D. H. Kim thanks the Department of Bioengineering at the University of Washington for the new faculty startup fund. D. H. Kim is also supported by the Perkins Coie Award for Discovery, the Wallace H. Coulter Foundation Translational Research Partnership Award, the Washington State Life Science Discovery Fund, and the American Heart Association Scientist Development Grant (13SDG14560076). J. Macadangdang and A. Jiao thank the support from the NIH Bioengineering Cardiovascular Training Grant Fellowship. &#160;Additional support for this work comes from the National Institutes of Health (NIH) grant R01HL111197 to M. Regnier.

כל הפעולות מבוצעות בטמפרטורת חדר (~ 23 ° C) אלא אם צוינו אחרת. 1. ייצור של הסיליקון מאסטר <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> פרוסות סיליקון נקיות עם 100% אתנול או קסילן ויבש תחת O 2 / N 2 גז. </li><li style=";text-align:right;direction:rtl"> מניחים פרוסה סיליקון בספין coater במהירויות סיבוב של 2,000-4,000 סל&quot;ד כדי לייצר סרט עבה 0.3-0.5 מיקרומטר. </li><li style=";text-align:right;direction:rtl"> תבנית סרט photoresist עם המידות הנכונות באמצעות מערכת photolithography </li><li style=";text-align:right;direction:rtl"> באופן מלא לטבול את פרוסות סיליקון מצופה photoresist דוגמת בנפח מתאים של פתרון photoresist המתפתח. </li><li style=";text-align:right;direction:rtl"> שוטפים את פרוסות סיליקון מצופה photoresist פיתחו עם מים deionized. </li><li style=";text-align:right;direction:rtl"> ליצירת מערכים של רכסים בקנה מידה תת מיקרון עם צד קירות ליד אנכיים, לחרוט יון תגובה עמוק הסיליקון החשוף באמצעות מערכת תחריט. </li><li style=";text-align:right;direction:rtl"> הסר את photoresist שנותר על ידי הנחת פרוסות סיליקון במערכת אשר פלזמה. </li><li style=";text-align:right;direction:rtl"> חותכים את סיליקון wafers עם חותך יהלום שקצה לאדוני סיליקון בגודל המתאים לדפוס העתק שלאחר מכן. </li></ol> 2. ייצור של עובש רשות החשמל מהסיליקון מאסטר הערה: נפח שיש להוסיף לאדון סיליקון לnanofabrication ישתנה בהתאם לאזור של המאסטר nanopatterned להיות משוכפל כמו גם הצמיגות של acrylate פוליאוריטן הפתרון (רשות החשמל). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> משטח נקי סיליקון הורים עם 100% אתנול או קסילן ויבש תחת O 2 / N 2 גז. </li><li style=";text-align:right;direction:rtl"> הנח בצד דפוס אדון סיליקון בצלחת פטרי. </li><li style=";text-align:right;direction:rtl"> לאדון סיליקון עם 2 ס&quot;מ X 2 סנטימטר משטח דפוס, פיפטה 40 μl של רשות החשמל על פני השטח התבנית. </li><li