This work was partly supported by NSF CBET 1227766, NSF CBET 1511759, and Byars-Tarnay Endowment. We gratefully acknowledge use of the West Virginia University Shared Research Facilities which are supported, in part, by NSF EPS-1003907.

שכפול 1. PDMS תבניות מתוך עובש EBL <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לפברק תבנית סיליקון 29 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מעיל ספין 200 methacrylate polymethyl μl (PMMA) פתרון על סיליקון ס&quot;מ 1 × 1 (Si) המצע ב 2500 סל&quot;ד עבור 1 דקות כדי ליצור סרט דק. </li><li style=";text-align:right;direction:rtl"> אופים את הסרט PMMA על פני המצע סי ב 180 מעלות צלזיוס למשך 2 דקות. </li><li style=";text-align:right;direction:rtl"> כתוב את nanopattern תוכנן על סרט PMMA באמצעות אלומת אלקטרונים ממוקדת במינון שטח של 300 μC / 2 סנטימטר. </li><li style=";text-align:right;direction:rtl"> לפתח את nanopattern PMMA ב מפתח עבור 80 שניות. </li><li style=";text-align:right;direction:rtl"> להפקיד את nanopattern PMMA בשכבת ניקל של 50 ננומטר עובי באמצעות המאייד E-קרן לעבר מתח המוצא של 10 קילו וולט, פליטה הנוכחי של 0.5 מילי-אמפר ושיעור בתצהיר של 0.5 A / sec. </li><li style=";text-align:right;direction:rtl"> הרם את החלק PMMA במסיר 20 מ&quot;ל ב 80 מעלות צלזיוס במשך 20 דקות. </li><li style=";text-align:right;direction:rtl"> לחרוט יון ריאקטיבי (ורי) את nanopattern לתוך המצע Si לקבל עובש Si של העומק הרצוי. הערה: גז תערובת של tetrafluoromethane (CF 4) / חמצן (O 2) (90% / 10%) תמורת פלזמה מצמידה אינדוקטיבי (ICP) כוח של 400 ואט ו ורי של 150 W משמש כדי לחרוט מצע Si עד לעומק של 560 ננומטר. </li></ol></li><li style=";text-align:right;direction:rtl"> Silanize Si עובש <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שים coverslip זכוכית עובש סי בצלחת 100 מ&quot;מ PS פטרי ולהעבירם ייבוש זכוכית הממוקם במנדף. </li><li style=";text-align:right;direction:rtl"> ירידה של כ -10 1H μl, 1H, 2H, 2H-Perfluorooctyltrichlorosilane על coverslip. זהירות: 1H, 1H, 2H, 2H-Perfluorooctyltrichlorosilane עלול לגרום לקורוזיה העור ופגיעה חמורה בעיניים. ללבוש ציוד מגן אישי מתאים (PPE). </li><li style=";text-align:right;direction:rtl"> מכסים את צלחת פטרי חלקית. </li><li style=";text-align:right;direction:rtl"> שמור על ייבוש תחת ואקום עבור 5 שעות במנדף להשלמת silanization של עובש סי. </li></ol></li><li style=";text-align:right;direction:rtl"> כן prepolymer PDMS <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשקול 10 גרם שרף PDMS וסוכן ריפוי 1.05 גרם בסירה במשקל הפנויה. </li><li style=";text-align:right;direction:rtl"> מערבבים את prepolymer PDMS ביסודיות באמצעות כפית פלסטיק. </li><li style=";text-align:right;direction:rtl"> דגת prepolymer PDMS ייבוש פלסטיק תחת ואקום במשך כ 20 דקות עד תערובת ברורה הוא ציין. </li></ol></li><li style=";text-align:right;direction:rtl"> לשכפל תבניות PDMS <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מכניסים את התבנית סי silanized בצלחת פטרי 60 מ&quot;מ. </li><li style=";text-align:right;direction:rtl"> יוצקים את prepolymer PDMS על עובש סי בצלחת פטרי. </li><li style=";text-align:right;direction:rtl"> מניחים את צלחת פטרי ייבוש פלסטיק דגה למשך כ -10 דקות עד שכל הבועות להיעלם. </li><li style=";text-align:right;direction:rtl"> מעבירים את צלחת פטרי פלטה חשמלית ולרפא את prepolymer PDMS ב 70 מעלות צלזיוס למשך 4 שעות. </li><li style=";text-align:right;direction:rtl"> לקלף עובש PDMS מהתבנית Si