Name: custom engineered tissue culture molds from laser-etched masters
Uploaded: 2018-05-21T13:00:00.000+00:00
Duration: 8 min 56 s
Description: Watch this Scientific Journal Video about Custom Engineered Tissue Culture Molds from Laser-etched Masters at JoVE.com

המחברים להכיר מימון NIH R00 HL115123, בראון אוניברסיטת בית הספר להנדסה. הם אסירי תודה גם סדנת עיצוב בראון, כריס בול הדרכה ותמיכה עם לייזר חותך.

<ol>
	<li>Qin, D., Xia, Y., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography+for+micro-+and+nanoscale+patterning.">Soft lithography for micro- and nanoscale patterning.</a> Nat. Protoc. 5, 491 (2010).</li><li>Rogers, J. A., Nuzzo, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+soft+lithography.">Recent progress in soft lithography.</a> Mater. Today. 8, 50-56 (2005).</li><li>Whitesides, G. M., Ostuni, E., Takayama, S., Jiang, X., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+Lithography+in+Biology+and+Biochemistry.">Soft Lithography in Biology and Biochemistry.</a> Annu. Rev. Biomed. Eng. 3, 335-373 (2001).</li><li>Isiksacan, Z., Guler, M. T., Aydogdu, B., Bilican, I., Elbuken, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+fabrication+of+microfluidic+PDMS+devices+from+reusable+PDMS+molds+using+laser+ablation.">Rapid fabrication of microfluidic PDMS devices from reusable PDMS molds using laser ablation.</a> J. Micromechanics Microengineering. 26, 035008 (2016).</li><li>Lee, K. Y., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+for+Tissue+Engineering.">Hydrogels for Tissue Engineering.</a> Chem. Rev. 101, 1869-1880 (2001).</li><li>Kloxin, A., Kloxin, C., Bowman, C., Anseth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+cellularly+responsive+hydrogels+and+their+experimental+determination.">Mechanical properties of cellularly responsive hydrogels and their experimental determination.</a> Adv. Mater. Deerfield Beach Fla. 22, 3484-3494 (2010).</li><li>Aubin, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+3D+cell+alignment+and+elongation+in+microengineered+hydrogels.">Directed 3D cell alignment and elongation in microengineered hydrogels.</a> Biomaterials. 31, 6941-6951 (2010).</li><li>Jaiswal, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vacancy-Driven+Gelation+Using+Defect-Rich+Nanoassemblies+of+2D+Transition+Metal+Dichalcogenides+and+Polymeric+Binder+for+Biomedical+Applications.">Vacancy-Driven Gelation Using Defect-Rich Nanoassemblies of 2D Transition Metal Dichalcogenides and Polymeric Binder for Biomedical Applications.</a> Adv. Mater. 29, (2017).</li><li>Lian, X., 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=Directed+cardiomyocyte+differentiation+from+human+pluripotent+stem+cells+by+modulating+Wnt/&#946;-catenin+signaling+under+fully+defined+conditions.">Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/&#946;-catenin signaling under fully defined conditions.</a> Nat. Protoc. 8, 162-175 (2013).</li><li>Boxshall, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+surface+treatments+to+modify+protein+adsorption+and+cell+attachment+properties+within+a+poly(dimethylsiloxane)+micro-bioreactor.">Simple surface treatments to modify protein adsorption and cell attachment properties within a poly(dimethylsiloxane) micro-bioreactor.</a> Surf. Interface Anal. 38, 198-201 (2006).</li><li>Pins, G. D., Christiansen, D. L., Patel, R., Silver, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+of+collagen+fibers.+Influence+of+fibrillar+alignment+and+decorin+on+mechanical+properties.">Self-assembly of collagen fibers. Influence of fibrillar alignment and decorin on mechanical properties.</a> Biophys. J. 73, 2164-2172 (1997).</li><li>Pipan, C. 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=Effects+of+antifibrinolytic+agents+on+the+life+span+of+fibrin+sealant.">Effects of antifibrinolytic agents on the life span of fibrin sealant.</a> J. Surg. Res. 53, 402-407 (1992).</li><li>Roberts, M. A., 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=Stromal+Cells+in+Dense+Collagen+Promote+Cardiomyocyte+and+Microvascular+Patterning+in+Engineered+Human+Heart+Tissue.">Stromal Cells in Dense Collagen Promote Cardiomyocyte and Microvascular Patterning in Engineered Human Heart Tissue.</a> Tissue Eng. Part A. 22, 633-644 (2016).</li><li>Ye, K. Y., Sullivan, K. E., Black, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+Cardiomyocytes+in+a+Fibrin+Hydrogel+for+Cardiac+Tissue+Engineering.">Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering.</a> JoVE. , (2011).</li><li>Zimmermann, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Engineering+of+a+Differentiated+Cardiac+Muscle+Construct.">Tissue Engineering of a Differentiated Cardiac Muscle Construct.</a> Circ. Res. 90, 223-230 (2002).</li><li>McCain, M. L., Agarwal, A., Nesmith, H. W., Nesmith, A. P., Parker, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromolded+Gelatin+Hydrogels+for+Extended+Culture+of+Engineered+Cardiac+Tissues.">Micromolded Gelatin Hydrogels for Extended Culture of Engineered Cardiac Tissues.</a> Biomaterials. 35, 5462-5471 (2014).</li><li>Hu, J. J., Chen, G. W., Liu, Y. C., Hsu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Specimen+Geometry+on+the+Estimation+of+the+Planar+Biaxial+Mechanical+Properties+of+Cruciform+Specimens.">Influence of Specimen Geometry on the Estimation of the Planar Biaxial Mechanical Properties of Cruciform Specimens.</a> Exp. Mech. 54, 615-631 (2014).</li><li>Munarin, F., Kaiser, N. J., Kim, T. Y., Choi, B. R., Coulombe, K. L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-Etched+Designs+for+Molding+Hydrogel-Based+Engineered+Tissues.">Laser-Etched Designs for Molding Hydrogel-Based Engineered Tissues.</a> Tissue Eng. Part C Methods. 23, 311-321 (2017).</li><li>Zhang, H., Chiao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-fouling+Coatings+of+Poly(dimethylsiloxane)+Devices+for+Biological+and+Biomedical+Applications.">Anti-fouling Coatings of Poly(dimethylsiloxane) Devices for Biological and Biomedical Applications.</a> J. Med. Biol. Eng. 35, 143-155 (2015).</li></ol>