style=";text-align:right;direction:rtl"> הנח גיליון של 4 סנטימטר סרט X 4 סנטימטר שקוף פוליאסטר (PET) על רשות החשמל חילק. </li><li style=";text-align:right;direction:rtl"> לחץ כלפי מטה על גיליון PET ולהפיץ את הרשות לשירותים הציבורית מתחת לסדין על פני התבנית באמצעות רולר או משטח קצוות שטוח (כגון כרטיס), כך שכלדפוס הוא מכוסה על ידי prepolymer רשות החשמל. </li><li style=";text-align:right;direction:rtl"> הנח מאסטר סיליקון, prepolymer, וPET כ 10 סנטימטר מתחת אור UV 20 ואט (115 V) (λ = 365 ננומטר) ל50 שניות. כדי להיות יעיל, אורך גל אור UV יכול להיות בכל מקום בין 310-400 ננומטר. עוצמת האור היא 10-15 mW / 2 סנטימטר על פני השטח של המצע. </li><li style=";text-align:right;direction:rtl"> לאחר הריפוי, להסיר סרט PET לאט עם מלקחיים. רשות לשירותים ציבוריים צריכה לצרף לסרט PET עם שלילי של nanopattern אדון סיליקון. </li><li style=";text-align:right;direction:rtl"> לרפא nanopatterns רשות החשמל / PET תחת UV לפחות 12 שעה לפני השימוש. חשיפה היא לא בעיה. </li><li style=";text-align:right;direction:rtl"> כדי לנקות אדוני סיליקון, למקם סרט נוסף של PET על גבי האדון ללא תוספת של רשות החשמל ולחשוף לאור UV (λ = 365 ננומטר) ל50 שניות ולהסיר סרט PET. זו תסיר את כל מונומרים unreacted. </li><li style=";text-align:right;direction:rtl"> יש לשטוף את אדון סיליקון עם 100% אתנול או קסילן ויבש תחת O 2 / N 2 גז. </li></ol> 3 א. פולימר פוליאוריתן Nanopatterning </p&gt; <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן 25 שקופיות זכוכית עגולות בקוטר מ&quot;מ על ידי הנחת בחדר טיפול באוזון ל10 דקות. </li><li style=";text-align:right;direction:rtl"> הנח שקופיות זכוכית שטופלה באוזון על גבי בלוק PDMS קטן לטיפול קל. </li><li style=";text-align:right;direction:rtl"> החל שכבה דקה של אמרגן הידבקות משטח עם מכחול לשקופיות זכוכית. שקופיות זכוכית אוויר יבשות למשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> מניחים את שקף הזכוכית על פיסת נייר במדפסת. </li><li style=";text-align:right;direction:rtl"> זרוק לוותר 10 μl של פוליאוריטן (PU) מראש פולימר (נועה 76) למרכז שקופית זכוכית. ודא שאין בועות נמצאות אחרי כן. </li><li style=";text-align:right;direction:rtl"> עובש רשות החשמל פלייס, דפוס עם הפנים כלפי מטה, לשקופית הזכוכית. לפזר את יחידת השיטור באופן אחיד על פני השטח של שקופית הזכוכית על ידי גלגול הרים גליל גומי לאורך עובש רשות החשמל. נייר מדפסת יספוג הצפת פולימר. </li><li style=";text-align:right;direction:rtl"> מנורת UV ואט. זמן פלמור של יחידת השיטור תלוי בכוחו של מקור קרינה. </li><li style=";text-align:right;direction:rtl"> הסר