This study was supported by a Singapore National Research Foundation (NRF) Grant NRF2012NRF-POC001-043.

1. ייצור Photomask <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> נקה רקיק זכוכית 4 &quot;עם פתרון פיראניה (H 2 SO 4 / H 2 O 2 ב 2: 1 יחס) במשך 20 דקות ב 120 מעלות צלזיוס על ידי טבילה בטנק קוורץ. </li><li style=";text-align:right;direction:rtl"> להפקיד רובד של כרום / זהב (30 ננומטר של Cr / 1 מיקרומטר של Au) שכבה על פרוסות סיליקון הזכוכית באמצעות מאייד 7 (איור 1 א) קורה אלקטרוני. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את הפרוסות במאייד קורה אלקטרוני. ברגע שהאבק מגיע 5 x 10 -6 Torr, להפעיל את מקור מתח הגבוה (10 קילו וולט). לשלוט בעובי של לוח בקרת צג. </li><li style=";text-align:right;direction:rtl"> טרום נקי כל חומר במשך 30 שניות באמצעות אקדח הקורה האלקטרוני, שמירה על התריס &quot;OFF&quot; (על מנת להימנע מהתצהיר על הוופלים). </li></ol></li><li style=";text-align:right;direction:rtl"> צור שכבת מיסוך photoresist Cr / Au לתחריט רטוב עמוק של הזכוכית. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> החל photoresist עבה 2 מיקרומטר ידי ספינינג 5 מיליליטר של הפתרון למשך 30 שניות בסל&quot;ד 3000 באמצעות מערכת coater ספין-ספריי.Prebake photoresist על פלטה חמה ב 100 מעלות צלזיוס במשך 1.5 דקות. </li><li style=";text-align:right;direction:rtl"> לחשוף וקשה לאפות photoresist ב 120 מעלות צלזיוס במשך 30 דקות על פלטה חשמלית. זה קריטי כדי ליצור משטח הידרופובי והדבקה חזקה של photoresist לשכבת המתכת. תבנית שכבת Cr / Au באמצעות etchants Cr וAu דרך photoresist להסוות 8,9. </li></ol></li><li style=";text-align:right;direction:rtl"> להגנה על משטח הזכוכית בדוגמת האו&quot;ם, באופן זמני אג&quot;ח רקיק הזכוכית לפרוסות סיליקון דמה. 9 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את פרוסות זכוכית על צלחת חמה ב 110 מעלות צלזיוס ולהמס את השעווה בצד השני של פרוסות סיליקון הזכוכית (באופן כזה שכל פני השטח של הרקיק מכוסה בשעווה). </li><li style=";text-align:right;direction:rtl"> מניחים פרוסות סיליקון דמה במגע עם רקיק הזכוכית ולחץ כדי להסיר את השעווה העודפת. על מנת להימנע משפיכת השעווה, למקם את נייר טישו חדר נקי על פלטה חשמלית. </li></ol></li><li style=";text-align:right;direction:rtl"> בצע תחריט איזוטרופיים של העדשה באמצעות חומצה הידרופלואורית מותאמת (49% V / V)פתרון וחומצה הידרוכלורית (37% V / V) (ביחס נפח של 10: 1). עם בוחש מגנטי עבור 8.5 דקות 10 נוכחות של HCl היא קריטית בהשגת איכות משטח טובה של עדשות שנוצרו. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ודא כי שיעור התחריט הוא 7 מיקרומטר / min; באמצעות נפח כולל של 200 מיליליטר של תמיסת תחריט. בצע תחריט במכל פלסטיק ולקחת אמצעי בטיחות לצעד זה עיבוד. </li><li style=";text-align:right;direction:rtl"> נקה את הרקיק במים ללא יונים (DI) על ידי שטיפה וייבוש נוסף ב RT. </li></ol></li><li style=";text-align:right;direction:rtl"> לאחר השלימה התהליך, להפריד את רקיק הזכוכית מפרוסות סיליקון דמה ולחמם את השעווה בעזרת צלחת חמה ב 100 מעלות צלזיוס במשך 15 שניות. כשעווה נמסה בטמפרטורה זו, לנתק את רקיק הזכוכית מפרוסות סיליקון דמה. </li><li style=";text-align:right;direction:rtl"> הסר שעווה שנותרה, photoresist ושכבות Cr / Au התלויים בקצוות של העדשות באמצעות ultrasonication עבור שעה 1 באמצעות -methyl-2-pyrrolidone N כממס על 80 מעלות צלזיוס בטנק קולי.