עבודה זו נתמכה על ידי הלאומי ללב, ריאות ודם המכון מעניק HL56237, המכון הלאומי לסוכרת ומחלות עיכול כליות מכון DK97391-03, הקרן הלאומית למדע (NSF-1,227,359 והרווח למניה-1,003,907).

<ol>
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יש המחברים שום אינטרסים מתחרים או אינטרסים מנוגדים לחשוף.

<html> במאמר זה, אנו מציגים פרוטוקולים מפורטים לפיתוח רשת microvessel התרבותי, האפיון של צמתי EC ו cytoskeleton הפצת F- תקטין, ואת מדידות כמותיות של EC [Ca 2 +] i ו NO ייצור באמצעות מכשיר microfluidic. מכשיר microfluidic perfused מספק מודל במבחנה המאפשר סימולציה קרובה של גיאומטריות כלי דם <em...

תאי האנדותל (EC) המצפים את דפנות כלי דם vivo חשופים כל זמן לזרום, אבל ECS בתרבית גדלה לעתים קרובות בתנאי סטטיים תערוכת פנוטיפ פרו-דלקתי 1,2. טכנולוגית מיקרופלואידיקה מאפשרת נוזל לשלוט באופן מדויק באמצעות microscale המוגבל גיאומטרי ערוצים (תת מילימטר) 3, אשר מספק את ההזדמנות עבור תאים בתרבית, במיוחד עבור כלי דם ECS, לגדול בתנאי זרימה רצויות. תכונות אלו הופכות את תנאי תרבית תאים קרובים in vivo מ בתרביות תאי 2D הקונבנציונליות, סטטי. הם מאוד חשובים כאשר מכשירי microfluidic משמשים מודל סוגים שונים של vasculatures וללמוד תגובות EC גירויים מכאניים ו / או כימיים. למרות היתרונות שמפגינים רשת microchannel על תרבית תאים סטטי, העיבוד ויישום של מיקרופלואידיקה ב- F ביוield להישאר מוגבל. דווח ע&quot;י בדיקה שנערכה לאחרונה, רוב הפרסומים בתחום זה (85%) עדיין בכתבי עת הנדסה 4. הביצועים של מכשירי microfluidic לא היו מספיק משכנע עבור רוב הביולוגים לעבור טכניקות נוכחיות כגון assay Transwell ואת מנת תרבות מאקרו בקנה המידה / זכוכית שקופית למכשיר מיניאטורי זה. מיקרופלואידיקה הוא שדה רב תחומי, המחייב שיתוף פעולה בינתחומי לנוע בתחום זה קדימה. מטרת מאמר טכני זה היא לצמצם את פערי הידע בין דיסציפלינות ולבצע את הליכי הייצור מובנים על ידי ביולוגים, תוך מתן יישום ביולוגי ואימות פונקציונלית של microvessels microfluidic. הפרוטוקולים הניסיונות דמיינו כוללים ייצור של שני מכשירים microfluidic והמקודדים הביולוגיים שלהם, אשר מייצג שיתוף פעולה הדוק בין מהנדסים וביולוגים. אנחנו דיווחנו לאחרונה כמהיישומים ביולוגיים באמצעות רשת microvessel במבחנה עם המכשיר microfluidic 5. על מנת לעצב את הממדים כראוי של רשת microchannel ולהחיל את לחץ הגזירה הרצוי, מודל מספרים נבנה עם תוכנה הידרודינמיקה חישובית להעריך את פרופיל הזרימה מקרוב. תאי אנדותל טבור וריד אדם מן המעלה הראשון (HUVECs) כי היו זורעים לתוך microchannels הגיע למפגש, כלומר כיסו את המשטחים הפנימיים כולו של microchannel, בתוך 3-4 ימים עם זלוף הרציף. היווצרות המחסום הנאותה הודגמה על ידי מכתים VE-cadherin בהשוואה לאלו שנוצרו בתנאי תרבית תאי סטטי ב microvessels ללא פגע. על ידי יישום פרוטוקולי הניסוי שפותח microvessels שלמים perfused בנפרד 6-8, אנחנו כמותית למדוד את השינויים EC [Ca 2 +] i ו תחמוצת החנקן (NO) בתגובה אדנוזין אדנוזין (ATP) עם ניאוןdicators ו confocal מיקרוסקופ פלואורסצנטי קונבנציונאלי. העליות נגרמות אגוניסט ב EC [Ca 2 +] i ו NO ייצור דווחו כאותות תאיים הכרחיים עקב השפעות מתווך דלקת בחדירות microvessel 6-15. למרות שמחקרים קודמים הראו כמה תמונות של DAF-2 DA מכשירי microfluidic טעונים 16,17, ניתוח ברזולוציה ונתונים המתאים טרם השיג 18. למיטב ידיעתנו, מחקר זה מדגים את מדידות כמותיות הראשונות של שינוי הדינמי הנגרמת אגוניסט ב האנדותל [Ca 2 +] i ו NO microfluidic ניצול ייצור מערכת מבוססת. יש טכניקות microfabrication את הגמישות לפברק למטה microchannels כדי מיקרונים אחדים ולאפשר פיתוח של תבניות מורכבות לחקות את הגיאומטריות של microvasculature in vivo. כאן הצגנו רשת microchannel טיפוסית עם שלוש רמות של הסתעפות. זֶהרשת הוא מפוברק על ידי השילוב של photolithography אשר מתבצע חדר נקי microfabrication ו ליתוגרפיה רכה.

