Funding for this work, GTK, TK, and JDM were provided by the Wisconsin Institute for Discovery and the Wisconsin Alumni Research Foundation.

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The authors declare that they have no competing financial interest.

מצעים אידיאליים עבור הנדסת רקמות יהיו bioinspired ובכך לשחזר את ההתפלגות המרחבית של ביו הליגנדים קריטיים נמצאים בתוך הרקמות המקומיות. הם גם היו בעלי תכונות דינמיות המאפשרות התאמות זמניות של הליגנדים ודפוסי המרחב שבו הם מוצגים כדי לאפשר המורפוגנזה רקמה ביימה ומרחבית מוג?...

היכולת של משטחים מורכבים-PEG להציג הליגנדים ביוכימיים קוולנטית כרוכים בו-זמנית, תוך שמירה על מאפייני עכירות שאינן טבועים להפוך אותם בחירה אידיאלית עבור סביבות מיקרוסקופיות מותאמות אישית הנדסה על מצעי תרבות 1,2,3. אינטראקציות biospecific מתווכות על ידי מברשות PEG יגנד מצומדות מאפשרת ניתוח מצמצם של ההשפעות של יוכימיים רמזים שנמצאו בתוך מתחם במייקרו-סביבות רקמות vivo על פנוטיפים תא בודדים. יתר על כן, ניתן להשתמש chemistries &quot;לחץ&quot; ביו-מאונך כדי להקל על חוסר תנועה כיוונית של הליגנדים, כך שהם מוצגים בתצורות ילידי 4-6. כך, דפוסים מרחביים מיקרוסקופים של PEG מברשות הוא כלי תכליתי ליצירת מעצב בנישות מבחנה לחקור איתות תא הנגרמת על ידי רמזים ביוכימיים משותקים 6,7. שיטה נפוצה ליצירת תבניות המרחביות של מ&quot;ק ביוכימייםes כרוך מצעים מצופים זהב הדפסת microcontact (μCP) עם דפוסי alkanethiols מצומדות PEG. ואז, monolayers micropatterned העצמי התאספו (טילי הקרקע-האוויר) של alkanethiols ylated-PEG מגביל ספיחה פיזית של מולקולות ביוכימיות, למשל, חלבונים, רק לאזורים בדוגמת שאינה של המצע 8,9. עם זאת, טילי הקרקע-האוויר שנוצרו על ידי טכניקה זו הן רגישות לחמצון בתרבית תאי תקשורת לטווח ארוך. כך, μCP&#39;d alkanethiol טילי הקרקע-האוויר לעתים קרובות מורכב עוד יותר עם ​​מברשות פולימר PEG באמצעות פילמור הרדיקלי העברת אטום ביוזמת פני השטח (SI-ATRP) כדי להגביר את היציבות שאינה מזהמת את האזור 10. באופן ספציפי, μCP של יוזם פילמור alkanethiol, bromoisobutyrate ω-meraptoundecyl, על משטחים מצופים זהב ואחריו SI-ATRP של פולי (אתילן גליקול) methacrylate אתר מתיל (PEGMEMA) מונומרים יוצר משטחים עם טווח ארוך micropatterned, יציב, ושאינה עכירות PEG מברשות. יתר על כן, אלה<html> הם מסוגלים להיות שונה יותר להציג moieties הכימית מגוון 11. ניצול של נכס זה, שא et. אל. פיתח שיטה להנדס מצעי תרבות עם מברשות PEGMEMA multicomponent הצגת chemistries &quot;לחץ&quot; מאונך. בשיטה זו, הם משתמשים בסדרה של צעדי μCP / SI-ATRP ביניהם אזיד הנתרן רציף, ethanolamine, וpropargylamine החלפות nucleophilic ליצור מצעי תרבות מציגים דפוסים מיקרוסקופים של הליגנדים משותקים מרובים 6. בעוד שהפוטנציאל של שימוש chemistries כזה בשיתוף עם μCP מדריך ללהנדס מצעי תרבות רומן הוא עצום, הוא מוגבל על ידי הדיוק ואת הדיוק שבה ניתן ליישר צעדי μCP מרובים על מצע אחד. רמה גבוהה של דיוק ודיוק היה נדרשת לייצור reproducibly מורכב בנישות מבחנה תוך שימוש בטכניקות מגוונות אלה. e_content &quot;&gt; כדי לטפל במגבלה זו, מספר מערכות μCP אוטומטית וחצי אוטומטי שנוצרו. צ&#39;אקרה et. al. פיתחו מערכת μCP בי בולים מותאמים אישית מונחים על מערכת רכבות והביאו במגע קונפורמי עם מגלשות מצופים זהב באמצעות מפעיל פנאומטי מבוקרת מחשב. עם זאת, שיטה זו דורשת ייצור המדויק של עיצובי בולים מותאמים אישית ומדווח 10 מיקרומטר דיוק ללא דו&quot;ח של הדיוק שהושג בעת ביצוע μCP מרובה השלבים 12. לאחרונה, שיטת ניצול מערכת צימוד kinematic משולבת דיוק שדווח להלן 1 מיקרומטר באמצעות דפוס בודד, אבל לא הצליחו ליישר במדויק דפוסים מרובים בשל חוסר השליטה מדויקת של תכונות חותמת מעובש לעצב 13. בנוסף, בשתי השיטות הקודמות דורשים מצע ליישארו קבועים בין צעדי דפוסים , וכך להגביל את מגוון chemistries שינוי פני השטח שיכול להיות משמעותימנוצל. כאן, אנו מתארים מערכת R-μCP אוטומטית מסוגל יישור מדויק ומדויק של צעדי μCP מרובים תוך מתן גמישות מירבית בעיצוב בולים וייצור. יתר על כן, מצעים בדוגמת ניתן להסיר שוב ושוב מהמערכת בין stampings, ובכך לאפשר את שימוש בchemistries שינוי המצע מגוון, כוללים החלפות nucleophilic רציפות. מצעים שהונדסו בשיטות chemistries כזה כבר נעשו שימוש בתרבית תאים בעבר על ידי שנינו 6,14 ואחרים 7. כך, יש לנו התמזגו R-μCP ותגובות התמר נוקלאופילית רציפות לפתח שיטה לייצור להרחבה של מצעי תרבות עם רמזים ביוכימיים מורכבים וmicropatterned.

<table border="0" cellpadding="0" cellspacing="0" width="740">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Name</td>
			<td style="width:185px;">Company</td>
			<td style="width:185px;">Catalog Number</td>
			<td style="width:185px;">Comments</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">SCARA&#160;</td>
			<td style="width:185px;">Epson</td>
			<td style="width:185px;">LS3-401ST</td>
			<td style="width:185px;">Higher end models with increased precision are available if desired.&#160;</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:185px;">(TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL)TRICHLOROSILANE</td>
			<td style="width:185px;">Gelest</td>
			<td style="width:185px;">SIT8174.0</td>
			<td style="width:185px;">CAUTION, Should only be handled in a chemical fume hood. When silanizing wafers no one should enter the hood until all silane has been evaporated.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:185px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:185px;">Ellsworth Adhesive Co</td>
			<td style="width:185px;">NC9020938</td>
			<td style="width:185px;">Thouroughly degass solutions via vacuum exposure before use. Alternative kits such as Kit 182 are acceptable.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:185px;">24mm X 50 mm #1 Cover Glass Slides</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:185px;">48393106</td>
			<td style="width:185px;">These can be purchased from a number of suppliers with varying dimensions to suit need.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">CHA-600 Telemark Electron Beam Evaporator</td>
			<td style="width:185px;">Telemark</td>
			<td style="width:185px;">SEC-600-RAP</td>
			<td style="width:185px;">Requries specialized training.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">EPSON LS3 SCARA</td>
			<td style="width:185px;">EPSON</td>
			<td style="width:185px;">LS3-401ST</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:185px;">&#969;-mertcaptoundecyl bromoisobutyrate</td>
			<td style="width:185px;">Prochimia</td>
			<td style="width:185px;">FT 015-m11-0.2</td>
			<td style="width:185px;">Store at -20&#176;C. Other ATRP initiators may be used as this R-&#956;CP platform is applicable to all micropatterning modalities.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Schlenk Tube Flask 50 mL</td>
			<td style="width:185px;">Synthware</td>
			<td style="width:185px;">60003-078</td>
			<td style="width:185px;">Requires rubber stoppers with diaphram.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">Poly(ethylene glycol) methyl ether methacrylate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">447943</td>
			<td style="width:185px;">Shipped containing MEHQ and BHT free readical inhibitors.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Methanol (Certified ACS)</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:185px;">A412-4</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Copper(II) Bromide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">437867</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">2&#39;,2-Bipyridine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">D216305</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Sodium L-Ascorbate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">A4034</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">20mL Borosilicate Glass Scintillation Vials</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:185px;">03-340-4E</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Sodium Azide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">S2002</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">N,N-dimethyformamide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">227056</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Ethanolamine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">398136</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Triethylamine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">T0886</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Dimethylsulfoxide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">276855</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Propargylamine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">P50900</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">200 Proof Ethanol</td>
			<td style="width:185px;">University of Wisconsin Material Distribution Services</td>
			<td style="width:185px;">2292</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Azide-PEG3-Biotin</td>
			<td style="width:185px;">ClickChemistryTools</td>
			<td style="width:185px;">AZ104-100</td>
			<td style="width:185px;">Solubilized in DMF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Copper(II) Sulfate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">C1297</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">678937</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">L-Ascorbic Acid</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">A7506</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Phosphate Buffer Saline</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:185px;">14190144</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:185px;">Donkey Serum</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">D9663</td>
			<td style="width:185px;">Donkey serum contaminated items are considered bio-hazardous material and should be disposed of accordingly. Various other compounds (e.g. BSA) are available and serve this purpose.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">12-Well Polystyrene Plate</td>
			<td style="width:185px;">Thermo Scientifit - NUNC</td>
			<td style="width:185px;">07-200-81</td>
			<td style="width:185px;">Plates can be purchased form a number of suppliers with varying dimensions.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">DBCO-PEG4-Biotin</td>
			<td style="width:185px;">Clickchemistytools</td>
			<td style="width:185px;">A105P4-10</td>
			<td style="width:185px;">Solubilized in DMF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Streptavidin, Alexa Fluor 488 Conjugate</td>
			<td style="width:185px;">Life Technologies</td>
			<td style="width:185px;">S-11223</td>
			<td style="width:185px;">Solubilized in PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Streptavidin, Alexa Fluor 546 conjugate</td>
			<td style="width:185px;">Life Technologies</td>
			<td style="width:185px;">S-11225</td>
			<td style="width:185px;">Solubilized in PBS</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:185px;">Nikon A1-R Confocal Microscope</td>
			<td style="width:185px;">Nikon</td>
			<td style="width:185px;">Nikon Eclipse Ti, A1R</td>
			<td style="width:185px;">An epifluorescent microscope is sufficient to image functionalized micropatterned substrates.</td>
		</tr>
	</tbody>
</table>