את הדגימה ממקור אור UV ולקלף בזהירות את תבנית רשות החשמל משקופיות זכוכית PU מצופה. פלמור של פו נחשב גomplete כאשר קליפות רשות החשמל העובש נקי מן המדגם ושקופיות זכוכית PU יש מראה ססגוני. </li><li style=";text-align:right;direction:rtl"> מקום דגימות סיימו בייבוש לאחסון במשך זמן רב ככל חודש. </li></ol> ב 3. Nanopatterning midi (lactide-Co-Glycolide) Hydrogel <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צור עובש PDMS שטוח ידי ערבוב נמרץ בסיס אלסטומר סיליקון וסוכן ריפוי אלסטומר סיליקון ביחס 10:1. </li><li style=";text-align:right;direction:rtl"> יוצקים מעורב PDMS מבשר פתרון לתוך צלחת פטרי, כך שמבשר PDMS מגיע 5 מ&quot;מ עד הקצה של המנה (כלומר, כך שPDMS הוא 5 מ&quot;מ עובי). </li><li style=";text-align:right;direction:rtl"> מניחים צלחת פטרי ומבשר PDMS בייבוש במשך שעה 1 לדגה. </li><li style=";text-align:right;direction:rtl"> להעביר צלחת פטרי ומבשר PDMS לתוך תנור 65 מעלות צלזיוס למשך לפחות 2 שעות כדי לרפא. </li><li style=";text-align:right;direction:rtl"> לאחר PDMS נרפא, השתמש בסכין גילוח כדי לחתוך את PDMS שטוח לתוך 3 סנטימטר חלקי ריבוע בגודל 3 סנטימטר. אלה יהיו תבניות PDMS השטוח השתמשו מאוחר יותר בתהליך הדפוסים. </li><li style=";text-align:right;direction:rtl"> שקופיות זכוכית עגולה בקוטר 25 מ&quot;מ נקי על ידי placing באלכוהול איזופרופיל ל30 דקות בsonicator מים. </li><li style=";text-align:right;direction:rtl"> מגלשות יבשים ניקו זכוכית תחת O 2 / N 2 גז. </li><li style=";text-align:right;direction:rtl"> זרוק לוותר על פתרון PLGA 100 μl (15% w / v PLGA בכלורופורם) על גבי שקופית זכוכית. </li><li style=";text-align:right;direction:rtl"> הנח עובש PDMS שטוח על גבי PLGA לוותר לספוג את הממס ולקבל משטח PLGA שטוח. החל לחץ קל (~ 10 kPa) על ידי הצבת משקל 200 גרם בחלק העליון של PDMS במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> לאט לאט לקלף עובש PDMS שטוח ומניח את מכסה הזכוכית על צלחת שחומם מראש (120 מעלות צלזיוס) במשך 5 דקות כדי להסיר ממס שיורי ולהגדיל את ההידבקות בין PLGA וכיסוי הזכוכית. </li><li style=";text-align:right;direction:rtl"> מניחים את התבנית בNP רשות החשמל על גבי PLGA השטוח ולהפעיל לחץ קבוע (~ 100 kPa) וחום (120 ° C) על ידי הצבת במשקל 1 קילוגרם על גבי עובש רשות החשמל בעת על צלחת החימום במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> הסר את המשקל מהשקופית לכסות PLGA ולאפשר לקרקע להתקרר לטמפרטורת חדר. אין להסיר עובש רשות החשמל עד לקרקע הייתהמקורר. </li><li style=";text-align:right;direction:rtl"> ברגע שהמצע התקרר מספיק, לקלף בזהירות