO 2 במשך 15 דקות ולעכל רקמה עד slurry עובר בקלות אם כי טפטפת מיליליטר סטרילי 5. הערה: ניתוק רקמות מושגים בדרך כלל על ידי עיכול אנזימטי בתוך 45-90 דקות. עיין בסעיף &#39;נציגי התוצאות &quot;לפרטים נוספים. </li><li style=";text-align:right;direction:rtl"> הוסף 2 כרכים של מדיום גידול סטריליים לרקמה ניתק, מסנן דרך מסננת 100 מיקרומטר תא מעל 50 מיליליטר תאי צינור וגלול חרוטי במשך 10 דקות ב 329 XG (~ 1,100 סל&quot;ד), בטמפרטורת חדר. אנא עיין בטבלת חומרים להרכב תקשורת. </li><li style=";text-align:right;direction:rtl"> Resuspend גלולה בנפח של 1 מדיום גידול סטרילי ולהוסיף 7 כרכים פתרון תמוגה תא דם אדום. סנן את הפתרון דרך מסננת תא 40 מיקרומטר, אז גלולה tהוא תאים במשך 10 דקות ב 329 XG, בטמפרטורת חדר. </li><li style=";text-align:right;direction:rtl"> ספירת התאים בhemocytometer וresuspend תאי BSA 0.5% 1x HBSS בריכוז של 1 x 10 6 תאים / μl 100. מניח בצד ~ 250,000 תאים בצינור אחד שישמשו כשלילית שליטה (בלא כתם). הגדר צינורות צד נוספים חד-צבע מוכתם שליטה ליודיד propidium ולCD56, הנדרשים לgating התקין של תאים חיוביים CD56 על ידי מיון FACS. אנא עיין בתא מדריכים למיון או להתייעץ עם FACS מיון מומחי מתקן ליבה כדי להבטיח בקרה נאותה כלולים. </li><li style=";text-align:right;direction:rtl"> כתם התאים להיות מסודרים עם 5μl / 10 6 תאים של נוגדן אנטי CD56. דגירה כל הדגימות (כולל בקרות) על קרח למשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> לשטוף דגימות ב 10 מיליליטר 1x HBSS ותאים גלולה במשך 10 דקות ב 329 XG (~ 1,100 סל&quot;ד) בצנטריפוגה בקירור, 4 ° C טמפרטורה. </li><li style=";text-align:right;direction:rtl"> להוסיף יודיד propidium בריכוז סופי של 1μg / מיליליטר המדגם להיות sorטד להרחקה של תאים מתים. לטהר תאים חיוביים myogenic CD56 מהתאים שאינם myogenic באמצעות סדרן תא הקרינה הופעלה. </li></ol></li><li style=";text-align:right;direction:rtl"> ניתוק של ביופסיה של עור יכול להיות מבודד fibroblasts Dermal מאגרוף עור מחולים כאשר ביופסיות שריר אינן זמינות: הערה. fibroblasts עורי יכול לשמש כחומר ביולוגי למחקרים רבים, כוללים התמרה עם Myod לייצר תאי myogenic. בנוסף, ניתן להשתמש fibroblasts עורי ליצירת תאי iPS, אשר יכול להיות מובחן לסוגי תאים שונים למחקר נוסף. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להעביר את הביופסיה של העור למעבדה במדיום תחבורה. לאחר המדגם הוא קיבל, לבצע את התרבות העיקרית בהקדם האפשרי. אם התרבות העיקרית לא ניתן לקבוע באותו היום, לאחסן המדגם בטמפרטורת חדר למשך לילה. </li><li style=";text-align:right;direction:rtl"> העבר את הביופסיה של העור לצלחת פטרי 35 מ&quot;מ סטרילי בזרימה למינרית. </li><li style=";text-align:right;direction:rtl"> יש לשטוף את הביופסיה של העור בצלחת פטרי עם 1 סטריליx PBS להסיר דם ופסולת. הסר את רקמת השומן עם אזמל סטרילי. </li><li style=";text-align:right;direction:rtl"> הוסף פתרון collagenase 2 מ&quot;ל ולקצץ את הרקמה עם אזמל, לדגור על 37 מעלות צלזיוס למשך 45 דקות לשעה 1, תלוי בגודל של הרקמה. </li><li style=";text-align:right;direction:rtl"> העברת הרקמה מתעכל לצינור חרוטי 15 מיליליטר, לשטוף את צלחת פטרי עם 2 מיליליטר של מדיום תרבות פיברובלסטים פעמיים, ולאסוף את המדיום באותו הצינור. </li><li style=";text-align:right;direction:rtl"> גלולה תאים ב XG 200 במשך 5 דקות בטמפרטורת חדר. </li><li style=";text-align:right;direction:rtl"> בטל supernatant ולשטוף את הכדור עם 3 מיליליטר של מדיום פיברובלסטים להסיר collagenase, אז תאי גלולה שוב. אנא עיין בטבלת חומרים להרכב תקשורת. </li><li style=";text-align:right;direction:rtl"> חזור על השלב 1.2.7 עוד פעם אחת. </li><li style=";text-align:right;direction:rtl"> להשעות מחדש גלולה בתאים בינוניים פיברובלסטים וצלחת 5 מיליליטר על בקבוק תרבות רקמת T25 סטרילי. דגירה הבקבוק ב 37 מעלות צלזיוס עם 5% CO 2. </li><li style=";text-align:right;direction:rtl"> להעריך את התרבות לקובץ מצורף פיברובלסטים וצמיחה מעל 1-3 הימים הקרובים. &lt;br /&gt; הערה: חלק פיסות רקמה קטנה עשויות גם לצרף הצלחת וfibroblasts להעביר את חתיכות רקמה אלה. </li><li style=";text-align:right;direction:rtl"> לשמור על התרבות באותם תנאים עד fibroblasts גדלים למפגש כ -80%. </li><li style=";text-align:right;direction:rtl"> לאסוף fibroblasts ידי trypsinization ולהעביר על גבי צלוחיות תרבות טריות להרחבה נוספת. בצע trypsinization ידי שטיפת התרבויות 3 פעמים ב1x PBS ללא Ca ++ Mg ++. להוסיף טריפסין-EDTA (ראו לוח חומרים) לתאים (2ml / בקבוק T25) למשך 2 דקות על 37 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> פיצול תאים מנותקים לתוך צלוחיות נוספות. אם כמה חתיכות רקמה נשארות צמודות לצלוחיות T25 המקוריות, להוסיף 5 מיליליטר מדיום תרבות הטרי לבקבוק זה ועוד fibroblasts יעבור את הרף. הערה: תרבות