</li><li style=";text-align:right;direction:rtl"> צור העתק עובש PDMS של microlenses המפוברק על photomasks 11. </li><li style=";text-align:right;direction:rtl"> לאפיין את ממדי photomask (אורך ורוחב) ועובש PDMS microlenses (עומק וקוטר) העתקים באמצעות מיקרוסקופ אלקטרונים סורק וסטראו בהתאמה. 12-14 </li></ol> ייצור 2. גלים MN <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צור חלל של 2.5 סנטימטר × 0.9 סנטימטר באמצעות שקופיות הזכוכית רכובה על שני צדדים של זכוכית. מספר שקופיות הזכוכית שנערמו על שני הצדדים יקבע את הגובה של החלל הידוע כעובי spacer (איור 1). </li><li style=";text-align:right;direction:rtl"> Secure כל שכבה של שקופיות זכוכית על ידי יישום שכבה דקה של פתרון prepolymer המכיל פולי (אתילן גליקול) diacrylate (PEGDA, MW = 258 Da) עם 0.5% w / w 2-2-מתיל-propiophenone הידרוקסי (HMP) על גבי שקופיות זכוכית ואחרי הקרנה של האור להגדיר עם אולטרה סגול בעוצמה הגבוהה (UV) למשך 2 שניות. </li><li style=";text-align:right;direction:rtl"> מקם את photoma<html> SK (מפוברק בעבר) עם משטחי Cr / Au המצופה מול הפנים של החלל. ודא שהצדדים של קירות החלל לא מסתירים את העדשות משובצות בphotomask. </li><li style=";text-align:right;direction:rtl"> למלא את החלל עם פתרון prepolymer עד המשטח המצופה כרום / Au הוא במגע עם הפתרון ללא כל בועות גלויות. </li><li style=";text-align:right;direction:rtl"> מכשיר את ההתקנה עם אור UV בעוצמה גבוהה של אינטנסיביות רצויה עבור 1 שניות במרחק של 3.5 סנטימטרים ממקור UV באמצעות תחנת ריפוי UV עם מגוון מסנן UV של 320-500 ננומטר. השתמש במתאם collimating עם חללית אור UV. </li><li style=";text-align:right;direction:rtl"> מדוד את עוצמת אור UV משמשת באמצעות מד קרינה. </li><li style=";text-align:right;direction:rtl"> בעקבות חשיפה לקרינת UV, להסיר את photomask עם המערך של MNS. יוצקים את פתרון prepolymer העודף שאינו polymerized בתהליך חזרה למכל המקורי שלה לשימוש חוזר. </li><li style=";text-align:right;direction:rtl"> לכמת את קוטר אורך וקצה MNS באמצעות סטראו לפי הוראות יצרן. </li></ol>class = &quot;jove_title&quot;&gt; 3. ייצור שכבת גיבוי MN <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עם מלקחיים, למקם את MNS (מפוברק בעבר) מצורף אל photomask בבאר של צלחת 24 גם כפי שמוצג באיור 1 ג. </li><li style=";text-align:right;direction:rtl"> הוספת נפח שצוין (300-400 μl) של פתרון prepolymer לתוך הבאר עד המחטים שקועות עד לגובה רצוי. כרך זה קובע את עובי שכבת גיבוי התוצאה. </li><li style=";text-align:right;direction:rtl"> מכשיר את ההתקנה עם אור בעוצמה גבוהה UV (15.1 W / 2 סנטימטר), 10.5 סנטימטרים ממקור UV למשך תקופה של 1 שניות. </li><li style=";text-align:right;direction:rtl"> הפרד את שכבת הגיבוי על מערך MN מphotomask באמצעות סכין חד. </li><li style=";text-align:right;direction:rtl"> לכמת את קוטר אורך, קוטר קצה ובסיס של MNS עם שכבת הגיבוי באמצעות סטראו לפי הוראות יצרן. </li></ol>