בזהירות באמצעות פינצטה. הערה: תבניות PDMS ניתן לאחסן בכל תנאי הסביבה עד שבוע אחד. לאחר ריפוי, יש כמה מולקולות PDMS שרף uncrosslinked וסוכן ריפוי שייר תבניות PDMS 30. מולקולות המשקל המולקולריות הנמוכות בהדרגה המפוזרת החוצה לצבור על פני השטח לאורך זמן. זה משפיע על תכונות טופוגרפיות מכאניות של פני שטח PDMS 31. DIFהיתוך אינו משמעותי בתוך שבוע. </li></ol></li></ol> 2. תפירה של PDMS תבניות לתוך תבנית גדולה, יחיד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן תבניות PDMS המרובה על ידי חזרה על שלב 1.4. הערה: לשקול אותה כמות של תערובת PDMS בכל פעם להשיג תבניות PDMS של עובי זהה. </li><li style=";text-align:right;direction:rtl"> לקבוע את הכיוון של nanopatterns PDMS איזוטרופי כגון nanogratings תחת מיקרוסקופ אופטי ולסמן אותה על הישבן של תבניות PDMS עם עט סימון. הערה: אין צורך לסמן את הכיוון של nanotopography איזוטרופיים כגון nanopillars. </li><li style=";text-align:right;direction:rtl"> נקו מצע Si עם אתנול במנדף ולייבש אותו עם אוויר דחוס. </li><li style=";text-align:right;direction:rtl"> חתוך את תחומי unpatterned של כל עובש PDMS עם להב. הערה: עבור תבניות PDMS כי תוצבנה בפריפריה של העובש התפור, רק באזורי unpatterned במגע עם אחרים צריכים להיות מטופח off. </li><li style=";text-align:right;direction:rtl"> מניחים את התבנית PDMS גזוז עם nanopattern עם הפנים כלפי מטה עלהצד במראה של מצע Si ולאחר מכן יישר תבניות אחרות קרובות אבל לא נוגעות עובש שמסביב (ים). </li><li style=";text-align:right;direction:rtl"> כן שכבת דבק PDMS <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עופרת 1 גרם degassed prepolymer PDMS (שרף PDMS ויחס סוכן ריפוי: 10: 1.05) בשקופית זכוכית נקי (7.5 ס&quot;מ × 2.5 ס&quot;מ) כדי ליצור שכבה 0.5 מ&quot;מ עובי. </li><li style=";text-align:right;direction:rtl"> אופים את שכבת PDMS ב 100 מעלות צלזיוס על פלטה חמה למשך 3-5 דקות. שימוש במחט לגעת השכבה ולהבטיח כי היא השכבה חלקית אך לא נרפא לגמרי. הערה: PDMS נרפא חלקית לא יכול לזרום כמו prepolymer PDMS דפוקה, אבל זה דביק לעומת PDMS נרפא. </li></ol></li><li style=";text-align:right;direction:rtl"> מניחים את השכבה PDMS על הישבן של תבניות PDMS מיושר ובמהירות להפוך הרכבה זה ולהעביר אותו אל הפלטה החמה. </li><li style=";text-align:right;direction:rtl"> החלת כוח דחיסה (5 kPa) באמצעות בלוק מתכת בחלק העליון של הרכבה כדי להבטיח מגע טוב בין שכבת דבק PDMS ואת הישבן של תבניות PDMS, ולרפא את PDMS שכבת דבק ב 100 מעלות צלזיוס במשך 1 שעה. &lt;br /&gt; הערה: בזהירות להתאים את המיקום של גוש המתכת כדי למנוע את ההטיה של ההרכבה. </li><li style=";text-align:right;direction:rtl"> הסר את גוש המתכת לקלף עובש PDMS היחיד, תפור מן מצע Si. </li></ol> דור 3. של עובש מאסטר על מצעי PS ההערה: עובש PDMS התפור המשותק בשקופית זכוכית יכול לשמש כדי ליצור עובש אמן על צלחת PS או סרט דק PS, שממנו עבודת מצעי nanopatterned יכולים להיות מיוצר. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צור עובש אמן על צלחת PS <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכינו צלחת PS <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לייבש את כדורי PS בתנור ואקום ב 80 מעלות צלזיוס במשך יומיים. </li><li style=";text-align:right;direction:rtl"> מחממי מכשיר משקולות ב 230 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> להרכיב צלחת אלומיניום, גיליון polytetrafluoroethylene (PTFE) ואת spacer אלומיניום בצו מלמטה למעלה. </li><li style=";text-align:right;direction:rtl"> כדורי PS טען 3.5 גרם ב spacer אלומיניום עם פתח מרובע של 3 ס&quot;מ (L) × 3 ס&quot;מ (W) × 0.3 ס&quot;מ (H). הערה: spacer הוא כ 0.1 ס&quot;מ עבה יותר תבניות PDMS, ובכך המצע PS nanopatterned הסופי עומד עבה 0.1 ס&quot;מ. </li><li style=";text-align:right;direction:rtl"> למקום אחר גיליון PTFE ולאחר מכן עוד צלחת אלומיניום על spacer אלומיניום. </li><li style=";text-align:right;direction:rtl"> הנח את המכלול במכונת העיתונות. </li><li style=";text-align:right;direction:rtl"> מחממים את כדורי PS ב 230 מעלות צלזיוס במשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> החלה לחץ דחיסה (0.1 MPA) שבמכלול למשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> שחרר את הלחץ ולאחר מכן החל מחדש לחץ דחיסה של 0.5 מגפ&quot;ס שבמכלול. </li><li style=";text-align:right;direction:rtl"> חזור על שלב 3.1.1.9 עם עלייה בלחץ של 0.5 מגפ&quot;ס עד הלחץ הרצוי של 1.5 מגפ&quot;ס הוא הגיע. </li><li style=";text-align:right;direction:rtl"> כבו את התנור של מכונת לחץ לקרר אותו מתחת ל -70 מעלות צלזיוס בלחץ מתמיד של 1.5 מגפ&quot;ס. </li><li style=";text-align:right;direction:rtl"> קח את המכלול החוצה ולאחסן את צלחת PS בתנור ואקום ב 80 ° C כדי למנוע לחות מלשוב אל צלחת PS. </li></ol></li><li style=";text-align:right;direction:rtl"> Nanoimprint עובש PDMS תפור לתוך צלחת PS <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את צלחת PS בתוך spacer אלומיניוםלהגדיר על פרוסות סיליקון Si 3 אינץ &#39;. הערה: הממדים הפנימיים של spacer הזהים לאלה של צלחת PS כך צלחת PS מתאימה ממש spacer. </li><li style=";text-align:right;direction:rtl"> מחמם את צלחת PS על פלטה חמה ב 250 מעלות צלזיוס למשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> מכניסים את התבנית PDMS תפור עם nanopatterns עם הפנים כלפי מטה על צלחת PS מותכת. הערה: צד אחד של עובש PDMS מושם על קשר עם פני השטח של צלחת PS הראשון לבין צד אחר הוא הוריד בהדרגה במגע עם המשטח PS כדי למנוע היווצרות של בועות אוויר על הממשק. זהירות: החלק הקדמי של פלטה חשמלית חם. תלבש thermogloves במהלך תהליך nanoimprinting. </li><li style=";text-align:right;direction:rtl"> מניחים צלחת אלומיניום בשקופית כוס עובש PDMS תפור. </li><li style=";text-align:right;direction:rtl"> החלת לחץ דחיסה (12.5 kPa) באמצעות גושי מתכות על לוחית אלומיניום ולחכות 3 דקות. הערה: ודא כי לוחית אלומיניום אינו מוטה. </li><li style=";text-align:right;direction:rtl"> רם ולהחליף את גוש המתכת מהצלחת האלומיניום ואניncrease לחץ הדחיסה 25 kPa. </li><li style=";text-align:right;direction:rtl"> חזור על שלב 3.1.2.6 עם הלחץ עלה ל 50 kPa. הערה: שלב זה הוא להסיר את האוויר שנלכד בין עובש PDMS ואת צלחת PS. </li><li style=";text-align:right;direction:rtl"> לשמור על הטמפרטורה של הפלטה החשמלית בין 240 לבין 250 מעלות צלזיוס בלחץ העקבי של 50 kPa במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> כבה את הפלטה החשמלית ו לצנן את ההתקנה כולה. הערה: מאוורר יכול לשמש כדי לזרז את תהליך הקירור. </li><li style=";text-align:right;direction:rtl"> הסר את גושי מתכות לאחר הטמפרטורה היא מתחת ל -50 מעלות צלזיוס, ובזהירות לקלף עובש PDMS תפור מהצלחת PS. הערה: מצע PS יש את nanopatterns ההפוכה והוא יכול לשמש עובש אמן לייצר מצעי PDMS עובדים. </li></ol></li></ol></li><li style=";text-align:right;direction:rtl"> צור עובש אמן על סרט PS דק <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנתי סרט PS דק <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ממיסים 1 PS גרם טולואן 10 מ&quot;ל במנדף. זהירות: טולואן עלול לגרום לעור irritation ונזק חמורה בעיניים, ועלול לגרום נזק לאיברים באמצעות