פיברובלסטים המורחבת אפשר להקפיא למטה כמו P1 ומאוחסנות בחנקן נוזלי לניסויים עתידיים. </li></ol></li></ol> 2. הנצחה של תאי myogenic <ol style=";text-align:right;direction:rtl"><li&gt; פלייט תאי 5 מיליון הפניקס Ecotropic אריזה (PE) לילה בצלחת תרבית רקמת 10 סנטימטרים סטרילי בDMEM ובינוני 199 על יחס של 4: 1, בתוספת 10% עגל בסרום. </li><li style=";text-align:right;direction:rtl"> תאי Feed 30 דקות לפני transfection עם 5 מיליליטר של תקשורת טרי בתוספת קפאין 10 מ&quot;מ. </li><li style=";text-align:right;direction:rtl"> Homogenize תערובת של 2 מיקרוגרם של ה- DNA פלסמיד מהכנת midi (פלסמיד cdk4 או hTERT) וPolyjet ודגירה בטמפרטורת חדר במשך 15 דקות, כפי שהומלץ על ידי היצרן. </li><li style=";text-align:right;direction:rtl"> מוסיף את תערובת פלסמיד / Polyjet לתאי PE הלילה. </li><li style=";text-align:right;direction:rtl"> תאים להאכיל עם מדיום חדש (DMEM ובינוני 199 על יחס של 4: 1, בתוספת 10% עגל בסרום). שתים עשרה שעות מאוחר יותר, לאסוף את supernatant המכיל וירוסים ולסנן אותה דרך מסנן גודל 0.45 מיקרומטר נקבובית. </li><li style=";text-align:right;direction:rtl"> השתמש 1 מיליליטר של supernatant נאסף להדביק לילה PA317 קו תא אריזת amphotropic 5 ולקבל שורת תאים לייצר וירוס יציב לאחר בחירה עם או 0.5 מ&quot;ג / מיליליטר נאומיצין (G418) לcdk4 או 0.5 מ&quot;ג / מיליליטר hygromycin לhTERT. </li><li style=";text-align:right;direction:rtl"> הכן עובד supernatants ויראלי על ידי גידול תאי אריזה היציב לconfluency הקרוב, ולאחר מכן קציר supernatant בכל בוקר, ערב ובוקר לשלושה יבולים. </li><li style=";text-align:right;direction:rtl"> סנן את supernatants ויראלי ובמישרין או להשתמש לחלק אותם ל1 aliquots מיליליטר ולאחסן ב -80 ° C לשימוש מאוחר יותר. שים לב שsupernatant ויראלי מאבד 50% יעילות הדבקה בכל להקפיא להפשיר. זכור כדי להלבין שטיפה לפני השלכת כל מה שנגע בחלקיקים הנגיפיים. הערה: שורת התאים-ייצור וירוס PA317 היציבה יכולה להיות קפוא ונשמרה ב-150 מעלות צלזיוס במשך אחסון קבוע (הקפאה בינונית היא DMSO 10%; סרום 90%). </li><li style=";text-align:right;direction:rtl"> מטוהר FACS צלחת תאי myogenic (מתואר בצעדים 1.1-1.9) בצפיפות של 5 x 10 4 תאים / גם ב6-גם צלחות מצופות ג&#39;לטין 0.1%. ודא כי תאים המחוברים לצלחת לפני שימשיכו עם זיהום ויראלי. </li><li style=";text-align:right;direction:rtl"> להוסיף 400 μl מסונן, freshly מיוצר supernatant הנגיפי או aliquots הקפוא לכל צלחת שש היטב לילה (לשמור 2 בארות כפקדים). </li><li style=";text-align:right;direction:rtl"> שנה בינונית על ידי האכלת תאים עם 2.5 מיליליטר / גם של תקשורת טרי שריר (4: 1 בינוני הנשר (DMEM) שונה Dulbecco ובינוני 199 בתוספת 15% בסרום שור עוברי; 0.02 M HEPES חיץ; ויטמין 1.4 מ&quot;ג / L B12; 0.03 מ&quot;ג / L ZnSO4, dexamethasone 0.055 מ&quot;ג / L, גורם גדילת hepatocyte / l 2.5 מיקרוגרם וגורם גדילת 10 מיקרוגרם / L פיברובלסטים בסיסיים). מחק את התקשורת וטפטפות המכילה חלקיקים הנגיפיים במכל אקונומיקה. לצאת מתאים באותו בינוני במשך 3 ימים להתאושש מזיהום, אז להתייחס לבחירה או באמצעות 400 מיקרוגרם / מיליליטר נאומיצין (זיהום cdk4) או 300 מיקרוגרם / מיליליטר hygromycin (זיהום hTERT). </li><li style=";text-align:right;direction:rtl"> שמור על תאים תחת בחירת תרופה עד שהתאים במנה למות שליטה (1-2 שבועות). </li><li style=";text-align:right;direction:rtl"> תאי מעבר לפני שהן הופכות מחוברות (confluency 60-80%; באמצעות 0.05% טריפסין EDTA), גם בselectiבתקופה. תאי Replate במנות 10 ס&quot;מ מרובים עם מדיום myoblast טרי (כמתואר ב2.11) בתוספת תרופת הבחירה. לשמור הנציחו תאים שנבחרו כאוכלוסייה הטרוגנית או לשכפל להשיג רקע הומוגנית לחלוטין גנטי (אותו החדרת transgene בכל תא). </li><li style=";text-align:right;direction:rtl"> לבצע בחירה משובט באמצעות השלבים הבאים: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> זרעי התאים בצפיפות נמוכה (למשל 300 עד 500 תאים 10 מנות סנטימטר) ולשמור אותם במשך כשבועיים, עד מושבות קטנות נוצרות (10-20 תאים). </li><li style=";text-align:right;direction:rtl"> הסר ביותר של המדיום בשלב זה, ומשאיר רק סרט דק כדי למנוע את התאים מהתייבשות. </li><li style=";text-align:right;direction:rtl"> מניחים טבעת שיבוט (עם קצה אחד טבול בשומן ואקום סיליקון) על כל שיבוט רצוי ולהוסיף כמה טיפות של טריפסין / EDTA. </li><li style=";text-align:right;direction:rtl"> קציר תאים ברגע שהם הופכים מעוגלים ידי aspirating בזהירות את התאים באמצעות קצה מיליליטר 1 או pipet פסטר והעביר לPLA multiwell הכי הקטןte (96 או 48, 24 או 12 צלחות גם). </li><li style=";text-align:right;direction:rtl"> להרחיב שיבוטים לפי צורך כדי למנוע confluency המקומי. </li></ol></li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+human+and+mouse+satellite+cells+really+the+same.">Are human and mouse satellite cells really the same.