את תבנית NP רשות החשמל, חושף את תשתית NP PLGA. תשתית NP PLGA צריכה מראה ססגוני. </li><li style=";text-align:right;direction:rtl"> מקום דגימות סיימו בייבוש לאחסון במשך זמן רב ככל חודש. </li></ol> 4. זריעת תאים ותרבות הערה: פרוטוקול זה מתאר את התרבות של myocytes ילוד חדרית העכברוש (NRVMs) ושריר לב H7 האנושי גזע עוברי שמקורם בתא (hESC-CMS), אבל מקורות של תאים אחרים עשויים להיות בשימוש. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צרף את coverslips ANFS (PU או PLGA) לצלחת קלקר 35 מ&quot;מ רקמת תרבות. פיפטה 20 μl של Norland האופטי דבק (NOA83H) לתחתית הצלחת ומניח בעדינות את coverslip ANFS על גבי נועה. לאפשר דבק להתפזר ולכסות את תחתית coverslip כולו. לרפא את נועה על ידי חשיפה למנת UV עבור 10 דקות. </li><li style=";text-align:right;direction:rtl"> לעקר ANFS על ידי שטיפה עם 2 מיליליטר של 70% אתנול פתרון מימיים במשך 5 דקות, פעמיים. הסר דוארthanol ידי שאיפה. לאפשר ANFS לחלוטין אוויר יבש ל~ 1 שעות מתחת למנורת עיקור UV (λ = 200-290 ננומטר) בארון הבטיחות הביולוגית. </li><li style=";text-align:right;direction:rtl"> הידבקות הסלולר היא משופרת על ידי ציפוי ANFS בפיברונקטין הלילה. לדלל פיברונקטין במים די ל5 מיקרוגרם / מיליליטר. פיפטה 2 מיליליטר של תמיסת פיברונקטין לתוך צלחת. הנח חממה ב 37 ° C ו-CO 2 לילה 5% (לפחות 6 שעות). </li><li style=";text-align:right;direction:rtl"> השג NRVMs, hESC-CMS, או תאי לב אחרים של עניין על פי פרוטוקולים קודמים 22. </li><li style=";text-align:right;direction:rtl"> מדגם תא צנטריפוגה ב 1,000 סל&quot;ד במשך 3 דקות עד גלולה התאים. </li><li style=";text-align:right;direction:rtl"> מוציא בזהירות את supernatant על ידי שאיפה. הקפד לא להפריע גלולה. </li><li style=";text-align:right;direction:rtl"> Resuspend תאים בתקשורת והתרבות המתאימה לריכוז של 4.6 x 10 6 תאים / מיליליטר. </li><li style=";text-align:right;direction:rtl"> זהירות פיפטה 200 μl של השעיה תא על ANFS מעוקר. ודא השעיה תא נשארת על coverslip. </li><li style=";text-align:right;direction:rtl"> מקום תאי חממה ב 37 ° C ו 5%CO 2 למשך 4 שעות כדי לאפשר לתאים לצרף לANFS. </li><li style=";text-align:right;direction:rtl"> הוסף 2 מיליליטר של תקשורת בתרבות החמה למנה ולהחליף את תאים בחממה באותם תנאים. </li><li style=";text-align:right;direction:rtl"> לאחר 24 שעות, להסיר את המדיה ולשטוף עם 2 מיליליטר של DPBS פעמיים כדי להסיר תאים עודפים. </li><li style=";text-align:right;direction:rtl"> הוסף 2 מיליליטר של תקשורת בתרבות החמה למנה ולהחליף את תאים בחממה באותם תנאים. תרבות התאים למפגש. החלף תקשורת בכל יום אחר. </li></ol>

