מחקר זה נתמך על ידי תכנית המחקר העירונית של המכון הלאומי לבריאות בארה&quot;ב, המכון הלאומי לסרטן, המרכז לחקר הסרטן.

ההערה: הפרוטוקול הבא מתאר את הצעדים הדרושים כדי לחסל תאים ספציפיים באמצעות NIR-PIT. בקרות ופרטים נוספים על כדאיות NIR-PIT ותא ניתן למצוא במקום אחר 18. 1. הצמיד של IR700 לנוגדנים חד-שבטיים (מב) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן מב עניין ב 2-5 מ&quot;ג / מ&quot;ל ב 0.1 M Na 2 HPO 4 (pH 8.6) פתרון. </li><li style=";text-align:right;direction:rtl"> מערבבים 6.8 nmol של מב עם 30.8 nmol של 10 מ&quot;מ IR700 ב 0.1 M Na 2 HPO 4 פתרון (pH 8.6) בצינור microcentrifuge ו דגירה ב RT עבור שעה 1, מכוסה בנייר אלומיניום. </li><li style=";text-align:right;direction:rtl"> לשטוף את העמודה PD-10 (ראו טבלה של ציוד חומרים /) עם 15 מ&quot;ל PBS, פעמיים. טען המדגם משלב 1.2. </li><li style=";text-align:right;direction:rtl"> לטהר את התערובת דרך עמודת PD-10 על ידי elution PBS על פי הוראות היצרן. הערה: כאן, elution היה לאורך צבע הלהקה של IR700. עבור מדגם 300 μl, חלק elution PBS הוא בדרך כלל בין 2.5 מ&quot;ל ל -4.4 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> לקבוע אתריכוז חלבון עם מכתים Coomassie על ידי מדידת הספיגה ב 595 ננומטר עם ספקטרופוטומטר 9. קבע את הריכוז של IR700 עם הקליטה 689 ננומטר כדי לאשר את המספר של מולקולות fluorophore מצומדות לכל מולקולה מב 9. הערה: חשוב לקבוע מספר נטיה אופטימלי של מולקולות IR700 ב 1 מב עם ספקטרופוטומטר. באופן כללי, כ -3 מולקולות IR700 במולקולת 1 מב הן אופטימליות עבור הן במבחנה בעבודת vivo. HPLC ושיטות SDS-PAGE ניתן להשתמש כדי לאשר את מב ו IR700 מאוגדים ובין אם לאו. </li><li style=";text-align:right;direction:rtl"> חנות ב 4 מעלות צלזיוס לאחר קביעת ריכוז החלבון. </li></ol> 2. הכנת תרבית תאים 3D מעורב (Mixed אליפטית) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> החל מים סטריליים (סביב 1 מיליליטר) לתוך קטע מאגר הצלחת לתלות צלחות טיפה. </li><li style=";text-align:right;direction:rtl"> הכן יחסים שונים של סוגי תאים של עניין - תאים A431-לוק-GFP ותאי 3T3-RFP -עם כל דגימה המכילה 5,000 תאים בסך הכל, המרחפים 50 μl של התקשורת והתרבות. הערה: כן 1: 100, 10: 100, 25: 100, 50: 100, וכו &#39;יחסים של סוגי תאים של עניין תקשורת והתרבות תלוי תאים מהסוג. </li><li style=";text-align:right;direction:rtl"> דגירת התמהיל 5-7 ימים בצלחת הירידה 96 גם תלוי חממת humidified על 37 מעלות צלזיוס, פחמן דו חמצני 5%. שנה את התקשורת והתרבות כל 2 ימים. הערה: כדי להפוך את 3D אליפטית בדיוק, להתמודד עם הצלחת בעדינות, כמו טיפות המכילות תאים ליפול בקלות לפני להרכיב צורות 3D. </li><li style=";text-align:right;direction:rtl"> שים את המורפולוגיה והגודל של spheroids באמצעות מיקרוסקופ brightfield הפוכה ב 10X - הגדלה 40X. הערה: למרות שזה תלוי סוגי תאים וגודל-Drop Hanging, להבטיח כי הקוטר של אליפטית הוא סביב 400-600 מיקרומטר לאחר 7 ימי דגירה של 96 גם תלוי צלחת טיפה. </li></ol> 3. במבחנה NIR-בור תרבית תאי 3D מעורבת <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שנה את התקשורת של חהnging צלחות ושחרר כדי המצומד 10 מיקרוגרם / מ&quot;ל ​​נוגדנים photoabsorber (APC) מדיה המכילה, דגירה במשך 6 שעות בתוך חממה humidified ב 37 ° C ופחמן דו חמצני 5%. </li><li style=";text-align:right;direction:rtl"> לאחר 6 דגירת hr, לשטוף את אליפטית פעמים עם תקשורת והתרבות הטריה (פנול האדום חינם). בעדינות להעביר את אליפטית לצלחת עם רצפת זכוכית 50 מ&quot;מ עם 100 μl פנול טריים התקשורת והתרבות אדום ללא באמצעות קצה פיפטה סטרילית 200 μl עם קצה לחתוך. מניחים אליפטית אחת בכל צלחת. </li><li style=";text-align:right;direction:rtl"> שים לב אליפטית עם מיקרוסקופ brightfield הפוך להבחין בשינוי של מורפולוגיה. כדי לבחון את הכתבים האופטיים (למשל, ה- GFP ו RFP) להשתמש מיקרוסקופ פלואורסצנטי עם גדרות המסנן הבאות: GFP - 469 מסנן עירור ננומטר, ואת מסנן פליטה 525 ננומטר; RFP - 559 מסנן עירור ננומטר, ו 630 מסנן פליטה ננומטר. </li><li style=";text-align:right;direction:rtl"> מניחים את אור דיודה (LED) מעל צלחת תחתית זכוכית עבור הקרנה. הערה: ניתן להיחשף Spheroids לנירלהדליק או על מיקרוסקופ או בשכונה למינרית. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מדוד את צפיפות ההספק של אור NIR עם מד כוח אופטי 9. על פי מדידה זו, מקרינים אור NIR באמצעות דיודות פולטות אור (LEDs) אשר פולטים אור באורכי גל של 670 עד 710 ננומטר 2 J / cm 2. הערה: הנורית יכול לחדור לעומק מרבי של כ 5 ס&quot;מ. השפעות ציטוטוקסיות של NIR-PIT תלויה רק אנרגיה נתון ללא קשר צפיפות הספק ומשך החשיפה 23. </li></ol></li><li style=";text-align:right;direction:rtl"> לאחר ההקרנה, להעביר את אליפטית לתוך צלחת Drop Hanging חדש עם 50 μl של התקשורת בתרבות טרי דגירה במשך יום 1 ב חממה humidified על 37 מעלות צלזיוס, פחמן דו חמצני 5%. </li><li style=";text-align:right;direction:rtl"> בעדינות להעביר את אליפטית לצלחת עם תחתית זכוכית עם 100 μl של התקשורת בתרבות טרי (פנול אדום חינם) באמצעות קצה פיפטה סטרילית 200 μl עם קצה לחתוך. שימו לב אליפטית עם יום פלואורסצנטי מיקרוסקופ 1 לאחר Fi באמצעות NIR-PITהגדרות LTER כמתוארות בשלב 3.3. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> זיהוי תאים מתים על ידי הוספת יודיד propidium לתקשורת בריכוז סופי של 2 מיקרוגרם / מ&quot;ל, או על ידי אובדן של GFP הקרינה cytoplasmic באמצעות מיקרוסקופ פלואורסצנטי (איור 2 ב) 14,18,22. </li></ol></li><li style=";text-align:right;direction:rtl"> חזור על שלבי 3.1-3.6 אם תאי יעד הם לא בוטלו לחלוטין. </li></ol>