המחברים אין לחשוף.

מותאם אישית גיאומטריות עובש PDMS התואמים לתרביות רקמה יש כלי נהדר ב כוונון מאפיינים חשובים רקמות מהונדסים, כגון יישור תא, קצב דיפוזיה יעילה נוקשות. בנוסף, הדגמים האלה הם מאוד שימושי עבור הכנת רקמות עבור יישומי ניתוח שבו הגיאומטריה חשוב, כגון מכונות בדיקה16,17. ה...

בשני העשורים האחרונים, הדפס אבן רכה שימש בהרחבה כמו טכניקה פבריקציה נוספת כדי לתמוך מחקר מדעי, במיוחד בתחומים של מיקרופלואידיקה חומרי מחקר, רקמות הנדסה1,2, 3. לכייר של עותק משוכפל, שבו נוצר אובייקט עם הצורה הרצויה תבנית בסיס שלילי, מציע במחיר נמוך ונוח שיטה לייצר חיובי ש-PDMS משכפל זה יכול לשמש הליהוק בצורת hydrogels. אולם, התבניות נדרש בסיס שלילית בדרך כלל מיוצרים בטכניקות מיקרו-מלאכותית כי הם יקרים, זמן רב, שגודל, מוגבל, נדרש שטח חדר נקי וכן ציוד מתוחכם. הדפסה תלת-ממדית מציעה חלופה פוטנציאלית, השירות שלה מוגבל במקצת בשל המגבלות ברזולוציה של מדפסות בעלות נמוכה, הגומלין הכימית בין פולימרים במדפסת תלת-ממד נפוצות PDMS זה יכול לעכב ריפוי.
מערכות לייזר חותך מסוגל גם חיתוך וגם תחריט חומרים כגון פלסטיק, עץ, זכוכית, מתכת הפכו לאחרונה באופן דרסטי פחות יקר, לכן לנגישה יותר עבור בדיית כלי מחקר. מסחרי כיתה לייזר מסוגלים בדיית אובייקטים בסולם סנטימטר עם מינימום שתכונות הקטנות מ מיקרומטר 25 ועוד יותר דורשים הכשרה מינימלית, המומחיות, הזמן להשתמש. בעוד לייזר אבלציה של PDMS בעבר שימש בייצור המכשירים מיקרופלואידיקה, לידע שלנו תיאר כתב היד לא תהליך על ידי איזה מ מ וסנטימטר בתבניות בקנה מידה יכול להיות מפוברק של לייזר לחתוך בתבניות בסיס שלילי4 .
השתמשנו בטכניקה זו בעיקר כדי לשנות את הצורה של רקמות מהונדסים על מנת לשפר את פיזור חומר מזין, יישור הסלולר ו תכונות מכניות5,6,7. עם זאת, הרב-גוניות של טכניקה זו מאפשרת הניצול בכל תחום היכן עי hydrogels של עניין, כמו סמים משלוח והחומרים המדע המחקר8. עם גישה בחותך לייזר, משכפל עובש PDMS יכול להתבצע עבור כמעט כל שינוי של גאומטריה ללא המסוכך (זה לעכב את הסרת ללא תבנית מרובה חלקים, אשר מעבר להיקף של כתב יד זה), זה מתאים לממדים של המיטה לייזר.