חשיפה ממושכת או חוזרת. תלבש PPE המתאים. </li><li style=";text-align:right;direction:rtl"> ספין-מעיל 1 מ&quot;ל פתרון PS על רשת 2-ב פרוסות סיליקון ב -2,500 סל&quot;ד דקות 1 כדי ליצור ~ 1 מיקרומטר סרט דק בעובי PS. </li><li style=";text-align:right;direction:rtl"> להתאדות טולואן מהסרט ידי הגדרת הסרט PS על Si רקיק במנדף במשך 3 ימים. </li><li style=";text-align:right;direction:rtl"> לחשל הסרט הדק PS בתנור ואקום ב 80 מעלות צלזיוס למשך לילה. </li></ol></li><li style=";text-align:right;direction:rtl"> Nanoimprint עובש PDMS על סרט דק PS <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מכניסים את התבנית PDMS תפור עם nanotopography עם הפנים כלפי מטה על סרט דק PS, אשר מוגדר על פלטה חמה. </li><li style=";text-align:right;direction:rtl"> החלת לחץ לחיצה של 12 kPa על עובש PDMS באמצעות גושי מתכות בצד כוס עובש PDMS. </li><li style=";text-align:right;direction:rtl"> העלה את הטמפרטורה של הפלטה החשמלית ל -180 מעלות צלזיוס ולתחזק אותו במשך 15 דקות. זהירות: סרט PS המותך יכול לתפקד כחומר סיכה. שימו לב כדי למנוע את אבני המתכת מפני החלקתי החוצה. </li><li style=";text-align:right;direction:rtl"> כבה את הפלטה החשמליתו לצנן את ההתקנה כולה. הערה: מאוורר יכול לשמש כדי לזרז את תהליך הקירור. </li><li style=";text-align:right;direction:rtl"> הסר את גושי מתכות לאחר הטמפרטורה יורדת מתחת ל -50 מעלות צלזיוס, ובזהירות לקלף עובש PDMS תפור מהסרט PS. הערה: סרט nanopatterned PS ישמש עובש אמן לייצר מצעי PDMS עובדים. </li></ol></li></ol></li></ol> 4. אפנון Nanotopographical של התנהגות התא הערה: תאי אפיתל האדם הם מתורבתים על nanotopographies נציג להפגין אפנון nanotopographical הפצת התא. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עופרת מצעי PDMS עבודת עובש אמן מופק השלב או 3.1 או 3.2 בהתאם ליישום. </li><li style=";text-align:right;direction:rtl"> באמצעות אגרוף קשת מתכת חלול, לחתוך את מצעי PDMS nanopatterned לתוך דיסקים כדי להתאים את התצורה של צלחת רבה גם ספציפית (למשל, 24 גם צלחת). </li><li style=";text-align:right;direction:rtl"> להשתמש בפינצטה כדי למקם את הדיסקים PDMS לתוך הבארות ofa צלחת רבה היטב. </li><li style=";text-align:right;direction:rtl"> לעקר את מצעי PDMS באמצעות 70% אתנול ולאחר מכן חשיפת UV, כל למשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> שטפו את מצעי PDMS עם בופר פוספט סטרילית 1x (PBS) שלוש פעמים. </li><li style=";text-align:right;direction:rtl"> מעיל מצעים PDMS עם חלבון תאי מטריקס (כלומר, 20 מיקרוגרם / מ&quot;ל פיברונקטין) למשך 30 דקות בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> יש לשטוף את מצעי PDMS שלוש פעמים עם PBS סטרילי, כל 5 דקות. </li><li style=";text-align:right;direction:rtl"> להשעות A549 תאים סרטני ריאה אנושית במדיום הנשר השונה של Dulbecco עם 10% בסרום שור עוברי לספור את התאים באמצעות hemocytometer. </li><li style=";text-align:right;direction:rtl"> פלייט התאים בצפיפות זריעה של 2,000 תאים / 2 ס&quot;מ על מצעים PDMS והתרבות אותם על 37 מעלות צלזיוס באווירה humidified המכיל 5% CO 2 למשך יום אחד. </li><li style=";text-align:right;direction:rtl"> שוטפים את התאים עם שלוש פעמים PBS. </li><li style=";text-align:right;direction:rtl"> תקנו את התאים בתערובת של 4% paraformaldehyde ו -2% glutaraldehyde PBS במשך 4 שעות ו מייבשי תאים באמצעות CO 2 PO הקריטימייבש int לסריקת תצפית מיקרוסקופית אלקטרונית 29. זהירות: Paraformaldehyde ו glutaraldehyde עלול לגרום לכוויות בעור חמור לנזק לעיניים. Operate במנדף כימי וללבוש PPE המתאים. </li></ol>

