Name: layer-by-layer collagen deposition in microfluidic devices for microtissue stabilization
Uploaded: 2015-09-29T14:00:00.000+00:00
Duration: 9 min 56 s
Description: Watch this Scientific Journal Video about Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization at JoVE.com

This work was supported by grants from the National Institutes of Health, including a microphysiological systems consortium grant from the National Center for Advancing Translational Sciences (UH2TR000503), a Ruth L. Kirschstein National Research Service Award Postdoctoral Fellowship (F32DK098905 for WJM) and pathway to independence award (DK095984 for AB) from the National Institute of Diabetes and Digestive and Kidney Diseases.

1. הכנת הפתרון המקומי מסיס קולגן <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן או לרכוש 200 מ&quot;ג של acidified, מסיס, סוג אני קולגן מזנבות עכברים ב1-3 מ&quot;ג / מיליליטר באמצעות פרוטוקולים סטנדרטיים בידוד, כגון שדווח על ידי et al Piez. 15 </li><li style=";text-align:right;direction:rtl"> קנה מידה של כמות חומר מוצא מבוסס על רצוי סוף הנפח של פתרונות קולגן שונה. כ לעשות 25-30 מיליליטר של מפוגל ו25-30 מיליליטר של פתרונות קולגן succinylated, כל 3 מ&quot;ג / מיליליטר, 200 מ&quot;ג משל קולגן ילידים המסיס. </li></ol> 2. מתילציה קולגן <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לדלל של הפתרון המקומי, acidified (pH 2-3) קולגן 100 מ&quot;ג לריכוז של 0.5 מ&quot;ג / מיליליטר עם מים סטריליים קרים כקרח ולשמור על הפתרון על קרח כדי למנוע gelation. </li><li style=";text-align:right;direction:rtl"> התאם את ה- pH של תמיסת קולגן ל9-10 עם כמה טיפות של 1 N NaOH ומערבבים על RT למשך 30 דקות. שים לב למשקע קולגן, גורם פתרון להפך מעונן. </li&gt; <li style=";text-align:right;direction:rtl"> ספין למטה פתרון קולגן זירז ב3,000 × גרם במשך 25 דקות. משקע ברור, כמו ג&#39;ל צריך להיות גלוי בחלק התחתון של הצינורות. לשאוב והשלכה נכונה של נוזל supernatant. </li><li style=";text-align:right;direction:rtl"> Resuspend קולגן זירז ב200 מיליליטר של מתנול עם 0.1 N HCl ולאפשר תגובת מתילציה להתרחש עם ערבוב על RT במשך 4 ימים. קולגן לא לפזר, אבל צריך להתפרק לחתיכות קטנות מאוד גלויות שיהפוך את פתרון מעונן. </li><li style=";text-align:right;direction:rtl"> לאחר מתילציה, צנטריפוגה הפתרון ב3,000 × גרם במשך 25 דקות עד גלולה קולגן מפוגל. לשאוב ולהיפטר supernatant מתנול acidified. </li><li style=";text-align:right;direction:rtl"> ממיסים את קולגן מפוגל ב 25 מיליליטר של PBS סטרילי, נותן ריכוז של כ 3 מ&quot;ג / מיליליטר, עם pipetting החוזר והנשנה, ולסנן את הפתרון דרך מסננת תא 60 מיקרומטר. התאם את ה- pH של הפתרון ל7.3-7.4 באמצעות 20 μl מרווחים של 1 N NaOH. </li><li style=";text-align:right;direction:rtl"> להעריך את concentration של הפתרון באמצעות ערכת ELISA קולגן העכברוש המסחרי או ערכת assay hydroxyproline. לדלל את הפתרון עד 3 מ&quot;ג / מיליליטר עם PBS סטרילי. </li><li style=";text-align:right;direction:rtl"> לעקר את פתרון קולגן מפוגל על ​​ידי העברתו לבקבוק זכוכית עם מכסה בורג, שכבות בזהירות 3 מיליליטר של כלורופורם בתחתית הבקבוק, לאפשר את הבקבוק להגדיר O / N ב 4 ° C, ולאחר מכן להסיר את הסביבה נקיה מחיידקים ולאחסן שכבה עליונה, שהוא קולגן מפוגל. </li><li style=";text-align:right;direction:rtl"> אחסן את הפתרון ב- C ° 4 לשימוש בתוך 1 חודש. </li></ol> 3. קולגן Succinylation <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לדלל של הפתרון המקומי, acidified (pH 2-3) קולגן 100 מ&quot;ג האחר לריכוז של 0.5 מ&quot;ג / מיליליטר עם מים סטריליים קרים כקרח ולשמור על הפתרון על קרח כדי למנוע gelation. </li><li style=";text-align:right;direction:rtl"> התאם את ה- pH של תמיסת קולגן ל9-10 עם כמה טיפות של 1 N NaOH ומערבבים על RT למשך 30 דקות. קולגן צריך לזרז, גורם פתרון להפך מעונן. </li><li style=";text-align:right;direction:rtl"> Dissolve 40 מ&quot;ג של sucאנהידריד cinic (0.4 מ&quot;ג לכל מ&quot;ג של קולגן) ב 10 מיליליטר של אצטון (1/20 ה הנפח של פתרון קולגן). לאט לאט (בכ 0.5 מיליליטר במרווחים) להוסיף את תערובת לפתרון קולגן עם ערבוב תוך מעקב pH ברציפות. לשמור על pH