<table><tbody><tr><td>&lt;strong&gt;Item&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bovine fibrinogen</td><td>Sigma</td><td>F8630-5G</td><td>Constructs</td></tr><tr><td>Bovine thrombin</td><td>Sigma</td><td>T6634-250UN</td><td>Constructs</td></tr><tr><td>Bovine aprotinin</td><td>Sigma</td><td>10820-25MG</td><td>Constructs</td></tr><tr><td>Rat tail collagen I, 4 mg/mL</td><td>Advanced Biomatrix</td><td>5153-100MG</td><td>Constructs</td></tr><tr><td>Sodim chloride</td><td>Fisher</td><td>BP358-10</td><td>Constructs</td></tr><tr><td>PBS</td><td>Life Technologies</td><td>14190-250</td><td>Constructs</td></tr><tr><td>Fine forceps</td><td>Fine Science Tools</td><td>11252-20</td><td>Constructs</td></tr><tr><td>Sylgard 184 silicone elastomer</td><td>Corning</td><td>4019862</td><td>PDMS Molds</td></tr><tr><td>Lab tape</td><td>Fisher</td><td>15-901-5R</td><td>PDMS Molds</td></tr><tr><td>Acrylic, 1/4&quot; thick</td><td>McMaster-Carr</td><td>8560K356</td><td>PDMS Molds</td></tr><tr><td>HEPES Buffer, 1 M</td><td>Sigma</td><td>H3537-100ML</td><td>Constructs</td></tr><tr><td>RPMI 1640 medium, powder</td><td>Fisher</td><td>31800-089</td><td>Constructs</td></tr><tr><td>Calcium chloride dihydrate</td><td>Fisher</td><td>AC423520250</td><td>Constructs</td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Fisher</td><td>M33 500</td><td>Constructs</td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P9541-500G</td><td>Constructs</td></tr><tr><td>Sodium phosphate dibasic heptahydrate</td><td>Sigma</td><td>S9390-500G</td><td>Constructs</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G5767-25G</td><td>Constructs</td></tr><tr><td>OCT</td><td>VWR</td><td>25608-930</td><td>Histology</td></tr><tr><td>Frozen block molds</td><td>VWR</td><td>25608-916</td><td>Histology</td></tr><tr><td>Hematoxylin</td><td>Fisher</td><td>3530 1</td><td>Histology</td></tr><tr><td>Eosin Y</td><td>Fisher</td><td>AC152880250</td><td>Histology</td></tr><tr><td>Fast green FCF</td><td>Fisher</td><td>AC410530250</td><td>Histology</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Illustrator</td><td>Adobe Systems</td><td></td><td>Vector Graphics</td></tr><tr><td>Inkscape</td><td>(Open Source)</td><td></td><td>Vector Graphics</td></tr><tr><td>UCP (Universal Control Panel)</td><td>Universal Laser Systems</td><td></td><td>Laser Cutter Interface</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PLS6.75 Laser Cutter</td><td>Universal Laser Systems</td><td></td><td>Laser Cutter</td></tr><tr><td>Micromechanical Analyzer</td><td>Aurora Scientific</td><td>1530A with 5 mN load cell</td><td>Mechanical Analysis</td></tr></tbody></table>

custom engineered tissue culture molds from laser-etched masters

כמו בתחום של הנדסת רקמות המשיכה להבשיל, היו עניין גדל במגוון רחב של פרמטרים רקמות, כולל רקמות צורה. מניפולציה רקמות צורה מיקרומטר לקנה המידה סנטימטר יכול לכוון יישור תא, לשנות תכונות מכניות יעיל, והכתובת מגבלות הקשורות דיפוזיה מזין בנוסף, הכלי שבו טישו מוכן להקנות אילוצים מכני על הרקמה, וכתוצאה מכך הלחץ ששדות נוספים יכולים להשפיע על מבנה התא והן מטריקס. בצורת רקמות עם ממדים מאוד לשחזור יש גם כלי עזר עבור במבחנה מבחני באיזו דגימה מידות הם קריטיים, כגון ניתוח מכני כל רקמות.
כתב יד זה מתאר שיטה חלופית פבריקציה נוספת ניצול שלילי בתבניות הבסיס שהוכנו אקריליק חרוט בלייזר: בתבניות אלה לבצע היטב עם polydimethylsiloxane (PDMS), היתר עיצובים עם ממדים על סולם סנטימטר, תכונה מידות קטנות יותר 25 מיקרומטר, ולא במהירות של מפוברק בעלות נמוכה ועם מומחיות מינימלי. זמן מינימלי ודרישות עלות מאפשרים חרוט בלייזר בתבניות כדי להיות במהירות iterated על עד עיצוב אופטימלי של נקבעת, ניתן להתאים בקלות בהתאם וזמינותו בכל עניין, כולל אלה מעבר בתחום של הנדסת רקמות.