<table><tbody><tr><td>ATP</td><td>Sigma-Aldrich</td><td>A2383</td><td></td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A929</td><td></td></tr><tr><td>Biopsy punch</td><td>Miltex</td><td>33-31 AA</td><td></td></tr><tr><td>Bovine Albumin</td><td>MP Biomedicals</td><td>810014</td><td></td></tr><tr><td>Bovine Brain Extract (BBE)</td><td>Lonza</td><td>CC-4098</td><td></td></tr><tr><td>Cover-slip</td><td>Fisher Scientific</td><td>12-542C</td><td></td></tr><tr><td>DAF-2 DA</td><td>Calbiochem</td><td>251505</td><td></td></tr><tr><td>Dextran</td><td>Sigma-Aldrich</td><td>31390</td><td></td></tr><tr><td>Donkey anti-Goat IgG (H+L) Secondary Antibody</td><td>Life technologies</td><td>A-11055</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Gibco</td><td>14190-250</td><td></td></tr><tr><td>DRAQ5 (nuclei staining)&amp;nbsp;</td><td>Cell Signaling Technology</td><td>4084</td><td></td></tr><tr><td>Endothelial Cell Growth Supplement (ECGS)</td><td>Sigma-Aldrich</td><td>E2759-15MG</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>Fibronectin</td><td>Gibco</td><td>PHE0023</td><td></td></tr><tr><td>Fluo-4 AM</td><td>Life technologies</td><td>F-14201</td><td></td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma-Aldrich</td><td>G1890-100G</td><td></td></tr><tr><td>Gentamicin (50 mg/ml)</td><td>Gibco</td><td>15750-078</td><td></td></tr><tr><td>Glass coverslip</td><td>Fisher Scientific</td><td>12-548B</td><td></td></tr><tr><td>Glass Pasteur pippette</td><td>VWR</td><td>14672</td><td></td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa</td><td>Sigma-Aldrich</td><td>H3393-10KU</td><td></td></tr><tr><td>HEPES Buffered Saline Solution</td><td>Lonza</td><td>CC-5024</td><td></td></tr><tr><td>Human umbilical vein endothelial cells (HUVECs)</td><td>Lonza</td><td>CC-2517</td><td></td></tr><tr><td>Isopropyl alcohol (IPA)</td><td>VWR</td><td>89125</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>Mammalian Ringer Solution Ingredient</td><td></td><td></td><td></td></tr><tr><td>NaCl (132 mM)</td><td>Fisher Scientific</td><td>S671-3</td><td></td></tr><tr><td>KCl (4.6 mM)</td><td>Fisher Scientific</td><td>P217-500</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; &amp;middot; 2H&lt;sub&gt;2&lt;/sub&gt;O (2.0 mM)</td><td>Fisher Scientific</td><td>C79-500</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt; &amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O (1.2 mM)</td><td>Fisher Scientific</td><td>M63-500</td><td></td></tr><tr><td>Glucose (5.5 mM)</td><td>Fisher Scientific</td><td>BP350-1</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt; (5.0 mM)</td><td>Fisher Scientific</td><td>S233-500</td><td></td></tr><tr><td>Hepes Salt (9.1 mM)</td><td>Research Organics</td><td>6007H</td><td></td></tr><tr><td>Hepes Acid (10.9 mM)</td><td>Research Organics</td><td>6003H</td><td></td></tr><tr><td>MCDB 131 