מעל 9.0 על ידי הוספת טיפות 1 או 2 של 1 N NaOH כpH 9.0 גישות. </li><li style=";text-align:right;direction:rtl"> ממשיך לבחוש בRT עבור 120 דקות לאחר הוספת כל אנהידריד succinic באצטון. שים לב לפתרון ליתברר כקולגן succinylated מתמוסס. מעת לעת לבדוק את ה- pH כדי להבטיח שהיא נשארת מעל 9.0. </li><li style=";text-align:right;direction:rtl"> התאם את ה- pH של התמיסה עד 4.0 עם 20 מרווחי μl של 1 N HCl. שים לב לפתרון מעונן שוב, כמו קולגן succinylated משקעים. </li><li style=";text-align:right;direction:rtl"> לאחר succinylation, צנטריפוגה הפתרון ב3,000 × גרם במשך 25 דקות עד גלולה קולגן succinylated. לשאוב ולהיפטר supernatant acidified עם אנהידריד succinic unreacted. </li><li style=";text-align:right;direction:rtl"> ממס את קולגן succinylatedב 25 מיליליטר של PBS סטרילי, נותן ריכוז של כ 3 מ&quot;ג / מיליליטר, עם pipetting החוזר והנשנה, ולסנן את הפתרון דרך מסננת תא 60 מיקרומטר. התאם את ה- pH של הפתרון ל7.3-7.4 באמצעות 20 μl מרווחים של 1 N NaOH. </li><li style=";text-align:right;direction:rtl"> להעריך את הריכוז של הפתרון באמצעות ערכת ELISA עכברוש קולגן מסחרי או ערכת assay hydroxyproline. לדלל את הפתרון עד 3 מ&quot;ג / מיליליטר עם PBS סטרילי. </li><li style=";text-align:right;direction:rtl"> לעקר את פתרון קולגן succinylated על ידי העברתו לבקבוק זכוכית עם מכסה בורג, שכבות בזהירות 3 מיליליטר של כלורופורם בתחתית הבקבוק, לאפשר את הבקבוק להגדיר O / N ב 4 ° C, ולאחר מכן להסיר את הסביבה נקיה מחיידקים ולאחסן שכבה עליונה, שהוא קולגן succinylated. </li><li style=";text-align:right;direction:rtl"> אחסן את הפתרון ב- C ° 4 לשימוש בתוך 1 חודש. </li></ol> 4. אימות של שינויי קולגן <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן 1 מיליליטר דגימות מכל אחת מפתרוני קולגן ילידים, מפוגלים, וsuccinylated ולדלל לקונצרט כלשהוentration של 0.1 מ&quot;ג / מיליליטר עם מים טהורים סטרילי לטיטרציה יון מימן. בהמשך לדלל החיץ לפחות 1,000 פי ידי דיאליזה נגד מים באמצעות 10 הפסקת membraneusing 3 שינויי פתרון kDa לפחות 4 שעות כל אחד. </li><li style=";text-align:right;direction:rtl"> התאם את ה- pH של הפתרונות ל7.3 עם כמויות קטנות של NaOH וHCl. באמצעות pH 7.3 כהתייחסות שרירותית, לבצע טיטרציה יון מימן על כל אחד מהדגימות כפי שתואר על ידי Tanford 16 כדי ליצור עקומות טיטרציה לפתרונות קולגן ילידים, מפוגלים, וsuccinylated. </li><li style=";text-align:right;direction:rtl"> עלילה השינוי ברמת חומציות לכל נפח של חומצה הוסיף לעומת המספר של H + יונים לכל מולקולה הקשורה. טווח &quot;האמין&quot; pH הגבוה צריך להראות שינוי בקולגן succinylated לעבר הנקודה הניטרלית (הפסד של קבוצות האמינים), והמגוון &quot;carboxyl&quot; pH הנמוך צריך להראות משמרת שמאלה בקולגן מפוגל (הפסד של קבוצות carboxyl ) ותזוזה ימינה בקולגן succinylated (רווח בקבוצת carboxylים), בהשוואה לקולגן הטבעי. </li><li style=";text-align:right;direction:rtl"> להעריך את היעילות של תגובת succinylation ידי קביעת% מקבוצות אמינו בקולגן הילידים הוחלף על ידי succinylation בשיטת חומצת 2,4,6-trinitrobenzenesulfonic (TNBA) colorimetric, הבאים פרוטוקולים סטנדרטיים 17,18. </li></ol> 5. ייצור של מכשירי microfluidic וזריעת תאים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לפברק מכשירי microfluidic תוך שימוש בשיטות סטנדרטית 9. השתמש בדפוס העתק של PDMS מSU-8 מאסטרים על סיליקון המוגדר באמצעות photolithography ליצור תאי תרבית תאי microfluidic עם 100 מיקרומטר גבוה, רחב .4-1.5 מ&quot;מ, ו1-10 מ&quot;מ ערוצים ארוכים לצמיחת תאים. </li><li style=";text-align:right;direction:rtl"> השתמש בשואב פלזמה לחמצן את המשטחים של המכשיר ושקופיות זכוכית, ולאחר מכן לחץ יחד כדי אג&quot;ח. לאחר חיטוי המכשיר על ידי חשיפה לאור UV במשך לפחות 30 דקות, למלא את התא עם 50 מיקרוגרם / מיליליטר פיברונקטין בPBS סטרילי ולדגור על 37 מעלות צלזיוס במשך 45 דקות. </li&gt; <li style=";text-align:right;direction:rtl"> זרעי המכשיר עם תאים, כגון חולדה ראשונית או hepatocytes אדם, אשר דורשים ג&#39;ל קולגן לייצוב של פנוטיפ או מדינת בידול. אנו משתמשים 20 μl של hepatocytes מבודד טרי עכברוש עיקרי 7,19 או hepatocytes האנושי הראשוני cryopreserved זמין מסחרי זרע בגיל 14 × 10 6 תאים / מיליליטר למכשיר. </li><li style=";text-align:right;direction:rtl"> אפשר התאים לצרף במשך 4-6 שעות, ולאחר מכן לשטוף את התאים פנויים ולהחליף את התקשורת עם ציפוי תקשורת צמיחה. דגירה O / N כדי להבטיח תא מלא מתפשט וליצור monolayer ומחוברות של תאים. </li></ol> 6. שכבה אחר שכבה קולגן הפקדת <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בשכונת תרביות רקמת זרימה למינרית, להכין כמויות מספיקות של פתרונות קולגן מפוגלים וsuccinylated במשך 10 יישומים של כל פתרון לכל מכשיר, כמו גם כמה MLS של תקשורת. שמור את הפתרונות על קרח. אנו משתמשים 20 μl (10-15 פעמים בסך הכל מכשיר נפח) של קולגן לכל שכבה לכל מכשיר. </li><li style=";text-align:right;direction:rtl"> להיותginning עם הפתרון מפוגל (polycationic), חלופי שטיפת המכשירים עם 20 μl של מפוגל ולאחר מכן succinylated פתרונות קולגן, מחכה דק &#39;1 בין כל יישום. לשטוף את המכשיר בסך הכל 10 פעמים בפתרון, שאמורה לקחת כ -20 דקות. לעבוד במהירות כדי לצמצם את כמות הזמן שהתאים ללא תקשורת. </li><li style=";text-align:right;direction:rtl"> שים לב קולגן לצבור לאט בכניסה / היציאה בהתאם לגודלה. אם ההתנגדות לזרימת נוזל עליות, לשטוף את המכשיר פעם או פעמיים עם תקשורת, ולאחר מכן להמשיך שכבות. </li><li style=";text-align:right;direction:rtl"> לאחר החלת כל השכבות, לשטוף את המכשיר פעמיים עם תקשורת טרי ולחזור לחממה. בהתאם לסוג התא, השינויים תאיים הנגרמת המטריצה ​​מורפולוגיים, כמו קיטוב משופר בhepatocytes, צריכים להיות גלויים בתוך כמה שעות. </li><li style=";text-align:right;direction:rtl"> הכן מכשירי נציג למיקרוסקופ אלקטרונים הילוכים באמצעות שיטות סטנדרטי 9 כדי לאמת את הנוכחות של מטריצת קולגןהרכבה על גבי התאים בתרבית. </li></ol> 7. ייצוב של הפנוטיפ תא ותפקוד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תמונת מורפולוגיה תא, כדאיות, וקוטביות בשיטות סטנדרטית לסוג התא. לhepatocytes, לאסוף תמונות מעל 14 ימים באמצעות מיקרוסקופ שלב, מכתים חי / מת כדאיות, וצבע CMFDA לקיטוב הפסגה כדי להדגים את ההשפעות של תצהיר קולגן. </li><li style=";text-align:right;direction:rtl"> למורפולוגיה תא, לשטוף את המכשיר דרך הכניסה על ידי פיפטה עם 20 μl של PBS לשטוף, להזריק 20 μl של PBS, ותמונת המכשיר באמצעות מיקרוסקופ לעומת שלב. </li><li style=";text-align:right;direction:rtl"> עבור מכתים חי / מת, שטוף את המכשיר דרך הכניסה על ידי פיפטה עם 20 μl של PBS לשטוף, להזריק 20 μl של פתרון בשידור חי / מת מכתים עם DAPI הכין הבאים הוראות היצרן, דגירה במשך 30 דקות ב 37 מעלות צלזיוס, לשטוף מכשיר שוב עם 20 μl של PBS, ותמונת המכשיר באמצעות מיקרוסקופ פלואורסצנטי. </li><li style=";text-align:right;direction:rtl"> לקנלי מרהצביעת culi, לשטוף את המכשיר דרך הכניסה על ידי פיפטה עם 20 μl של PBS לשטוף, להזריק 20 μl של 2 פתרון מכתים מיקרומטר CMFDA עם DAPI הכין הבאים הוראות היצרן, דגירה במשך 30 דקות ב 37 מעלות צלזיוס, לשטוף את המכשיר שוב עם 20 μl של PBS, ותמונת המכשיר באמצעות מיקרוסקופ פלואורסצנטי. </li><li style=";text-align:right;direction:rtl"> לאסוף את התקשורת בילתה ולמדוד מוצרי חילוף חומרים תאיים בנקודות זמן מתאימות על משך התרבות כדי לקבוע את תפקוד התא. לhepatocytes, למדוד את הכמות של אלבומין ואוריאה בתקשורת בילתה. </li><li style=";text-align:right;direction:rtl"> לבצע ניתוחים תפקודיים נקודות קצה בתאים עצמם, כגון רמות הערכת אנזים פעילות, מכתים נוגדן, או ניתוח RNA. לhepatocytes, לעורר ולמדוד את פעילות אנזים ציטוכרום P450 או שלב S-transferase גלוטתיון האנזים השני הצמידה. </li></ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilineage+differentiation+of+rhesus+monkey+embryonic+stem+cells+in+three&#8208;dimensional+culture+systems.">Multilineage differentiation of rhesus monkey embryonic stem cells in three&#8208;dimensional culture systems.</a> Stem Cells. 21 (3), 281-295 (2003).</li><li>Whelan, M. C., Senger, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+I+initiates+endothelial+cell+morphogenesis+by+inducing+actin+polymerization+through+suppression+of+cyclic+AMP+and+protein+kinase+A.">Collagen I initiates endothelial cell morphogenesis by inducing actin polymerization through suppression of cyclic AMP and protein kinase A.</a> J Biol Chem. 278 (1), 327-334 (2003).