<ol>
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The authors have nothing to disclose.

אנו מציגים שיטה פשוטה, זולה עדיין צדדית כדי ליצור שטח גדול של מצע nanopatterned. כדי להרחיב נאמן מאוד nanopatterns מוגדר, תשומת לב רבה צריך להיות משולם על כמה שלבים קריטיים. הראשון הוא לחתוך את תבניות PDMS המרובות. באזורים unpatterned של תבניות PDMS צורך להסירו. בנוסף, הצדדי של התבניות יש לח?...

A number of recent findings reveal that substrate nanotopography has pronounced influence on cell behavior, from cell adhesion, spreading and migration, to proliferation and differentiation1-6. For instance, a smaller cell size and lower proliferation rate have been observed in cells cultured on deep nanogratings, even leading to apoptosis although the cell alignment, elongation and migration were enhanced, compared with the flat controls2,7-10. Moreover, nanotopography has been shown to facilitate the differentiation of stem cells into certain lineages such as neuron2,11,12, muscle13, and bone3,4. In addition, because of increasing concerns on the toxicity of engineered nanomaterials14,15, there is a need to incorporate nanotopography into physiologically relevant in vitro models for accurate risk assessment of nanomaterials. To fulfil the biochemical and molecular biology analyses, enough cells are needed to be grown on a large area of nanopatterned substrate. However, conventional nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area.
Self-assembly including colloid lithography16 and polymer demixing17 can readily generate large-area nanostructures at low costs. Because self-assembly relies on interactions between the assembling elements such as colloidal particles and macromolecules, and possible interactions between these elements and substrate, it cannot be a stand-alone method of producing nanostructures with precise spatial positioning and arbitrary shapes18. The accompanied high density of defects is also a drawback. Precise spatial control of nanopatterns can be achieved by employing templated self-assembly, which uses top-down lithographic approaches to provide the topographical and/or chemical template to guide the bottom-up assembly of the assembling elements19-21. Alternative nanofabrication techniques such as step-and-flash lithography22 and a roll-to-roll nanoimprinting lithography23 have been developed but have limited use because of their sophisticated process or the requirement of specialized equipment. Nevertheless, a template or a master mold with defined nanoscale patterns is needed for templated or alternative nanofabrication techniques. 
Such templates and master molds are conventionally generated by using focused electron, ion, or photon beam lithography. For instance, electron beam lithography (EBL)24 and focused ion beam lithography25 can generate defined patterns with a sub-5 nm resolution. Two-photon lithography has demonstrated a feature size as small as 30 nm26. Although the focused beam lithography techniques enable generation of well-defined nanoscale structures, the capital investment and the time-consuming, costly process restrict their widespread use in academic research27. Therefore, it is highly desirable to develop enabling yet affordable techniques to produce a large area of nanopatterned surfaces with high fidelity.
We have reported a simple, cost-effective stitch technique for generating a large area of nanopatterned surface from a small well-defined mold28. This protocol provides step-by-step procedure from replication of poly(dimethylsiloxane) (PDMS) molds using an EBL-written pattern, to assembly of multiple PDMS molds into a single large mold, to generation of a master mold on polymeric such as polystyrene (PS) substrates, to production of working substrates. With the expanded nanopatterned substrates, we demonstrated nanotopographical modulation of cell spreading.