</a> J Histochem Cytochem. 58, 941-955 (2010).</li><li>Belles-Isles, 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=Rapid+selection+of+donor+myoblast+clones+for+muscular+dystrophy+therapy+using+cell+surface+expression+of+NCAM.">Rapid selection of donor myoblast clones for muscular dystrophy therapy using cell surface expression of NCAM.</a> Eur J Histochem. 37, 375-380 (1993).</li><li>Meng, J., Adkin, C. F., Xu, S. W., Muntoni, F., Morgan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+human+muscle-derived+cells+to+skeletal+muscle+regeneration+in+dystrophic+host+mice.">Contribution of human muscle-derived cells to skeletal muscle regeneration in dystrophic host mice.</a> PLoS One. 6, e17454 (2011).</li><li>Zhu, C. H., 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=Cellular+senescence+in+human+myoblasts+is+overcome+by+human+telomerase+reverse+transcriptase+and+cyclin-dependent+kinase+4:+consequences+in+aging+muscle+and+therapeutic+strategies+for+muscular+dystrophies.">Cellular senescence in human myoblasts is overcome by human telomerase reverse transcriptase and cyclin-dependent kinase 4: consequences in aging muscle and therapeutic strategies for muscular dystrophies.</a> Aging Cell. 6, 515-523 (2007).</li><li>Mamchaoui, 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=Immortalized+pathological+human+myoblasts:+towards+a+universal+tool+for+the+study+of+neuromuscular+disorders.">Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders.</a> Skelet Muscle. 1, 34 (2011).</li><li>Sigmund, C. D., Stec, 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=.">.</a> Genetic Manipulation of the Renin-Angiotensin System Using Cre-loxP-Recombinase. Methods Mol Med. 51, 53-65 (2001).</li><li>Ludlow, A. 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=Quantitative+telomerase+enzyme+activity+determination+using+droplet+digital+PCR+with+single+cell+resolution.">Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution.</a> Nucleic Acids Res. 42, e104 (2014).</li><li>Jankowski, R. J., Haluszczak, C., Trucco, M., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+characterization+of+myogenic+cell+populations+obtained+via+the+preplate+technique:+potential+for+rapid+isolation+of+muscle-derived+stem+cells.">Flow cytometric characterization of myogenic cell populations obtained via the preplate technique: potential for rapid isolation of muscle-derived stem cells.</a> Hum Gene Ther. 12, 619-628 (2001).</li><li>Gharaibeh, 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=Isolation+of+a+slowly+adhering+cell+fraction+containing+stem+cells+from+murine+skeletal+muscle+by+the+preplate+technique.">Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique.</a> Nat Protoc. 3, 1501-1509 (2008).</li><li>Qu, Z., 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=Development+of+approaches+to+improve+cell+survival+in+myoblast+transfer+therapy.">Development of approaches to improve cell survival in myoblast transfer therapy.</a> J Cell Biol. 142, 1257-1267 (1998).</li><li>Choi, J., 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=MyoD+converts+primary+dermal+fibroblasts,+chondroblasts,+smooth+muscle,+and+retinal+pigmented+epithelial+cells+into+striated+mononucleated+myoblasts+and+multinucleated+myotubes.">MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes.</a> Proc Natl Acad Sci U S A. 87, 7988-7992 (1990).</li><li>Huard, 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=Transplantation+of+dermal+fibroblasts+expressing+MyoD1+in+mouse+muscles.">Transplantation of dermal fibroblasts expressing MyoD1 in mouse muscles.</a> Biochem Biophys Res Commun. 248, 648-654 (1998).</li><li>Lattanzi, 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=High+efficiency+myogenic+conversion+of+human+fibroblasts+by+adenoviral+vector-mediated+MyoD+gene+transfer.+An+alternative+strategy+for+ex+vivo+gene+therapy+of+primary+myopathies.">High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies.</a> J Clin Invest. 101, 2119-2128 (1998).</li><li>Park, J. I., 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=Telomerase+modulates+Wnt+signalling+by+association+with+target+gene+chromatin.">Telomerase modulates Wnt signalling by association with target gene chromatin.</a> Nature. 460, 66-72 (2009).</li></ol>

