Name: microfluidic genipin deposition technique for extended culture of micropatterned vascular muscular thin films
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Description: Watch this Scientific Journal Video about Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films at JoVE.com

We acknowledge financial support from the American Heart Association Scientist Development Grant, 13SDG14670062 (PWA) and the University of Minnesota Doctoral Dissertation Fellowship (ESH). We also acknowledge the microfabrication resources of the Minnesota Nano Center (MNC) and the image processing resources of the University Imaging Centers (UIC), both at the University of Minnesota. Parts of this work were carried out in the Characterization Facility, University of Minnesota, which receives partial support from NSF through the MRS program.

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The authors have nothing to disclose.

כאן, אנו מציגים פרוטוקול שבונה על טכנולוגיה שפותחה בעבר vMTF, מאפשרים פעמים ניסוי מורחבות טיפוסיות יותר של מסלולי מחלת כלי דם כרוניים 1,23,24. כדי להשיג זאת, אנו micropattern genipin, אשר בעבר הוכח לספק functionalization לטווח הארוך של מצעי PDMS 11, תוך שימוש בטכניקה בתצהיר microfluidic ל?...

מחלות כלי דם, כגון vasospasm המוחית 1,2, יתר לחץ דם 3, וטרשת עורקת 4, לפתח לאט, הן בדרך כלל כרוניים בטבע, וכרוכים בכוח-דור מתפקד על ידי תאי שריר חלק של כלי דם (VSMCs). אנו שואפים ללמוד בתפקוד אלה איטיים מתקדמים של כלי הדם באמצעות בשיטות מבחנה עם שליטה עדינה יותר של תנאי ניסוי מאשר בin vivo מודלים. סרטים שפותחנו בעבר בכלי דם שרירי דק (vMTFs) למדידת התכווצות פונקציונלית של רקמות מהונדסות במבחנה לב וכלי דם 5, אך שיטה זו הוגבלה למחקרים יחסית לטווח קצר. כאן, אנו מציגים טכניקת שינוי מצע שמתרחבת טכניקת vMTF הקודמת שלנו למדידות לטווח ארוך. בעוד האנדותל הוא גם קריטי בתפקוד כלי דם כללי, lamellae עורקים מהונדסים לספק מערכת מודל שימושית להערכת שינויים בכלי דםהתכווצות במהלך התקדמות מחלה. להנדס מודל פונקציונלי של כלי דם ברקמת מחלה, גם את המבנה ותפקוד של lamella העורקים, יחידת ההתכווצות הבסיסית של כלי השיט, יש סכמה עם איכות גבוהה. lamellae עורקים הם גיליונות קונצנטריים, circumferentially מיושר של VSMCs ההתכווצות מופרדת על ידי גיליונות של אלסטין 6. הדפסת microcontact של חלבוני מטריצה ​​תאית (ECM) על גבי מצעי polydimethylsiloxane (PDMS) כבר השתמשה בעבר כדי לספק רמזי הדרכה לארגון רקמה לחקות מיושרים רקמת לב וכלי דם 5,7-10. עם זאת, רקמות דוגמת באמצעות הדפסת microcontact יכול לאבד יושרה לאחר 3-4 ימים בתרבות, הגבלת תחולתם בלימודים כרוניים. פרוטוקול זה מספק פתרון לבעיה זו על ידי החלפת טכניקות הדפסת microcontact קודמות עם טכניקה בתצהיר microfluidic חדשה. Genchi et al. מצעי PDMS שונה עם genipin וFound ממושך כדאיות של myocytes עד חודש בתרבות 11. כאן, אנו משתמשים בגישה דומה להארכת תרבות של תאי שריר בדוגמת כלי דם חלקים על PDMS. Genipin, נגזר hydrolytic טבעית של הפירות גרדניה, הוא מועמד רצוי לשינוי מצע בשל רעילותה הנמוכה יחסית בהשוואה לסוכני crosslinking דומים והשימוש הגובר שלה כביולוגי בתחומי תיקון רקמות 12,13 ושינוי ECM 14, 15. בפרוטוקול זה, פיברונקטין הוא מנוצל כרמז הדרכת תא, כמו בשיטות הדפסת microcontact קודמות; עם זאת, genipin מופקד על גבי מצעים לפני דפוסי פיברונקטין PDMS. לפיכך, כתאים לבזות את המטריצה ​​בדוגמת, ECM מסונתז חדש מVSMCs המצורף יכול להיקשר למצע PDMS המצופה genipin. פרוטוקול זה משתמש בהתקן משלוח microfluidic לgenipin שני שלבים ותצהיר ECM. העיצוב של microco מחקה מכשיר microfluidicדפוסי הדפסת ntact משמשים לlamellae עורקים מהונדסים במחקרים קודמים 16. לפיכך, אנו מצפים פרוטוקול זה להניב מחקה lamellae עורקים שבהצלחה לשחזר מזדהים ביותר במבנה ותפקוד vivo התכווצות של lamellae עורקים. אנו גם מעריכים התכווצות רקמות כדי לאשר genipin שהיא תרכובת שינוי המצע מתאימה לטווח ארוך בדגמי מחלת כלי דם במבחנה.