<table><tbody><tr><td>JEOL field emission SEM</td><td>JEOL</td><td>JSM-7600F</td><td>EBL</td></tr><tr><td>E-beam evaporator</td><td>Kurt J. Lesker</td><td>Model: LAB 18 e-beam evaporator</td><td>nickel deposition</td></tr><tr><td>Trion Minilock III ICP/RIE</td><td>Trion technology</td><td>Model: Minilock-phantom III</td><td></td></tr><tr><td>Press machine</td><td>PHI Hydraulic Press</td><td>Molde: SQ-230H</td><td></td></tr><tr><td>Spin coater</td><td>Laurell Technologies</td><td>Modle: WS-400A-6NPP-LITE</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; critical dryer</td><td>Tousimis</td><td>Modle: Autosamdri-815</td><td></td></tr><tr><td>Silicon wafer</td><td>University Wafer</td><td>1080</td><td></td></tr><tr><td>Aluminum plates</td><td>McMaster-carr</td><td>9057K123</td><td></td></tr><tr><td>Teflon sheets</td><td>McMaster-carr</td><td>8711K92</td><td></td></tr><tr><td>100 mm Petri dish</td><td>FALCON</td><td>353003</td><td></td></tr><tr><td>60 mm Petri dish</td><td>FALCON</td><td>353004</td><td></td></tr><tr><td>Glass coverslip</td><td>Fisher Scientific</td><td>12-542-B</td><td></td></tr><tr><td>Glass slide</td><td>Fisher Scientific</td><td>12-550-34</td><td></td></tr><tr><td>Disposable weighing boats</td><td>Fisher Scientific</td><td>13-735-743</td><td></td></tr><tr><td>Glass desiccator</td><td>Fisher Scientific</td><td>02-913-360</td><td></td></tr><tr><td>Plastic desiccator</td><td>Bel-Art Products</td><td>F42025-000</td><td></td></tr><tr><td>Hotplate</td><td>Fisher Scientific</td><td>1110049SH</td><td></td></tr><tr><td>Tweezer</td><td>Ted Pella, inc.</td><td>5726</td><td></td></tr><tr><td>Blade</td><td>Fisher Scientific</td><td>S17302</td><td></td></tr><tr><td>Metal blocks</td><td>McMaster-carr</td><td></td><td></td></tr><tr><td>Punch</td><td>Brettuns Village Leather Craft Supplies</td><td>Arch punch</td><td></td></tr><tr><td>Poly(methyl methacrylate)</td><td>MicroChem</td><td>495 PMMA A4</td><td></td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>Sylgard 184 kit</td><td></td></tr><tr><td>Polystyrene</td><td>Dow Chemical</td><td>Styron 685D</td><td></td></tr><tr><td>1H,1H,2H,2H-perfluorooctylmethyldichlorosilane</td><td>Oakwood Chemical</td><td>7142</td><td></td></tr><tr><td>Developer</td><td>MicroChem</td><td>MIBK/IPA at 1: 3 ratio</td><td></td></tr><tr><td>Remover</td><td>MicroChem</td><td>Remover PG</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>BP2818500</td><td></td></tr><tr><td>Toluene</td><td>Fisher Scientific</td><td>T324-500</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma Aldrich</td><td>D8537</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified eagle medium</td><td>Sigma Aldrich</td><td>D5796</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Atlanta Biologicals</td><td>S11550</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electron Microsopy Science</td><td>15712-S</td><td></td></tr><tr><td>Glutaraldehyde&amp;nbsp;</td><td>Fisher Chemical</td><td>G151-1</td><td></td></tr><tr><td>Fibronectin</td><td>Corning</td><td>356008</td><td></td></tr><tr><td>A549 cells</td><td>ATCC</td><td>ATCC CCL-185</td><td></td></tr></tbody></table>