</li><li>Dunn, J. C., Tompkins, R. G., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vitro+function+of+adult+hepatocytes+in+a+collagen+sandwich+configuration.">Long-term in vitro function of adult hepatocytes in a collagen sandwich configuration.</a> Biotechnol Prog. 7 (3), 237-245 (1991).</li><li>Decher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fuzzy+Nanoassemblies:+Toward+Layered+Polymeric+Multicomposites.">Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites.</a> Science. 277, 1232-1237 (1997).</li><li>McCarty, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+ultrathin+collagen+nanolayer+assembly+for+3-D+microtissue+engineering:+Layer-by-layer+collagen+deposition+for+long-term+stable+microfluidic+hepatocyte+culture.">A novel ultrathin collagen nanolayer assembly for 3-D microtissue engineering: Layer-by-layer collagen deposition for long-term stable microfluidic hepatocyte culture.</a> TECHNOLOGY. 2 (01), 67-74 (2014).</li><li>Swierczewska, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+response+to+nanoscale+elastin-like+polypeptide+polyelectrolyte+multilayers.">Cellular response to nanoscale elastin-like polypeptide polyelectrolyte multilayers.</a> Acta Biomater. 4 (4), 827-837 (2008).</li><li>Kim, Y., Larkin, A. L., Davis, R. M., Rajagopalan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+of+in+vitro+liver+sinusoid+mimics+using+chitosan-hyaluronic+acid+polyelectrolyte+multilayers.">The design of in vitro liver sinusoid mimics using chitosan-hyaluronic acid polyelectrolyte multilayers.</a> Tissue Eng Pt A. 16 (9), 2731-2741 (2010).</li><li>Larkin, A. L., Rodrigues, R. R., Murali, T., Rajagopalan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+a+Multicellular+Organotypic+3D+Liver+Model+with+a+Detachable,+Nanoscale+Polymeric+Space+of+Disse.">Designing a Multicellular Organotypic 3D Liver Model with a Detachable, Nanoscale Polymeric Space of Disse.</a> Tissue Eng Pt C. 19 (11), 875-884 (2013).</li><li>Kidambi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+Co&#8208;Culture+of+Primary+Hepatocytes+and+Fibroblasts+Using+Polyelectrolyte+Multilayer+Templates.">Patterned Co&#8208;Culture of Primary Hepatocytes and Fibroblasts Using Polyelectrolyte Multilayer Templates.</a> Macromol Biosci. 7 (3), 344-353 (2007).</li><li>Janorkar, A. V., Rajagopalan, P., Yarmush, M. L., Megeed, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+elastin-like+polypeptide-polyelectrolyte+complexes+to+control+hepatocyte+morphology+and+function+in+vitro.">The use of elastin-like polypeptide-polyelectrolyte complexes to control hepatocyte morphology and function in vitro.</a> Biomaterials. 29 (6), 625-632 (2008).</li><li>Piez, K. A., Eigner, E. A., Lewis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Chromatographic+Separation+and+Amino+Acid+Composition+of+the+Subunits+of+Several+Collagens*.">The Chromatographic Separation and Amino Acid Composition of the Subunits of Several Collagens*.</a> Biochemistry. 2 (1), 58-66 (1963).</li><li>Tanford, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interpretation+of+hydrogen+ion+titration+curves+of+proteins.">The interpretation of hydrogen ion titration curves of proteins.</a> Adv Protein Chem. 17, 69-165 (1962).</li><li>Cayot, P., Tainturier, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quantification+of+protein+amino+groups+by+the+trinitrobenzenesulfonic+acid+method:+a+reexamination.">The quantification of protein amino groups by the trinitrobenzenesulfonic acid method: a reexamination.</a> Anal Biochem. 249 (2), 184-200 (1997).</li><li>Kakade, M. L., Liener, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+available+lysine+in+proteins.">Determination of available lysine in proteins.</a> Anal Biochem. 27 (2), 273-280 (1969).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods Cell Biol. 13, 29-83 (1976).</li></ol>