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The authors declare no conflict of Interest.

הפרוטוקול שתואר לעיל עבור ייצור של מערך MNS הוצג לפברק מערך MNS של ~ 1 סנטימטר 2. ניתן לשנותם על ידי יצירת מערכי חלל גודל גדול ובאמצעות photomask גדול יותר. גודל החלל המוגבר ניתן ליצור על ידי הגדלת הרוחב בין מפרידי שני צדדים. למרות שכל צעד לפברק מערכי MN בפרוטוקול היה חשוב, ...

העברת תרופות דרך העור מציעה גישה חלופית אטרקטיבית למתן תרופה, במיוחד למולקולות ביולוגיות, המנוהלים באופן כמעט בלעדי על ידי זריקות מזרק. עם זאת, העור, במיוחד את השכבה העליונה (השכבה הקרנית), הוא מחסום אדיר מניעת מולקולות אקסוגניים מלהיכנס לגוף האדם. לאחרונה, מכשירי MN צמחו כמאפשרים כלים כדי לספק תרופות דרך עור. מכשירי MN ליצור נקבוביות זמני בתוך השכבה הקרנית לאפשר המעבר של מולקולות תרופה כדי להשיג את הפעילות הפיזיולוגית הרצויה עם תאימות מטופל השתפרה ונוחות 1-3. שיטות ייצור שונות אומצו לפברק MNS פולימריים 4. עם זאת, הם בדרך כלל כרוכים בתהליכים מסובכים ומרובים צעד דורשים פעמים רבות ו / או טמפרטורות גבוהות לפברק מערכי MNS. 4 כדי לפשט את תהליך הייצור, באמצעות תהליך ללא עובש צעד אחדphotomask פותח לאחרונה 5,6. עם זאת, בשיטה זו, מפוברק היה MNS טיפים מחט בוטים, כפי שאין מנגנון היה במקום לשנות את נתיב אור אולטרה סגול (UV) המעורב בphotolithography. במחקר זה, microlenses המוטבע בphotomask הוצעו להגדיר את הגיאומטריה של MNS. הפרוטוקול לפברק photomasks בהיקף של microlenses המשובץ ולאחר מכן MN ייצור עם טיפים חדים באמצעות photomask מדווחים.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Poly(ethylene glycol) diacrylate (PEGDA Mn=258)</td>
			<td>SIGMA&#160;</td>
			<td>475629-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>2-hydroxy-2-methyl-propiophenone (HMP)</td>
			<td>SIGMA&#160;</td>
			<td>405655-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine collagen type 1, FITC conjugate&#160;</td>
			<td>SIGMA&#160;</td>
			<td>C4361</td>
			<td></td>
		</tr>
		<tr>
			<td>UV curing station&#160;&#160;&#160;</td>
			<td>EXFO Photonic Solutions Inc., Canada</td>
			<td>OmniCure S200-XL</td>
			<td></td>
		</tr>
		<tr>
			<td>Collimating Adaptor&#160;</td>
			<td>EXFO Photonic Solutions Inc., Canada</td>
			<td>EXFO 810-00042</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon SMZ 1500 stereomicroscope&#160;</td>
			<td>Nikon, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dillon GL-500 digital force gauge&#160;</td>
			<td>Dillon, USA</td>
			<td></td>
			<td></td>
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polymeric microneedle array fabrication by photolithography

כתב יד זה מתאר את הייצור של מערכי microneedle פולימרים (MN) על ידי photolithography. זה כרוך בתהליך ללא עובש פשוט באמצעות photomask המורכב ממייקרו-עדשות מוטבעות. מיקרו-עדשות משובצות נמצאו להשפיע גיאומטריה MN (חדות). מערכי MN חזקים בקטרים ​​הנעים בין קצה 41.5 מיקרומטר ± 8.4 מיקרומטר ו71.6 מיקרומטר ± 13.7 מיקרומטר, עם שני אורכים שונים (1,336 מיקרומטר ± 193 מיקרומטר ומיקרומטר 957 ± 171 מיקרומטר) היו מפוברקים. מערכי MN אלה עשויים לספק יישומים פוטנציאליים באספקה ​​של תרופות מולקולריות וmacromolecular נמוכות דרך עור.

הגיאומטריה של MNS יכולה להיות מושפעת באופן משמעותי על ידי מאפייני photomask וmicrolens המוטבעים. התואר של שבירה משפיע על נתיב העברת קרני UV, שהשפיעה על הגיאומטריה MN (איור 2 א). כל microlens נמצא כי קוטר 350 מיקרומטר, 130 מיקרומטר שיטח משטח קמור, ו62.3 מיקרומטר עומק (איור 2-D).</strong...

Watch this Scientific Journal Video about Polymeric Microneedle Array Fabrication by Photolithography at JoVE.com

Polymeric Microneedle Array Fabrication by Photolithography

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

ייצור מערך microneedle פולימרית יתוגרפיה

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

polymeric-microneedle-array-fabrication-by-photolithography

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

12.0K Views.  National University of Singapore. Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography. 

Video: Polymeric Microneedle Array Fabrication by Photolithography

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 ...

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

גיבוש חלקיקים פולימריים Diblock באמצעות טכניקה Nanoprecipitation

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

חיישן הולו Microneedle מבוסס על מרובב דרך העור אלקטרוכימי חישה

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

הערכת משלוח גן פולימרית חלקיקים על ידי Nanoparticle מעקב ניתוח וזרימת cytometry תפוקה גבוהה

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

תאי גזע תכנות טיפולי אנגיוגנזה שימוש אורגניים פולימריים חלקיקים

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

ייצור מיקרופלואידי של סיבים פולימריים וביוהיברידים עם גודל וצורה מעוצבים מראש

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

ייצור Biomembrane ידי השומנים bilayer (SALB) השיטה בסיוע מרכך

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

בודה חומרים פולימריים עבור יישומים ביו-הידרופובי

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

פוטו-ליתוגרפיה גובה רב שלבי משתנה עבור Valved רב שכבתי מכשירים microfluidic

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

ייצור של מערך מרובבת MicroEnvironment הסלולר מלאכותי

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...