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Authors have nothing to disclose.

רקמות לב תפקודית בוגרות חסרות לשניהם in vivo ו במבחנת יישומים של הנדסת רקמות לב. שיטות nanofabrication CFL שתוארו כאן הן טכניקות חזקות להשגת יישור סלולארי והשפעה על תפקוד רקמות מקרוסקופית עקב יכולת ההרחבה של המערכת. יכולים בקלות להיות בדוגמת שטחים גדולים ומשמשים לתרבי...

מחלות לב וכלי דם היא הסיבה המובילה לתחלואה ותמותה בעולם ולהציג את נטל כלכלי וחברתי כבדות משקל על 1,17 מערכת הבריאות העולמי מתוח כבר. יש הנדסת רקמות לב שתי מטרות נפרדות: (1) כדי להתחדש שריר הלב פגוע לאחר מחלה או קרדיומיופתיה איסכמית או (2) ליצור מודל נאמנות גבוה של הלב במבחנת הקרנת סמים או דוגמנות מחלה. הלב הוא איבר מורכב שחייבים לעבוד כדי לספק דם לגוף כל הזמן. מבני מינרית בצפיפות של שריר לב ורקמות התומכות מסודרים בדפוסי סליל לאורך קיר לב 18,19. הלב הוא גם בשילוב Electromechanically 20 באופן מתואם מאוד כדי להוציא דם בצורה יעילה לגוף 21. כמה מכשולים עיקריים יישארו לטפל, עם זאת, לפני העיצוב המורכב של הטבע יכול להיות סכם באופן מהימן במבחנה.ראשית, למרות ששיטות בידול cardiomyocyte חזקות תמשיך להיות מפותחות 22, hPSC-CMS עדיין תערוכת פנוטיפים ולא בשלה. מאפייני אלקטרו והמורפולוגיה להתאים באופן הדוק ביותר רמות עובריות 23. שנית, כאשר כל הזמן בתנאי תרבות מסורתיים, שניהם נגזרים תאי הגזע ושריר לב עיקרי לא יצליחו להרכיב למבני ילידים, כמו רקמות. במקום זאת, התאים הופכים לכיוון אקראי ואינו מציגים את המראה בצורת המוט התאגדו של שריר הלב מבוגר 24. הסביבה תאית מטריקס (ECM) שבה תאי אינטראקציה משחקת תפקיד משמעותי בתהליכים תאיים רבים 11,13,25. ECM מורכב מרמזים מורכבים, מוגדרים היטב מולקולריים וטופוגרפיים המשפיעים על המבנה ותפקוד של תאים 6,26 באופן משמעותי. בתוך הלב, יישור סלולארי עוקב מקרוב סיבי ECM קנה מידה ננומטרי הבסיסית 2. ההשפעה של nanotopograph אלהרמזי iCal בתא ותפקוד רקמות, עם זאת, הוא רחוק מלהיות מובן לחלוטין. מחקרים ראשוניים של אינטראקציה תא ביולוגי בקנה מידה ננומטרי לציין את החשיבות הפוטנציאלית ואת ההשפעה של רמזים טופוגרפיים תת מיקרון לתא איתות 27, הידבקות 28-30, 31 בצמיחה, ובידול 32,33. עם זאת, בשל הקושי בפיתוח מצעי nanofabricated לשעתק וניתן להרחבה, מחקרים כאלה לא יכולים לשחזר את ההשפעות סלולריות רב היקף של המתחם בסביבת ECM vivo. בפרוטוקול זה, טכניקת nanofabrication פשוטה וחסכונית כדי לייצר פיגומי תרבית תאים מחקה יישור ECM לב סיבי ילידים מתואר, המאפשרת מגוון רחב של חקירות רומן של אינטראקציות cardiomyocyte-ביולוגי. הבנה כיצד שריר לב באינטראקציה עם סביבת ECM ננו עלולה לאפשר את היכולת לשלוט בהתנהגות סלולרית באופן הדוק יותר לחקות תפקוד רקמת ילידtion. יתר על כן, monolayers התא הוא מערכת ניסיונית פשוטה בהשוואה למבני 3D, אך עדיין מפגין התנהגות רב תאית מורכבת לחקירות מעמיקות והקרנה פונקציונלית 2,34-36. לבסוף, יכולים לשמש פיגומים כאלה כדי לשפר את תפקוד שתל סלולארי כאשר מושתלים לתוך הלב למטרות משובי 37.