Culture Medium</td><td>Life technologies</td><td>10372-019</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Phalloidin (F-actin staining)</td><td>Sigma-Aldrich</td><td>P1951</td><td></td></tr><tr><td>Phosphate Buffered Saline&amp;nbsp;</td><td>Life technologies</td><td>14040-133</td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning Corporation</td><td>Sylgard 184</td><td></td></tr><tr><td>Scalpel</td><td>Exel Int</td><td>29552</td><td></td></tr><tr><td>Scotch tape</td><td>3M</td><td>34-8711-3070-3</td><td></td></tr><tr><td>Silicon wafer</td><td>VWR</td><td>14672</td><td></td></tr><tr><td>SU-8 photoresist</td><td>MicroChem</td><td>SU-8 2050 Y111072</td><td></td></tr><tr><td>SU-8 developer</td><td>MicroChem</td><td>Y020100</td><td></td></tr><tr><td>tissue culture flasks</td><td>Sigma-Aldrich</td><td>Z707503-100EA</td><td></td></tr><tr><td>Triton X-100</td><td>Chemical Book</td><td>T6878</td><td></td></tr><tr><td>Trypsin Neutralizer solution</td><td>Gibco</td><td>R-002-100</td><td></td></tr><tr><td>Trypsin/EDTA Solution (TE)</td><td>Gibco</td><td>R-001-100</td><td></td></tr><tr><td>Tubing</td><td>Cole-Parmer</td><td>PTFE microbore tubing, 0.012&quot; ID x 0.030&quot; OD</td><td></td></tr><tr><td>VE-cadherin</td><td>Santa Cruz Biotechnology</td><td>SC-6458</td><td></td></tr><tr><td>&lt;strong&gt;Name of Equipment&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>Biosafety Laminar hood</td><td>NuAire</td><td>NU-425 Class II, Type A2</td><td></td></tr><tr><td>CCD camera</td><td>Hamamatsu</td><td>ORCA</td><td></td></tr><tr><td>Confocal microscope</td><td>Leica</td><td>TCS SL</td><td></td></tr><tr><td>Desiccator</td><td>Bel-Art</td><td>F42022</td><td></td></tr><tr><td>Hotplate</td><td>Wenesco</td><td>HP-1212</td><td></td></tr><tr><td>Incubator</td><td>Forma Scientific</td><td>3110</td><td></td></tr><tr><td>Isotemp oven</td><td>Barnstead</td><td>3608-5</td><td></td></tr><tr><td>Lithography bench</td><td>Karl Suss</td><td>MA6 Contact Lithography</td><td></td></tr><tr><td>Optical microscope</td><td>Nikon</td><td>L200 ND &amp;amp; Diaphod 300</td><td></td></tr><tr><td>Shutter for the CCD camera</td><td>Sutter Instrument</td><td>Lambda 10-2</td><td></td></tr><tr><td>Plasma cleaner</td><td>PVA TePla/Harrick plasma</td><td>M4L/PDC-32G</td><td></td></tr><tr><td>Spin coater</td><td>Brewer Science</td><td>Cee 200X</td><td></td></tr><tr><td>Syringe pump system</td><td>Harvard Apparatus</td><td>703005</td><td></td></tr><tr><td>&lt;strong&gt;Name of Software&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>CAD software</td><td>Autodesk</td><td>AutoCAD 2015</td><td></td></tr><tr><td>CFD simulation software</td><td>COMSOL</td><td>COMSOL Multiphysics 4.0.0.982</td><td></td></tr><tr><td>Images acquire and analyse for NO production&amp;nbsp;</td><td>Universal Imaging</td><td>Metafluor</td><td></td></tr></tbody></table>