expanding nanopatterned substrates using stitch technique for nanotopographical modulation of cell behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

<html> טכניקת התפר יכולה ליצור שטח גדול של מצעי nanopatterned עם איכות גבוהה. איור 1 א ו 1- להציג את השטח הגדול של nanopatterns הועבר מתבנית PDMS תפורה על צלחת PS וקולנוע PS הדק על מצע Si, בהתאמה. ההשוואה בין עובש EBL בכתב המקורי (איור 1 ג &#39;) ואת PDMS ה...

Watch this Scientific Journal Video about Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior at JoVE.com

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

הרחבת טכניקת תפר באמצעות מצעי nanopatterned עבור Nanotopographical אפנון של התנהגות תא

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

expanding-nanopatterned-substrates-using-stitch-technique-for

Research

JoVE Journal

Bioengineering

6.5K Views.  West Virginia University. A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

Video: Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

סטנסיל micropatterning של תאי גזע האנושי pluripotent עבור גשוש הארגון המרחבי של גורל הבידול

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

Developmental Biology

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

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 ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

Microfabrication של דפוסי זהב Nanoporous לסלולרי מהותי מחקרי אינטראקציה

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

תא דפוסים על photolithographically מוגדר Parylene-C: SiO<sub&gt; 2</sub&gt; מצעים

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

מתיחת תאי micropatterned בממברנה PDMS

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

פשוט ליתוגרפיה-ללא מיקרוהדפס תא יחיד באמצעות סטנסילים לייזר לחתוך

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

שיטה צדדית של חלבוני דפוסי תאים

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular reactions. However, it is difficult to determine the relation between the size of a nanostructure and the responses of cells owing to the lack of proper screening tools. Here, we show the development of reproducible and cost-effective gradient nanopattern plates for the manipulation of cellular responses. Using anodic aluminum oxide (AAO) as a master mold, gradient nanopattern plates with nanopillars of increasing diameter ranges [120-200&#8239;nm (GP 120/200), 200-280&#8239;nm (GP 200/280), and 280-360&#8239;nm (GP 280/360)] were fabricated by a thermal imprinting technique. These gradient nanopattern plates were designed to mimic the various sizes of nanotopography in the ECM and were used to screen the responses of human endothelial colony-forming cells (hECFCs). In this protocol, we describe the step-by-step procedure of fabricating gradient nanopattern plates for cell engineering, techniques of cultivating hECFCs from human peripheral blood, and culturing hECFCs on nanopattern plates.

Here, we present a protocol for the fabrication of gradient nanopattern plates via thermal nanoimprinting and the method of screening responses of human endothelial progenitor cells to the nanostructures. By using the described technology, it is possible to produce a scaffold that can manipulate cell behavior by physical stimuli.

ייצור של מעבר צבע Nanopattern על ידי טכניקת Nanoimprinting תרמית והקרנת התגובה של תאים אנושיים המושבה יוצרי אנדותל

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

שליטה בגיאומטריית התא באמצעות מיקרופטרנינג בסיוע לייזר אינפרא אדום

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

יצירת תבניות למיקרוסקופיית מתיחה מיקרופטרן

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...