fabricating complex culture substrates using robotic microcontact printing (r-&#181;cp) and sequential nucleophilic substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

השימוש בטכניקות μCP היישור ידניים להנדס מצעי תרבות עם מערכים של מברשות שהושתלו PEG פונקציונליות עם מאונך &quot;לחץ&quot; chemistries הוכח בעבודות קודמות 6. עם זאת, זה מציע שליטה מינימאלית של התמצאות דפוס ולעתים קרובות תוצאות חפיפה של התחומים פונקציונליות. כאן, מערכת R-μCP רומ?...

Watch this Scientific Journal Video about Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R-ÂµCP and Sequential Nucleophilic Substitution at JoVE.com

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &amp;#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

בודה מצעים מורכבים תרבות באמצעות רובוטית microcontact הדפסה (R-μCP) והתמר נוקלאופילית סדרתית

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

fabricating-complex-culture-substrates-using-robotic-microcontact

Research

JoVE Journal

Bioengineering

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

10.4K Views.  University of Wisconsin, Madison. Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands. 

Video: Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

יצירת דו מימדי מצעים Patterned עבור החזקת חלבון ו-Cell

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

דור של רקמות שריר לב פונקציונליות מזדהות באמצעות הדפסת microcontact

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

ייצור רקמת סחוס אנושי באמצעות שלושה ממדי הזרקת דיו טכנולוגיית הדפסה

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

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 ...

Fabricating Cotton Analytical Devices

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.

To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.

בודה כותנה התקנים אנליטית

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

הרחבת טכניקת תפר באמצעות מצעי nanopatterned עבור Nanotopographical אפנון של התנהגות תא

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

בדיית כליות קורטקס חוץ-תאית מטריקס-Derived הידרוג

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Four-Dimensional CT Analysis Using Sequential 3D-3D Registration

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data remains difficult because segmentation in all volumetric frames is time-consuming. We aimed to analyze joint kinematics using a sequential 3D-3D registration technique to provide the kinematics of the moving bone with respect to the fixed bone semiautomatically using 4DCT DICOM data and existing software. Surface data of the source bones are reconstructed from 3DCT. The trimmed surface data are respectively matched with surface data from the first frame in 4DCT. These trimmed surfaces are sequentially matched until the last frame. These processes provide positional information for target bones in all frames of the 4DCT. Once the coordinate systems of the target bones are decided, translation and rotation angles between any two bones can be calculated. This 4DCT analysis offers advantages in kinematic analyses of complex structures such as carpal or tarsal bones. However, fast or large-scale motions cannot be traced because of motion artifacts.

We analyzed joint kinematics from four-dimensional computed tomography data. The sequential 3D-3D registration method semiautomatically provides the kinematics of the moving bone with respect to the subject bone from four-dimensional computed tomography data.

4-מימדי ניתוח CT באמצעות 3D סדרתית-3D הרישום

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data ...

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous lipid tubules found in eukaryotic cells. However, in vivo LNTs are highly non-equilibrium structures that require chemical energy and molecular motors to be assembled, maintained, and reorganized. Furthermore, the composition of in vivo LNTs is complex, comprising of multiple different lipid species. Typical methods to extrude LNTs are both time- and labor-intensive, and they require optical tweezers, microbeads, and micropipettes to forcibly pull nanotubes from giant lipid vesicles. Presented here is a protocol for the gliding motility assay (GMA), in which large scale LNT networks are rapidly generated from giant unilamellar vesicles (GUVs) using kinesin-powered microtubule motility. Using this method, LNT networks are formed from a wide array of lipid formulations that mimic the complexity of biological LNTs, making them increasingly useful for in vitro studies of lipid biophysics and membrane-associated transport. Additionally, this method is capable of reliably producing LNT networks in a short time (&#60;30 min) using commonly used laboratory equipment. LNT network characteristics such as length, width, and lipid partitioning are also tunable by altering the lipid composition of the GUVs used for fabricating the networks.

This protocol describes a process for fabricating lipid nanotube networks using gliding kinesin motility in conjunction with giant unilamellar lipid vesicles.

ייצור רשתות ננו-צינוריות ליפידים מרובות רכיבים באמצעות בדיקת תנועתיות Kinesin Gliding

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous ...

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

מניפולציה של תאי גזע עצביים יחיד ונוירונים בפרוסות מוח באמצעות Microinjection רובוטי

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...