The authors have nothing to disclose.

ניתן להפקיד מכלולי קולגן הטהור Ultrathin על תאים טעונים או surfaces חומר באמצעות תצהיר שכבה אחר שכבה של collagens שונה. תוצאות מחקר זה מראות כי מתילציה וsuccinylation של קולגן ילידים ליצור פתרונות קולגן polycationic וpolyanionic טעון (איור 1) שניתן להשתמש בו עם טכניקת שכבה אחר שכבה להפקיד ?...

Although microfluidics allows for the exquisite control of the cellular microenvironment, culturing cells, especially primary cells, within microfluidic devices can be challenging. Many traditional cell culture techniques have been developed to sustain and stabilize cell function when cultured in tissue culture plates, but translating those techniques to microfluidic devices is often difficult. 
One such technique is the culture of cells on or sandwiched between collagen gels as a model of the physiological 3D cell environment.1 Type I collagen is one of the most frequently used proteins for biomaterials applications because of its ubiquity in extracellular matrix, natural abundance, robust cell attachment sites, and biocompatibility.2 Many cells benefit from 3D culture with collagen, including cancer cells3,45, microvascular endothelial cells6, and hepatocytes7, among others. While the use of collagen gels is easy in open formats, such as tissue culture plates, the limited channel dimensions and enclosed nature of microfluidic devices makes the use of liquids that gel impractical without blocking the entire channel.
To overcome this problem, we combined the layer-by-layer deposition technique8 with chemical modifications of native collagen solutions to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These layers can stabilize cell morphology and function similar to collagen gels and can be deposited on cells in microfluidic devices without blocking the channels with polymerized matrix. The goal of this method is to modify native collagen to create polycationic and polyanionic collagen solutions and to stabilize cells in microfluidic culture by depositing thin collagen matrix assemblies onto the cells. This technique has been used to stabilize the morphology and function of primary hepatocytes in microfluidic devices.9
Although layer-by-layer deposition has previously been reported with natural and synthetic polyelectrolytes10 to cover hepatocytes in plate culture11,12 and as a seeding layer for hepatocytes in microfluidic devices13,14, this method describes the deposition of a pure collagen layer on top of hepatocytes, mimicking the 3D collagen culture techniques. In this protocol, we use hepatocytes as example cells that can be maintained using 3D collagen layers. The many other types of cells that benefit from 3D culture in collagen may similarly benefit from culture after layer-by-layer deposition of an ultrathin collagen matrix assembly.