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			<td class="xl63" height="43" style="height:32.25pt;width:66pt" width="88">Name</td>
			<td class="xl63" style="width:66pt" width="88">Company</td>
			<td class="xl63" style="width:66pt" width="88">Catalogue number</td>
			<td class="xl64" style="width:66pt" width="88">Comments (optional)</td>
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		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Fibronectin</td>
			<td class="xl65" style="width:66pt" width="88">BD Biosciences</td>
			<td class="xl65" style="width:66pt" width="88">354008</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">NOA 76</td>
			<td class="xl65" style="width:66pt" width="88">Norland Products, Inc.</td>
			<td class="xl65" style="width:66pt" width="88">7606B</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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			<td class="xl65" height="106" style="height:79.5pt;width:66pt" width="88">Surface Adhesion Promotor (Glass Primer)</td>
			<td class="xl65" style="width:66pt" width="88">Minuta Tech</td>
			<td class="xl65" style="width:66pt" width="88"></td>
			<td class="xl66" style="width:66pt" width="88"></td>
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			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">PUA</td>
			<td class="xl65" style="width:66pt" width="88">Minuta Tech</td>
			<td class="xl65" style="width:66pt" width="88">MINS-311RM</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Soft Rubber Roller</td>
			<td class="xl65" style="width:66pt" width="88">Speedball</td>
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			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">Silicon Wafers</td>
			<td class="xl65" style="width:66pt" width="88">NOVA Electronic Materials</td>
			<td class="xl65" style="width:66pt" width="88">FA01-9900</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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			<td class="xl65" height="22" style="height:16.5pt;width:66pt" width="88">Photoresist</td>
			<td class="xl65" style="width:66pt" width="88">Shipley</td>
			<td class="xl65" style="width:66pt" width="88">SPRT510</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Photoresist Developer</td>
			<td class="xl65" style="width:66pt" width="88">Shipley</td>
			<td class="xl65" style="width:66pt" width="88">MF320</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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			<td class="xl65" height="85" style="height:63.75pt;width:66pt" width="88">Electron-Beam Lithography System</td>
			<td class="xl65" style="width:66pt" width="88">JEOL</td>
			<td class="xl65" style="width:66pt" width="88">JBX-9300FS</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">Etching System</td>
			<td class="xl65" style="width:66pt" width="88">Surface Technology Systems</td>
			<td class="xl65" style="width:66pt" width="88">NP10 8UJ</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">Plasma Asher System</td>
			<td class="xl65" style="width:66pt" width="88">BMR Technology Co.</td>
			<td class="xl65" style="width:66pt" width="88">DSF-200</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Ozone Cure System</td>
			<td class="xl65" style="width:66pt" width="88">Minuta Tech</td>
			<td class="xl65" style="width:66pt" width="88">MT-UV-O- 08</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Fusion Cure System</td>
			<td class="xl65" style="width:66pt" width="88">Minuta Tech</td>
			<td class="xl65" style="width:66pt" width="88">MT-UV-A 11</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">NOA 83H</td>
			<td class="xl65" style="width:66pt" width="88">Norland Products, Inc.</td>
			<td class="xl65" style="width:66pt" width="88">8301</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Spin Coater</td>
			<td class="xl65" style="width:66pt" width="88">Laurel Technology</td>
			<td class="xl65" style="width:66pt" width="88">WS-400-6NPP</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Skyrol PET Film</td>
			<td class="xl65" style="width:66pt" width="88">SKC Co., Ltd.</td>
			<td class="xl65" style="width:66pt" width="88">23038-59-9</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">25mm Glass Slides</td>
			<td class="xl65" style="width:66pt" width="88">Corning</td>
			<td class="xl65" style="width:66pt" width="88">2948</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="85" style="height:63.75pt">
			<td class="xl65" height="85" style="height:63.75pt;width:66pt" width="88">Sylgard 184 Silicone Elastomer Kit</td>
			<td class="xl65" style="width:66pt" width="88">Dow Corning</td>
			<td class="xl67" style="width:66pt" width="88">6/5/2553</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">Poly(D,L-lactide-co-glycolide)</td>
			<td class="xl65" style="width:66pt" width="88">Sigma-Aldrich</td>
			<td class="xl65" style="width:66pt" width="88">P2191-1G</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Chloroform</td>
			<td class="xl65" style="width:66pt" width="88">Sigma-Aldrich</td>
			<td class="xl65" style="width:66pt" width="88">372978-1L</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">500g Weights</td>
			<td class="xl65" style="width:66pt" width="88">Global Insustrial</td>
			<td class="xl65" style="width:66pt" width="88">T9FB503120</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Isopropyl Alcohol</td>
			<td class="xl65" style="width:66pt" width="88">EMD Millipore</td>
			<td class="xl65" style="width:66pt" width="88">PX1835-2</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl65" height="22" style="height:16.5pt;width:66pt" width="88">Hot Plate</td>
			<td class="xl65" style="width:66pt" width="88">Corning</td>
			<td class="xl65" style="width:66pt" width="88">PC-420D</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl65" height="22" style="height:16.5pt;width:66pt" width="88">Sonicator</td>
			<td class="xl65" style="width:66pt" width="88">Branson</td>
			<td class="xl65" style="width:66pt" width="88">B2510MTH</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
	</tbody>
</table>