development and characterization of in vitro microvessel network and quantitative measurements of endothelial [ca2+]i and nitric oxide production

תאי האנדותל (EC) המצפים את דפנות כלי דם vivo חשופים כל זמן לזרום, אבל ECS בתרבית גדלה לעתים קרובות בתנאי סטטיים תערוכת פנוטיפ פרו-דלקתי. למרות הפיתוח של מכשירי microfluidic כבר אומץ על ידי מהנדסים למעלה משני עשורים, היישומים הביולוגיים שלהם יישארו מוגבלים. מודל microvessel במבחנה רלוונטי יותר פיסיולוגי אומת על ידי יישומים ביולוגיים חשוב לקדם את התחום ולגשר על הפערים בין in vivo ו במבחנה. כאן, אנו מציגים נהלים מפורטים לפיתוח רשת microvessel בתרבית באמצעות מכשיר microfluidic עם יכולת זלוף לטווח ארוך. אנחנו גם מדגימים ושימושיה מדידות כמותיות של שינויים המושרה אגוניסט ב EC [Ca 2 +] i ו תחמוצת החנקן (NO) בזמן אמת באמצעות confocal מיקרוסקופ פלואורסצנטי קונבנציונאלי. רשת microvessel נוצרהעבודה עם זלוף הרציף הראתה צמתים מפותחות בין ECS. VE-cadherin הפצה היה קרוב יותר לזה שנצפה microvessels השלם מ monolayers EC בתרבית באופן סטטי. ATP-induced עלייה זמנית EC [Ca 2 +] i ו NO ייצור נמדדו כמותית ברמות תאים בודדות, אשר תוקפות את הפונקציונליות של microvessels התרבותי. מכשיר microfluidic זה מאפשר ECS לגדול תחת זרם רלוונטי מבוקר היטב, מבחינה פיזיולוגית, מה שהופך את סביבת תרבית תאים קרוב in vivo מזה בתרבויות 2D הקונבנציונליות, סטטי. עיצוב רשת microchannel הוא תכליתי מאוד, ותהליך הייצור הוא פשוט דיר. המכשיר ניתן לשלב בקלות למערכת המיקרוסקופית confocal או קונבנציונלית המאפשרת הדמיה ברזולוציה גבוהה. והכי חשוב, כי רשת microvessel התרבותית יכולה להיוצר על ידי ECS אדם מן המעלה הראשונה, גישה זו תשמש ככלי שימושי כדי לחקור כיצדפתולוגית מרכיבי דם שינו ממדגמים חולים להשפיע ECS אדם ולספק תובנה בנושאים קליניים. כמו כן ניתן לפתח כפלטפורמת הקרנת סמים.

סעיף זה מציג כמה מהתוצאות שהושגו עם רשת microvessel בתרבית שפותחה עם פרוטוקול זה. דפוס microchannel הוא רשת הסתעפות שלוש רמות (איור 1 א). בתכנון זה, סניפי ערוץ אמא רחבים 159 מיקרומטר לשתי 126 מיקרומטר ערוצים רחבים, וענפים שוב לארבעה 100 מיקרומטר ערוצי בת רחבי...

Watch this Scientific Journal Video about Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production at JoVE.com

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

פיתוח ואפיון של<em&gt; במבחנה</em&gt; כלי דם קטן רשת מדידות כמותיות של האנדותל [Ca<sup&gt; 2 +</sup&gt;]<sub&gt; i</sub&gt; וייצור Nitric Oxide

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

development-characterization-vitro-microvessel-network-quantitative

Research

JoVE Journal

Biology

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

8.5K Views.  College of Medicine, Penn State University. Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Video: Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

מיקרופלואידיקה Endothelialized לחקר אינטראקציות כלי הדם במחלות המטולוגיות

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Bioengineering

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune cells, and the coordination with lymphatic vessels and nerves. The multi-cell, multi-system interactions necessitate the investigation of angiogenesis in a physiologically relevant environment. Thus, while the use of in vitro cell-culture models have provided mechanistic insights, a common critique is that they do not recapitulate the complexity associated with a microvascular network. The objective of this protocol is to demonstrate the ability to make time-lapse comparisons of intact microvascular networks before and after angiogenesis stimulation in cultured rat mesentery tissues. Cultured tissues contain microvascular networks that maintain their hierarchy. Immunohistochemical labeling confirms the presence of endothelial cells, smooth muscle cells, pericytes, blood vessels and lymphatic vessels. In addition, labeling tissues with BSI-lectin enables time-lapse comparison of local network regions before and after serum or growth factor stimulation characterized by increased capillary sprouting and vessel density. In comparison to common cell culture models, this method provides a tool for endothelial cell lineage studies and tissue specific angiogenic drug evaluation in physiologically relevant microvascular networks.