<table><tbody><tr><td>collagen type I, rat tail</td><td>Life Technologies</td><td>A1048301</td><td>option for concentrated rat tail collagen</td></tr><tr><td>collagen type I, rat tail</td><td>Sigma-Aldrich</td><td>C3867-1VL</td><td>option for concentrated rat tail collagen</td></tr><tr><td>collagen type I, rat tail</td><td>EMD Millipore</td><td>08-115</td><td>option for concentrated rat tail collagen</td></tr><tr><td>collagen type I, rat tail</td><td>R%D Systems</td><td>3440-100-01</td><td>option for concentrated rat tail collagen</td></tr><tr><td>succinic anhydride</td><td>Sigma-Aldrich</td><td>239690-50G</td><td>succinylation reagent</td></tr><tr><td>anhydrous methanol</td><td>Sigma-Aldrich</td><td>322415-100ML</td><td>methylation reagent</td></tr><tr><td>sodium hydroxide</td><td>Sigma-Aldrich</td><td>S5881-500G</td><td>pH precipitation reagent</td></tr><tr><td>hydrochloric acid</td><td>Sigma-Aldrich</td><td>320331-500ML</td><td>pH precipitation reagent</td></tr><tr><td>rat collagen type I ELISA</td><td>Chondrex</td><td>6013</td><td>option for detecting collagen content</td></tr><tr><td>hydroxyproline assay kit</td><td>Sigma-Aldrich</td><td>MAK008-1KT</td><td>option for detecting collagen content</td></tr><tr><td>hydroxyproline assay kit</td><td>Quickzyme Biosciences</td><td>QZBtotcol1</td><td>option for detecting collagen content</td></tr></tbody></table>