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החוקרים אין לי מה לחשוף.

אנו מדגימים שיטת חיסול תאים ספציפיים מתוך תרבית תאים 3D מעורבת ללא נזק לתאים היעד הלא באמצעות NIR-PIT. עד כה, לא קיימת שיטה מעשית של חיסול תאים פעם הרקמה מוקמת או לאחר השתלה. לכן, ניר-PIT הוא שיטה מבטיחה לעשות זאת. טכניקה זו יכולה גם להיות מנוצלת in vivo 18,22, מאז נג...

ביטול תאים ספציפיים מבלי לפגוע בתאים אחרים קשה מאוד, במיוחד ברקמה הוקמה, ויש צורך דחוף שיטת חיסול תא בתחום הנדסת רקמות. כיום בתחום של רפואה רגנרטיבית, תרביות רקמה באמצעות תאי גזע עובריים (ES), תאי גזע פלוריפוטנטיים (PSCs), או תאי גזע מושרים (iPS) הם חומרים מבטיח 1 - 3. למרות התחדשות רקמות זו נראית מבטיחה, זיהום עם תאים לא רצויים הוא דאגה עיקרית. יתר על כן, קיים חשש לשלומם של tumorigenicity לאחר ההשתלה 4,5. למרות מחקרים רבים התמקדו בנושאים אלה לחסל תאים ספציפיים, במיוחד ברפואה רגנרטיבית 6 - 8, אין שיטה מעשית פותחה. photoimmunotherapy אינפרא אדום קרוב (NIR-PIT) הוא טיפול המבוסס על conjugat נוגדן-photoabsorberדואר (APC). נגמ&quot;ש מורכב של נוגדנים חד שבטיים תא ספציפי (מב) וכן photoabsorber, IR700. IR700 היא נגזרת סיליקה-phthalocyanine הידרופילי לא לגרום phototoxicity בעצמה 9. IR700 הוא מצומדות קוולנטית הנוגדן באמצעות שאריות אמידות על שרשרת הצד של מולקולות ליזין. הנגמ&quot;ש נקשר מולקולות מטרה על קרום התא ולאחר מכן גורם כמעט תא נימק מיידי לאחר חשיפה לאור NIR ב 690 ננומטר. במהלך החשיפה NIR-אור, נקרע קרום הסלולר המובילה לתא פטירה 9 - 14. NIR-PIT הוכיח כיעיל עם נוגדנים מרובים או שברי נוגדנים, כולל אנטי EGFR, אנטי HER2, אנטי PSMA, אנטי CD25, אנטי מזותלין, אנטי GPC3, ואנטי-CEA 15 - 21. לכן, ניר-PIT יכול לשמש נגד מגוון רחב של מולקולות מטרה. יתר על כן, ניר-PIT הוא טיפול מבוקר היטב המאפשר טיפול סלקטיבית של אזורים ספציפיים על ידי הגבלת-תאורת NIRt הקרנה 18,22. כאן, אנו מציגים שיטה של ​​חיסול תאים ספציפיים באמצעות NIR-PIT מתרבויות 3D מעורבת.

<table border="0" cellpadding="0" cellspacing="0" width="1083">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:424px;">IRDye 700DX Ester Infrared Dye</td>
			<td style="width:364px;">LI-COR Bioscience (Lincoln, NE, USA)</td>
			<td style="width:191px;">929-70011</td>
			<td style="width:104px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>SIGMA-ALDRICH (St. Louis, MO, USA)</td>
			<td>S9763</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex G25 column (PD-10)&#160;</td>
			<td>GE Healthcare (Piscataway, NJ, USA)</td>
			<td>17-0851-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coomassie (bradford) Plus protein assay</td>
			<td>Thermo Fisher Scientific Inc (Waltham, MA, USA)</td>
			<td>PI-23200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perfecta3D 96-Well hanging Drop Plates</td>
			<td>3D Biomatrix Inc (Ann Arbor, MI, USA)</td>
			<td>HDP1096-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optical power meter</td>
			<td>Thorlabs (Newton, NJ, USA)</td>
			<td>PM100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LED: L690-66-60</td>
			<td>Marubeni America Co. (Santa Clara, CA, USA)</td>
			<td>L690-66-60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectibix (panitumumab)</td>
			<td>Amgen (Thousand Oaks, CA, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35mm glass bottom dish, dish size 35mm, well size 10mm</td>
			<td>Cellvis (Mountain View, CA, USA)</td>
			<td>D35-10-0-N</td>
			<td></td>
		</tr>
	</tbody>
</table>

selective cell elimination from mixed 3d culture using a near infrared photoimmunotherapy technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

כדי אופטית לפקח על השפעת NIR-PIT, שורת תאים A431, אשר overexpresses EGFR, שונה גנטית גם להביע GFP ו בלוציפראז (A431-לוק-GFP). כתוצאה היעד אי NIR-PIT, הקו הסלולרי Balb / 3T3 שונה אופטית להביע RFP (3T3-RFP). הנגמ&quot;ש, וקטיביקס-IR700 (פאן-IR700), היה מסונתז. Spheroids מעורבת, אשר הורכבו יחסים שונים של ...

Watch this Scientific Journal Video about Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique at JoVE.com

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

ביעור Cell סלקטיבי מן תרבות 3D מעורבת באמצעות טכניקה האדומה קרוב Photoimmunotherapy

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

selective-cell-elimination-from-mixed-3d-culture-using-near-infrared

Research

JoVE Journal

Bioengineering

7.8K Views.  National Cancer Institute. Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT). 

Video: Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

תא פשוט תלוי תרבות Drop פרוטוקול הדור של Spheroids 3D

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

סיבוב תרבית תאים מערכות התרבות תאים אנושיים: תאים Trophoblast האדם כמודל

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

הכנת 3D הפיברין פיגומים יישומים סלולריים גזע תרבות

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

שיטה חדשה ללוקליזציה חרוזי ניאון כתב סמוך לפני שטח תרבית תאים לגרירה מיקרוסקופית כוח

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

הקרינה-מרווה של Fluorophore הקרוב אינפרא אדום-כמוס liposomal ככלי ל<em&gt; In vivo</em&gt; דימות אופטי

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

באמצעות דפוסים דבקים כדי לבנות התקני 3D נייר Microfluidic

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

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 ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

הידרוג מתכוונן מ מטריקס ריאתי עבור תרבית תאים 3D

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting של הסחוס, תחליפי רקמת העור ניצול פסיבים הרומן ערבוב טכניקה יחידה עבור Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

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

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

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

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

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