The authors have nothing to disclose.

תאים כמשאב שימושי הבידוד והתרבות של אוכלוסיות תאי myogenic הוא מאוד שימושי כאשר הקמת פנוטיפים מחלה או במודלים חוץ גופית של מחלה. הליך בידוד תא myogenic המתואר כאן מאפשר הבידוד של myoblasts ופיברובלסטים מדגימות שרירי שלד, אז מה שיכול להי?...

A correction was made to the authors section in: Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling
One of the authors names was corrected from:
Stacy C. Cossette
to:
Stacy A. Cossette

A correction was made to the authors section in: Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

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

Molecular diagnostics has dramatically evolved in the past 20 years. Genomic DNA is now routinely isolated from sputum or cheek swab, while in the past it required a blood draw. With the current fast turnaround time and ease of gene sequencing, many disease mutations are routinely identified with no need of additional testing. In the case of muscle disease diagnostics, identification of dozens of new genes in the past decade responsible for either muscular dystrophy or myopathy have dramatically changed the ways these diseases are diagnosed 1,2. Currently, there are dozens of genes that have been identified as causes of muscular dystrophy and congenital myopathy, although the mechanisms by which many of these genes produce disease remain unclear. In particular, rare diseases constitute a challenge due to the small size of the patient populations. For these cases, as well as for more common diseases, the generation of stable tools that facilitate studies on the mechanism of pathogenesis and screening of therapeutic drugs is highly desirable. 
Despite dramatic progress in DNA diagnostics, muscle biopsies are still performed to establish the primary diagnosis in many patients in whom primary metabolic or muscle disease is suspected. When a muscle biopsy is necessary, it offers the opportunity for additional diagnostic and research tissue collection with minimal additional morbidity risk for the patient. As there are a number of uses for each tissue specimen, it is highly desirable to establish techniques for primary cell culture using surgical tissue that are straightforward, efficient, and require minimal amounts of tissue. Proper triage of muscle or skin biopsies is required to maximize isolation of primary cells from tissue and long-term storage of live material. Additionally, stem cell research and drug screening holds great promise for developing therapies for many diseases using cell-based assays 3,4.
We herein describe methods for primary cell isolation from human muscle or skin biopsies. Additionally, we include a protocol for immortalization of myogenic cells, which is useful for generating large numbers of cells from an individual. These cells can be used for downstream applications, such as custom drug-screenings, which are otherwise unachievable with the overall low number of cells obtained from primary tissue.