layer-by-layer collagen deposition in microfluidic devices for microtissue stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

קולגן ילידים יכול להיות שונה באמצעות מתילציה וsuccinylation ליצור פתרונות קולגן polycationic וpolyanionic לשימוש בתצהיר שכבה אחר שכבה. Succinylation משנה את קבוצות ε-אמינו של קולגן יליד עם קבוצות succinyl, ומתילציה משנה את קבוצות carboxyl של קולגן יליד עם קבוצה מתיל (איור 1 א). שינויים אלה ל?...

Watch this Scientific Journal Video about Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization at JoVE.com

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

קולגן שכבת הפקדת-ידי שכבה במכשירי microfluidic לMicrotissue ייצוב

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

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Research

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Bioengineering

9.3K Views.  Massachusetts General Hospital, Harvard Medical School. The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Video: Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

ייצור של מיקרו-רקמות באמצעות מודולים של קולגן ג&#39;ל התאים המכילים

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

קולגן אני Assay Organotypic: פלטפורמה חשילה לבחינת התנהגות תא בהקשר 3-Dimensional

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

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. ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

טכניקת microfluidic Genipin הפקדת לתרבות מורחבת של כלי דם micropatterned השרירים Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

הכנת ג&#39;לי 3D קולגן microchannels לחקר אינטראקציות 3D<em&gt; In vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning והרכבת 3D microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Vascularized Microfluidic Bioprinting עבור הנדסת רקמות, Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

פלטפורמת מיקרופלואידיג לעירור כונדרוציטים עם דחיסה דינמית

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

פיתוח רקמת לב אנושית מאורגנת תלת מימדית בתוך פלטפורמה מיקרופלואידית

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark of cardiac and musculoskeletal tissues. To study cell response to aligned microenvironments in vitro, several protocols have been developed to generate COL1 matrices with defined fiber alignment, including magnetic, mechanical, cell-based, and microfluidic methods. Of these, microfluidic approaches offer advanced capabilities such as accurate control over fluid flows and the cellular microenvironment. However, the microfluidic approaches to generate aligned COL1 matrices for advanced in vitro culture platforms have been limited to thin "mats" (&lt;40 &#181;m in thickness) of COL1 fibers that extend over distances less than 500 &#181;m and are not conducive to 3D cell culture applications. Here, we present a protocol to fabricate 3D COL1 matrices (130-250 &#181;m in thickness) with millimeter-scale regions of defined fiber alignment in a microfluidic device. This platform provides advanced cell culture capabilities to model structured tissue microenvironments by providing direct access to the micro-engineered matrix for cell culture.

This protocol demonstrates the use of a microfluidic channel with changing geometry along the fluid flow direction to generate extensional strain (stretching) to align fibers in a 3D collagen hydrogel (&lt;250 &#181;m in thickness). The resulting alignment extends across several millimeters and is influenced by the extensional strain rate.

מיקרו-הנדסה הידרוג'ל קולגן תלת-ממדית עם יישור סיבים לטווח ארוך

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark ...

קולגן שכבת הפקדת-ידי שכבה במכשירי microfluidic לMicrotissue ייצוב

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ייצור של מיקרו-רקמות באמצעות מודולים של קולגן ג'ל התאים המכילים