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

<em&gt; Ex Vivo</em&gt; שיטת הדמיה זמן לשגות של רשתות כלי הדם Mesenteric עכברוש מתורבתות

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

Developmental Biology

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

הליך הפיתוח של Multi-עגול עומק חתך Endothelialized microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis has become a central readout of endothelial progenitor cell functionality, and therefore, several attempts have been made to improve both in vitro and in vivo vasculogenesis models. However, standard methods are limited in scope, with static measurements failing to capture many aspects of this highly dynamic process. Therefore, the goal of developing this novel protocol was to assess the kinetics of in vitro vasculogenesis in order to quantitate rates of network formation and stabilization, as well as provide insight into potential mechanisms underlying vascular dysfunction. Application of this protocol is demonstrated using fetal endothelial colony forming cells (ECFCs) exposed to maternal diabetes mellitus. Fetal ECFCs were derived from umbilical cord blood following birth, cultured, and plated in slides containing basement membrane matrix, where they underwent vasculogenesis. Images of the entire slide wells were acquired using time-lapse phase contrast microscopy over 15 hours. Images were analyzed for derivation of quantitative data using an analysis software called Kinetic Analysis of Vasculogenesis (KAV). KAV uses image segmentation followed by skeletonization to analyze network components from stacks of multi-time point phase contrast images to derive ten parameters (9 measured, 1 calculated) of network structure including: closed networks, network areas, nodes, branches, total branch length, average branch length, triple-branched nodes, quad-branched nodes, network structures, and the branch to node ratio. Application of this protocol identified altered rates of vasculogenesis in ECFCs obtained from pregnancies complicated by diabetes mellitus. However, this technique has broad implications beyond the scope reported here. Implementation of this approach will enhance mechanistic assessment and improve functional readouts of vasculogenesis and other biologically important branching processes in numerous cell types or disease states.

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Here, we present a protocol for time-lapse imaging and analysis of vasculogenesis in vitro using phase contrast microscopy coupled with the open source software, Kinetic Analysis of Vasculogenesis. This protocol can be applied to quantitatively assess the vasculogenic potential of numerous cell types or experimental conditions that model vascular disease.

ניתוח קינטי של Vasculogenesis מכמתת את הדינמיקה של Vasculogenesis ואנגיוגנזה במבחנה

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of ...

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

מודל תלת מימד וצינור חישוב מתורבת לכמת את הפוטנציאל הוושלתי של iPSC-נגזר של התגובה האנטי-מגנטית

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

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 ...

התמיינות וגירוי מכני של תאי אנדותל שמקורם ב-hiPSC

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides transporting oxygen and nutrients to ensure fetal growth, fetal circulation controls many crucial developmental events taking place within the endothelial layer through mechanical cues. Biomechanical signals induce blood vessel structural changes, establish arteriovenous specification, and control the development of hematopoietic stem cells. The inaccessibility of the developing tissues limits the understanding of the role of circulation in early human development; therefore, in vitro models are pivotal tools for the study of vessel mechanobiology. This paper describes a protocol to differentiate endothelial cells from human induced pluripotent stem cells and their subsequent seeding into a fluidic device to study their response to mechanical cues. This approach allows for long-term culture of endothelial cells under mechanical stimulation followed by&#160;retrieval of the endothelial cells for phenotypical and functional characterization. The in vitro model established here will be instrumental to elucidate&#160;the intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.

מחבר זרקור: חקר תאי אנדותל שמקורם ב-hiPSC בתרבית תחת גירוי מכני בתיווך פלואיד 

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.

במבחנה מודל של כלי דם אנושיים עובריים על שבב לחקר מכנוביולוגיה התפתחותית

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Assay היווצרות צינור תא האנדותל ל<em&gt; במבחנה</em&gt; חקר אנגיוגנזה

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

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מיקרופלואידיקה Endothelialized לחקר אינטראקציות כלי הדם במחלות המטולוגיות

הליך הפיתוח של Multi-עגול עומק חתך Endothelialized microchannels-on-a-chip

ניתוח קינטי של Vasculogenesis מכמתת את הדינמיקה של Vasculogenesis ואנגיוגנזה במבחנה

מודל תלת מימד וצינור חישוב מתורבת לכמת את הפוטנציאל הוושלתי של iPSC-נגזר של התגובה האנטי-מגנטית

Micropatterning והרכבת 3D microvessels

במבחנה מודל של כלי דם אנושיים עובריים על שבב לחקר מכנוביולוגיה התפתחותית

במבחנה מודל של כלי דם אנושיים עובריים על שבב לחקר מכנוביולוגיה התפתחותית

במבחנה מודל של כלי דם אנושיים עובריים על שבב לחקר מכנוביולוגיה התפתחותית

Assay היווצרות צינור תא האנדותל ל