<table border="0" cellpadding="0" cellspacing="0" width="1197">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;"><a name="RANGE!A1:D52">Name of Material/ Equipment</a></td>
			<td style="width:187px;">Company</td>
			<td style="width:207px;">Catalog Number</td>
			<td style="width:484px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Equipment</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Tissue&#160;culture biosafety hood</td>
			<td style="width:187px;">Baker Company, Inc.</td>
			<td style="width:207px;">Model SterilGuard Hood</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:320px;">Benchtop centrifuge, such as Beckman Model&#160;</td>
			<td style="width:187px;">Beckman Coulter</td>
			<td style="width:207px;">Model Allegra 6R</td>
			<td>If cell sorting is performed, a centrifuge with refrigeration is preferred</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Microscope</td>
			<td style="width:187px;">Nikon</td>
			<td style="width:207px;">Model Eclipse TS100</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:320px;">Tissue culture incubator, connected to a CO2 source</td>
			<td style="width:187px;">Forma Scientific&#160;</td>
			<td style="width:207px;">Series II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Fluorescence-activated cell sorter (FACS)</td>
			<td style="width:187px;">Becton Dickinson</td>
			<td style="width:207px;">Model Aria</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:320px;">Reagents for isolation of primary myoblasts from tissue</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dispase II</td>
			<td style="width:187px;">Roche Applied Science</td>
			<td style="width:207px;">#04942078001</td>
			<td>Prepare a sock solution of 2.4U/ml in DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase D&#160;</td>
			<td style="width:187px;">Roche Applied Science</td>
			<td style="width:207px;">#088882001</td>
			<td>Prepare a stock solution of 10 mg/ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">1x Sterile Hank&#8217;s Balanced Saline Solution (HBSS), calcium and magnesium free</td>
			<td style="width:187px;">GIBCO Life Technologies</td>
			<td style="width:207px;">#14185-052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin, fraction V</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:207px;">#05470</td>
			<td>Prepare a sterile&#160; solution of 1X HBSS 0.5% BSA for FACS sorting</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Sterile growth medium for myoblasts: Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) with high glucose (4.5g) supplemented with 30% fetal bovine serum</td>
			<td style="width:187px;">GIBCO Life Technologies</td>
			<td style="width:207px;">11965-092</td>
			<td>Contains L- glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">RBC lysis solution</td>
			<td style="width:187px;">Qiagen</td>
			<td style="width:207px;">158904</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Propidium Iodide stock 10mg/mL</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:207px;">P4170</td>
			<td>Prepare the stock solution by diluting the powder in sterile distilled water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Anti-human CD56 antibody for flow cytometry</td>
			<td style="width:187px;">Biolegend</td>
			<td>318310</td>
			<td>APC-conjugated antibody, other labels are avaialable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Reagents for immortalization of primary myoblasts</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Ecotropic packaging cell line PE</td>
			<td style="width:187px;">Cell Biolabs</td>
			<td style="width:207px;">RV-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Amphotropic packaging cell line PA3174</td>
			<td style="width:187px;">Cell Biolabs</td>
			<td style="width:207px;">RV-102</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">Pig skin gelatin</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:207px;">G1890-500g</td>
			<td style="width:484px;">Prepare a stock solution of 0.1% gelatin in water. Coat the dish with the solution at 37&#176;C for one hour. Remove the solution and add medium.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">PolyJet&#160;</td>
			<td style="width:187px;">Signagen</td>
			<td>SL100688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">G418</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:207px;">345812</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Hygromycin</td>
			<td style="width:187px;">EMD Biosciences</td>
			<td style="width:207px;">400051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">pBabe plasmids containing mCDK4 and hTERT</td>
			<td style="width:187px;">not commercially available</td>
			<td style="width:207px;"></td>
			<td>Stadler et al, 2013</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Qiagen plasmid midiprep kit</td>
			<td style="width:187px;">Qiagen</td>
			<td>12143</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Medium 199</td>
			<td style="width:187px;">Life Technologies&#160;</td>
			<td>Medium 199 (31150022)</td>
			<td style="width:484px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Dulbecco&#8217;s modified Eagle medium (DMEM)</td>
			<td style="width:187px;">Life Technologies&#160;</td>