המערכת האופטית של החותך לייזר יגרמו חרוט למקומות מאוד מעט פחתו מידות כמו תצריב עומק בעליות, תוצאות עובש בקירות עם שפוע מאוד עדין, בשל כדי מתחדדת קרן הלייזר. זה יעזור להקל על הסרת התבניות PDMS יצוקה, אבל יש לשקול בזהירות אם עמוק חרוט בתבניות בסיס שלילי (> 6 מ מ) הם הנדרש (<strong class=...

Watch this Scientific Journal Video about Custom Engineered Tissue Culture Molds from Laser-etched Masters at JoVE.com

Custom Engineered Tissue Culture Molds from Laser-etched Masters

במסמך זה אנו מציגים שיטה מהירה, נתיישב ועלות נמוכה בדיית בתבניות polydimethylsiloxane מותאם אישית יכול לשמש לייצור המבוסס על הידרוג רקמות מהונדסים עם לסימולציה. בנוסף, אנו מתארים תוצאות הערכות מכני, היסטולוגית שנערכו על רקמות הלב מהונדסים המיוצר בעזרת טכניקה זו.

מותאם אישית מהונדסים תרביות רקמה בתבניות של מאסטרים נחרטו לייזר

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue ...

custom-engineered-tissue-culture-molds-from-laser-etched-masters

Research

JoVE Journal

Bioengineering

6.4K Views.  Brown University. במסמך זה אנו מציגים שיטה מהירה, נתיישב ועלות נמוכה בדיית בתבניות polydimethylsiloxane מותאם אישית יכול לשמש לייצור המבוסס על הידרוג רקמות מהונדסים עם לסימולציה. בנוסף, אנו מתארים תוצאות הערכות מכני, היסטולוגית שנערכו על רקמות הלב מהונדסים המיוצר בעזרת טכניקה זו.

Video: Custom Engineered Tissue Culture Molds from Laser-etched Masters

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

פלטפורמות microfabricated לתרבות Cell דינמי מכאני

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

בימוי נייד הרכבה עצמית לפברק Cell-נגזר טבעות רקמות לניתוח biomechanical הנדסת רקמות

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

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

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

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

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

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

Medicine

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

שיפוע צפיפות רבה שכבתי פלמור (DGMP): שיטה חדשנית ליצירת תא בעל מספר, פיגומים להנדסת רקמות להתאמה אישית

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Microfabrication של שבב בגודל פיגומים לעיבוד תלת ממדי נייד

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Biology

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

תא Multi-CNS-Neuron גליה שותף תרבות Microfluidic רציף

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

ייצור של בארות Agarose מותאם אישית עבור תא זריעה וטבעת רקמות הרכבה עצמית תוך שימוש בתבניות מודפס 3D

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

ייצור מכלולי גידי עכבר לחקר אינטראקציות תאיות במחלות

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. 
In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. 
The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease- and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.

מחבר זרקור: קידום מחקר הגידים על ידי פיתוח מכלולי עכברים להבנת מנגנונים תאיים

Here, we present an assembloid model system to mimic tendon cellular crosstalk between the load-bearing tendon core tissue and an extrinsic compartment containing cell populations activated by disease and injury. As an important use case, we demonstrate how the system can be deployed to probe disease-relevant activation of extrinsic endothelial cells.

מותאם אישית מהונדסים תרביות רקמה בתבניות של מאסטרים נחרטו לייזר

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פלטפורמות microfabricated לתרבות Cell דינמי מכאני

בימוי נייד הרכבה עצמית לפברק Cell-נגזר טבעות רקמות לניתוח biomechanical הנדסת רקמות

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

שיפוע צפיפות רבה שכבתי פלמור (DGMP): שיטה חדשנית ליצירת תא בעל מספר, פיגומים להנדסת רקמות להתאמה אישית

Microfabrication של שבב בגודל פיגומים לעיבוד תלת ממדי נייד

תא Multi-CNS-Neuron גליה שותף תרבות Microfluidic רציף

ייצור של בארות Agarose מותאם אישית עבור תא זריעה וטבעת רקמות הרכבה עצמית תוך שימוש בתבניות מודפס 3D

הנדסת מכלולי גידים לבדיקת הצלבה תאית במחלה ותיקון

הנדסת מכלולי גידים לבדיקת הצלבה תאית במחלה ותיקון

הנדסת מכלולי גידים לבדיקת הצלבה תאית במחלה ותיקון