<table><tbody><tr><td>Coverslip staining rack</td><td>Electron Microscopy Sciences</td><td>&lt;a href=&quot;http://www.emsdiasum.com/&quot;&gt;www.emsdiasum.com/&lt;/a&gt;</td><td>72239-04</td></tr><tr><td>Microscope cover glass - 25 mm</td><td>Fisher Scientific, Inc.</td><td>&lt;a href=&quot;http://www.fishersci.com/&quot;&gt;www.fishersci.com&lt;/a&gt;</td><td>12-545-102</td></tr><tr><td>Poly(N-iso-propylacrylamide) (PIPAAm)</td><td>Polysciences, Inc.</td><td>&lt;a href=&quot;http://www.polysciences.com/&quot;&gt;www.polysciences.com/&lt;/a&gt;</td><td>#21458</td></tr><tr><td>1-butanol</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;www.sigmaaldrich.com&lt;/a&gt;</td><td>360465</td></tr><tr><td>Spincoater</td><td>Specialty Coating Systems, Inc.</td><td>&lt;a href=&quot;http://www.scscoatings.com/&quot;&gt;www.scscoatings.com&lt;/a&gt;</td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Ellsworth Adhesives (Dow Corning)</td><td>&lt;a href=&quot;http://www.ellsworth.com/&quot;&gt;www.ellsworth.com&lt;/a&gt;</td><td>184 SIL ELAST KIT 0.5KG</td></tr><tr><td>Fluorescent microbeads</td><td>Polysciences, Inc.</td><td>&lt;a href=&quot;http://www.polysciences.com/&quot;&gt;www.polysciences.com/&lt;/a&gt;</td><td>17151</td></tr><tr><td>Silicon wafers</td><td>Wafer World, Inc.</td><td>&lt;a href=&quot;http://www.waferworld.com/&quot;&gt;www.waferworld.com&lt;/a&gt;</td><td>2398</td></tr><tr><td>Photoresist&amp;nbsp;</td><td>MicroChem Corp.</td><td>&lt;a href=&quot;http://www.microchem.com/&quot;&gt;www.microchem.com&lt;/a&gt;</td><td></td></tr><tr><td>Contact mask aligner</td><td>Suss MicroTec</td><td>&lt;a href=&quot;http://www.suss.com/&quot;&gt;www.suss.com&lt;/a&gt;</td><td></td></tr><tr><td>Developer</td><td>MicroChem Corp.</td><td>&lt;a href=&quot;http://www.microchem.com/&quot;&gt;www.microchem.com&lt;/a&gt;</td><td></td></tr><tr><td>Tridecafluro-trichlorosilane</td><td>UCT Specialties, Inc.</td><td>&lt;a href=&quot;http://www.unitedchem.com/&quot;&gt;www.unitedchem.com&lt;/a&gt;</td><td>T2492</td></tr><tr><td>Surgical biopsy punch</td><td>Integra LifeSciences Corp.</td><td>&lt;a href=&quot;http://www.miltex.com/&quot;&gt;www.miltex.com&lt;/a&gt;</td><td>33-31AA-P/25</td></tr><tr><td>Genipin</td><td>Cayman Chemical</td><td>&lt;a href=&quot;http://www.caymanchem.com/&quot;&gt;www.caymanchem.com&lt;/a&gt;</td><td>10010622</td></tr><tr><td>1X phosphate buffered saline</td><td>Mediatech, Inc.</td><td>&lt;a href=&quot;http://www.cellgro.com/&quot;&gt;www.cellgro.com&lt;/a&gt;</td><td>21-031-CV</td></tr><tr><td>Fibronectin</td><td>Corning, Inc.</td><td>&lt;a href=&quot;http://www.corning.com/&quot;&gt;www.corning.com&lt;/a&gt;</td><td>356008</td></tr><tr><td>Penicillin/streptomycin</td><td>Life Technologies, Inc.</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/&quot;&gt;www.lifetechnologies.com&lt;/a&gt;</td><td>15140-122</td></tr><tr><td>Umbillical artery smooth muscle cells</td><td>Lonza</td><td>&lt;a href=&quot;http://www.lonza.com/&quot;&gt;www.lonza.com&lt;/a&gt;</td><td>CC-2579</td></tr><tr><td>Tyrode&#039;s solution components</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;www.sigmaaldrich.com&lt;/a&gt;</td><td>various</td></tr><tr><td>Stereomicroscope</td><td>Zeiss</td><td>&lt;a href=&quot;http://www.zeiss.com/&quot;&gt;www.zeiss.com&lt;/a&gt;</td><td>4350020000000000</td></tr><tr><td>Temperature-controlled platform</td><td>Warner Instruments</td><td>&lt;a href=&quot;http://www.warneronline.com/&quot;&gt;www.warneronline.com&lt;/a&gt;</td><td>641659; 640352; 641922</td></tr><tr><td>Endothelin-1</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;www.sigmaaldrich.com&lt;/a&gt;</td><td>E7764-50UG</td></tr><tr><td>HA-1077</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;www.sigmaaldrich.com&lt;/a&gt;</td><td>H139-10MG</td></tr></tbody></table>

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.

המטרה העיקרית של עבודה זו הייתה להאריך את הכדאיות של VSMCs micropatterned על מצעי PDMS הידרופובי. הדבר זה הושג על ידי שילוב מערכת מסירת microfluidic להפקיד genipin בדוגמת ופיברונקטין על PDMS (איור 1). בתצהיר של חלבוני ECM באמצעות משלוח microfluidic הניב העברת נאמנות גבוהה של דפוס הערוץ עם PD...

Watch this Scientific Journal Video about Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films at JoVE.com

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

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 ...

microfluidic-genipin-deposition-technique-for-extended-culture

Research

JoVE Journal

Bioengineering

7.6K Views.  University of Minnesota. 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.

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

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 ...

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

המצאה של הידרוג micropatterned למערכות התרבות עצבית באמצעות photolithography דינמי הקרנה מסכת

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

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 ...

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, ...

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.

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 ...

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 ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

תמונה מונחה, ייצור מבוסס-לייזר של רשתות Microfluidic הנגזרות דם

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

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 ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

רשת כלי הדם perfusable עם דגם רקמה במכשיר Microfluidic

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

מודל מיקרופלואידי לחקות אירוע ראשוני של ניאו-סקולריזציה

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...