			<td>DMEM (11965-092)</td>
			<td style="width:484px;">Mix 4:1 DMEM:199</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:320px;">Myoblast growth medium: 4:1 Dulbecco&#8217;s modified Eagle medium (DMEM) and Medium 199 supplemented with 15% fetal bovine serum; 0.02 M HEPES buffer; 1.4 mg/l vitamin B12; 0.03 mg/l ZnSO4, 0.055 mg/l dexamethasone, 2.5 &#956;g/l hepatocyte growth factor and 10 &#956;g/l beta fibroblast growth factor.</td>
			<td style="width:187px;">Life technologie (DMEM, F199); Atlanta Biological (FBS); Invitrogen (Hepes); Fisher (ZincSulfate); Sigma (Vit.B12, Dexamethasone); Chemicon international (HGF); Biopioneer (betaFGF)&#160;</td>
			<td style="width:207px;">#15630-080 (Hepes); #Z68-500 (ZnSO); #V2876.#D4902 (Vit.B12, Dex); GF116 (HGF); HRP-0011 (bFGF).&#160;</td>
			<td style="width:484px;">Prepare media and stock solution Vit. B12 (20mg/ml); ZincSulfate (60&#181;g/ml); Dex (55&#181;g/&#181;l) separately for easier use. HGF stock solution (5&#181;g/ml) and FGF (20&#181;g/ml) should be added freshly every week at the final working concentration.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">TrypLE express</td>
			<td style="width:187px;">GIBCO Life Technologies</td>
			<td style="width:207px;">12605-010</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Myosin heavy chain antibody for immunostaining</td>
			<td style="width:187px;">Developmental Hybridoma Bank</td>
			<td style="width:207px;">MF20</td>
			<td>Clone MF20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Desmin Antibody for immunostaining</td>
			<td style="width:187px;">Thermo Scientific</td>
			<td>MS-376-S0</td>
			<td>Clone D33</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Horse serum</td>
			<td style="width:187px;">Invitrogen</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Reagents for primary skin fibroblast isolation</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">Transport medium: RPMI 1640 supplemented with 10% fetal bovine serum and 0.2% penicillin/streptomycin</td>
			<td style="width:187px;">RPMI medium 1640: GIBCO Life Technologies</td>
			<td style="width:207px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">Human primary fibroblast culture medium: RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin</td>
			<td style="width:187px;">Fetal bovine serum: Thermo Scientific</td>
			<td style="width:207px;">SH30071.03</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">Collagenase solution: Collagenase type 2 (100mg) resuspended in 12.5 ml of fibroblast culture medium and filter-sterilized</td>
			<td style="width:187px;">Worthington</td>
			<td style="width:207px;">4176</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Sterile 1X Phosphate Buffer Saline (PBS) calcium and magnesium free</td>
			<td style="width:187px;">Lonza</td>
			<td style="width:207px;">17-516F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials for isolation of primary myoblasts</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">50ml and 15ml sterile conical tubes</td>
			<td style="width:187px;">GeneMate/Bioexpress</td>
			<td style="width:207px;">C-3394-4 (50ml) ; C3394-1 (15ml))</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile scalpels</td>
			<td style="width:187px;">Aspen Surgical</td>
			<td style="width:207px;">372610</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer</td>
			<td style="width:187px;">Hausser Scientific</td>
			<td style="width:207px;">1492</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile 5, 10 and 25 ml pipettes</td>
			<td>Bellco glass</td>
			<td>1226-05010 (5ml); 1200-10010 (10ml) ;1228-25050 (25ml)</td>
			<td>Reusable pipettes are washed, cotton plugged and autoclaved before use</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile tissue culture-treated plastic dishes (10cm)</td>
			<td style="width:187px;">BD Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile nylon cell strainers (100&#181;m and 40&#181;m size)</td>
			<td style="width:187px;">BD Falcon</td>
			<td colspan="2">352340 (40&#181;m); 352360 (100&#181;m)</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">&#160;Materials for myoblast immortalization</td>
			<td style="width:187px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile 0.45&#181;m filters</td>
			<td>Millipore</td>
			<td>SLHV013SL</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Cloning rings</td>
			<td style="width:187px;">Corning</td>
			<td>#3166-8</td>
			<td>To be cleaned and autoclaved before and after use</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Materials for fibroblast cell lines&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile 35mm tissue culture-treated plastic dishes</td>
			<td>Greiner bio-one</td>
			<td>628160</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile scalpels</td>
			<td>DeRoyal</td>
			<td>D4510A</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile T25 tissue culture flasks</td>
			<td>Techno Plastic Product, TPP</td>
			<td>90026</td>
			<td>sold in the USA by MIDSCI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">TrypLE express</td>
			<td style="width:187px;">GIBCO Life Technologies</td>
			<td style="width:207px;">12605-010</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