capillary force lithography for cardiac tissue engineering

מחלות לב וכלי דם נשארת הגורם המוביל למוות בכל העולם 1. הנדסת רקמות לב טומנת בחובו הבטחה רבה כדי לספק תגליות רפואיות פורצות עם המטרות של פיתוח רקמות פונקציונליות להתחדשות לב, כמו גם מבחני מיון במבחנה. עם זאת, את היכולת ליצור מודלים באיכות גבוהה של רקמת לב הוכיחה קשה. של הלב מטריצה ​​תאית (ECM) היא מבנה מורכב בהיקף של שני האותות ביוכימיים ויומכנית החל מיקרו לקנה המידה ננומטרי 2. לאחרונה מקומי תנאי העמסה מכאנית ואינטראקציות התא-ECM הוכרו כמרכיב חיוני בהנדסת רקמות לב 3-5. חלק גדול מECM הלב מורכב של סיבי קולגן מיושרים עם קטרים ​​בקנה מידה ננו שמשפיע על מבנה רקמה וצימוד אלקטרו 2 באופן משמעותי. למרבה הצער, כמה שיטות havהיה דואר מסוגל לחקות את הארגון של סיבי ECM עד לקנה המידה ננומטרי. הפיתוחים אחרונים בטכניקות nanofabrication, לעומת זאת, מאפשרים לעיצוב וייצור של פיגומי מדרגי המחקים את in vivo רמזי קשיחות מבניים ומצע של ECM בלב 6-9. כאן אנו מציגים את הפיתוח של שני לשחזור, חסכוני, ותהליכי nanopatterning להרחבה עבור היישור התפקודי של תאי לב באמצעות פולי ביולוגית הפולימר (lactide-co-glycolide) (PLGA) 8 ופוליאוריטן (PU) פולימר מבוסס. מצע nanofabricated anisotropically אלה (ANFS) לחקות את ECM הבסיסי של רקמות מאורגנות היטב, מיושרות והוא יכול לשמש כדי לחקור את תפקידה של nanotopography על מורפולוגיה תא ותפקודו 10-14. באמצעות אב סיליקון nanopatterned (NP) כתבנית, עובש acrylate פוליאוריטן (רשות החשמל) הוא מפוברק. עובש רשות החשמל זה משמש לאחר מכן לרשותttern הידרוג&#39;ל PU או PLGA באמצעות סיוע UV או ליתוגרפיה בתיווך ממס כוח הנימים (CFL), בהתאמה 15,16. בקצרה, PU או PLGA מראש פולימר הוא טיפה לוותר על coverslip זכוכית ועובש רשות החשמל מונח על גבי. לCFL בסיוע UV, PU מכן נחשף לקרינת UV (λ = 250-400 ננומטר) לריפוי. לCFL בתיווך ממס, PLGA מוטבע באמצעות חום (120 מעלות צלזיוס) ולחץ (100 kPa). לאחר הריפוי, עובש רשות החשמל הוא קילף, והשאיר מאחורי ANFS לתרבית תאים. תאים ראשוניים, כגון myocytes ילוד חדרית העכברוש, כמו גם שריר לב שמקורם בתאים אנושי pluripotent גזע, יכולים להישמר בANFS 2.

איור 1 הוא סקירה סכמטי של תהליך הייצור לשתי שיטות הייצור. בשל התאבכות של אור הנגרם על ידי הטופוגרפיה ננומטרי, nanopatterning צריך לגרום ססגוני משטח לANFS. איור 2 מתאר ססגוני משטח זה על coverslip דוגמת היטב 25 מ&quot;מ NP-PU (איור 2 א) עם רכס 800 ננומטר וגרוב רוחב <stro...

Watch this Scientific Journal Video about Capillary Force Lithography for Cardiac Tissue Engineering at JoVE.com

Capillary Force Lithography for Cardiac Tissue Engineering

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

נימי חיל יתוגרפיה להנדסת רקמות לב

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

capillary-force-lithography-for-cardiac-tissue-engineering

Research

JoVE Journal

Bioengineering

12.3K Views.  University of Washington. In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Video: Capillary Force Lithography for Cardiac Tissue Engineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

ייצור של חומרים בזריקות נגזר ביולוגית של הנדסת רקמות שריר הלב

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

PGS אלסטומרי פיגומים להנדסת רקמות העורקים

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Surface פלזמה ליתוגרפיה דפוסים של יצירת רשתות Cell

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Encapsulation של cardiomyocytes ב Hydrogel הפיברין עבור הנדסת רקמות לב

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Postproduction עיבוד Fibres Electrospun עבור הנדסת רקמות

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

הנדסת רקמות של 3D אדם<em&gt; במבחנה</emמערכת מבחן גידול&gt;

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

הנדסת ג&#39;ל קולגן Cellularized 3D לשחזור רקמות וכלי דם

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

הכנת Thermoresponsive Nanostructured משטחי הנדסת רקמות

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

הנדסת רקמות ידי עצמותי כלי דם בתוך<em&gt; In vivo</em&gt; הנדסת רקמות קאמרית

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

פרוטוקול עבור Decellularizing Cochleae העכבר עבור הנדסת רקמות האוזן הפנימית

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...