איור 1 מדגים כמה מהשלבים העיקריים המעורבים בניתוק הרקמה העיקרי: כמות הרקמה המדויקת נשקלה בצלחת פטרי תרבית רקמה סטריליים (איור 1 א &#39;, ב&#39;). הרקמה אז דק טחון באמצעות אזמלים סטריליים, עד slurry רקמה מתקבל (איור 1 ג, ד). בעקבות תוספת של אנזימי העיכול...

Watch this Scientific Journal Video about Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling at JoVE.com

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

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.

בידוד והנצחה של שורות תאים שמקורם בחולה מביופסיה של שריר למחל דוגמנות

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

isolation-immortalization-patient-derived-cell-lines-from-muscle

Research

JoVE Journal

Medicine

16.4K Views.  UT Southwestern Medical Center. 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.

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

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

מידול והדמיה 3-Dimensional Cell קיבוציים Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

שרירי השלד מין Dimorphism מ Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

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.

הבידוד של גרעיני Cardiomyocyte מרקמות שלאחר המוות

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.

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 ...

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

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

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

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

תרבות מטופל נגזר סלולרי ובידוד של CD133<sup&gt; +</sup&gt; תאי גזע סרטני משוערים מלנומה

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

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&amp;#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

נהלי ביופסיה של שריר שלד אנושי באמצעות טכניקת Bergstrom השתנה

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

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, ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

ההכנה וRespirometric הערכה של המיטוכונדריה מבודדת מרקמת שריר שלד מתקבלת על ידי Percutaneous מחט ביופסיה

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

דימות תהודה מגנטית כמותי של מחלת שריר שלד

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

הקמתו של ארגון Human אפיתל Enteroids וColonoids מרקמות שלמות וביופסיה

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...