Name: a customizable chamber for measuring cell migration
Uploaded: 2017-03-12T14:00:00
Description: Watch this Scientific Journal Video about A Customizable Chamber for Measuring Cell Migration Video at JoVE.com

המחברים מודים תוכנית הודעה Creative של אוניברסיטת קלמסון NSF CBET1254609 למתן המימון לפרויקט זה.

1. הפקת לשכת microchannel ליצור מפל ריכוזים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חיתוך חתיכת עובש אקריליק <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השג חתיכת אקרילי בעובי הרצוי. משתמשים בדרך כלל 1/16 אינץ &#39;בעובי גליונות אקריליק. גיליונות עבים ק?...

<ol>
	<li>Sia, S. K., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+fabricated+in+poly+(dimethylsiloxane)+for+biological+studies.">Microfluidic devices fabricated in poly (dimethylsiloxane) for biological studies.</a> Electrophoresis. 24 (21), 3563-3576 (2003).</li><li>Lin, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+dynamic+temporal+and+spatial+concentration+gradients+using+microfluidic+devices.">Generation of dynamic temporal and spatial concentration gradients using microfluidic devices.</a> Lab Chip. 4 (3), 164-167 (2004).</li><li>Darby, I., Hewitson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+Differentiation+in+Wound+Healing+and+Fibrosis.">Fibroblast Differentiation in Wound Healing and Fibrosis.</a> Int Rev Cytol. 257, 143-179 (2007).</li><li>Thampatty, B. P., Wang, J. H. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+study+fibroblast+migration.">A new approach to study fibroblast migration.</a> Cell Motil Cytoskel. 64, 1-5 (2007).</li><li>Discher, D., Mooney, D., Zandstra, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+Factors,+Matrices,+and+Forces+Combine+and+Control+Stem+Cells.">Growth Factors, Matrices, and Forces Combine and Control Stem Cells.</a> Science. 324, 1673-1677 (2009).</li><li>Zengel, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#181;-Slide+Chemotaxis:+A+new+chamber+for+long-term+chemotaxis+studies.">&#181;-Slide Chemotaxis: A new chamber for long-term chemotaxis studies.</a> BMC Cell Biol. , (2011).</li><li>Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boyden+Chamber+Assay.">Boyden Chamber Assay.</a> Methods Molec Biol. 2, 15-22 (2005).</li><li>Safferling, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+revised:+A+novel+reepithelialization+mechanism+revealed+by+in+vitro+and+in+silico+models.">Wound healing revised: A novel reepithelialization mechanism revealed by in vitro and in silico models.</a> JCB. 203 (4), 691-709 (2013).</li><li>Saadi, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+stable+concentration+gradients+in+2D+and+3D+environments+using+a+microfluidic+ladder+chamber.">Generation of stable concentration gradients in 2D and 3D environments using a microfluidic ladder chamber.</a> Biomed Microdevices. 9, 627-635 (2007).</li><li>Cammer, M., Cox, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotactic+Responses+by+Macrophages+to+a+Directional+Source+of+a+Cytokine+Delivered+by+a+Micropipette.">Chemotactic Responses by Macrophages to a Directional Source of a Cytokine Delivered by a Micropipette.</a> Methods Mol Biol. , 125-135 (2014).</li><li>Zicha, D., Dunn, G. A., Brown, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+direct-viewing+chemotaxis+chamber.">A new direct-viewing chemotaxis chamber.</a> J Cell Sci. 99 (4), 769-775 (1991).</li><li>Wells, C. M., Ridley, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cell+migration+using+the+Dunn+chemotaxis+chamber+and+time-lapse+microscopy.">Analysis of cell migration using the Dunn chemotaxis chamber and time-lapse microscopy.</a> Methods Mol Biol. , 31-41 (2005).</li><li>Liang, C. C., Park, A. Y., Guan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+scratch+assay:+a+convenient+and+inexpensive+method+for+analysis+of+cell+migration+in+vitro.">In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.</a> Nat. Protoc. 2, 329-333 (2007).</li><li>Chen, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+migration+chip+for+chemotaxis-based+microfluidic+selection+of+heterogeneous+cell+populations.">Single-cell migration chip for chemotaxis-based microfluidic selection of heterogeneous cell populations.</a> Sci. Rep. 5, (2015).</li><li>Wright, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-chip+open+microfluidic+devices+for+chemotaxis+studies.">On-chip open microfluidic devices for chemotaxis studies.</a> Microsc. Microanal. 18 (04), 816-828 (2012).</li><li>Adler, M., Polinkovsky, M., Gutierrez, E., Groisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+oxygen+gradients+with+arbitrary+shapes+in+a+microfluidic+device.">Generation of oxygen gradients with arbitrary shapes in a microfluidic device.</a> Lab Chip. 10 (3), 388-391 (2010).</li><li>Huang, Y., Agrawal, B., Sun, D., Kuo, J. S., Williams, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics-based+devices:+New+tools+for+studying+cancer+and+cancer+stem+cell+migration.">Microfluidics-based devices: New tools for studying cancer and cancer stem cell migration.</a> Biomicrofluidics. 5 (1), 013412 (2011).</li><li>Taylor, A. M., Rhee, S. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+chambers+for+cell+migration+and+neuroscience+research.">Microfluidic chambers for cell migration and neuroscience research.</a> Microfluid Tech: Rev Protoc. , 167-177 (2006).</li><li>Tang, S. K., Whitesides, G. M., Fainman, Y., Lee, L., Psaltis, D., Yang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic microfluidic and soft lithographic techniques. Optofluidics: Fundamentals, Devices and Applications. , (2010).</li><li>Taylor, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+multicompartment+device+for+neuroscience+research).">Microfluidic multicompartment device for neuroscience research).</a> Langmuir. 19 (5), 1551-1556 (2003).</li><li>Aidelberg, G., Goldshmidt, Y., Nachman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Microfluidic+Device+for+Studying+Multiple+Distinct+Strains.">A Microfluidic Device for Studying Multiple Distinct Strains.</a> JoVE. (69), (2012).</li></ol>

תא microchannel שלנו עשוי לשמש מספר רב של מטרות, כולל קביעת שיעור נדידת תאים בתגובה גורמי גדילה chemoattractants ומדידת קצב הדיפוזיה של תרופה ממטריקס פולימר. אפשר לנצל תא microchannel שלנו לגדל תאים ומקום chemoattractant בקצה אחד של החדר. התאים גדלים בתגובה chemoattractant, ואת נדידת תאים ניתן לכמת על ?...

In order for vital processes such as wound healing, immune responses, and embryonic development to occur, cell migration must take place. Cell migration involves the interaction between cells and neighboring cells, the extracellular matrix, and soluble chemical cues (attractants or repellants). As an example, in the process of wound healing, fibroblasts play an integral role in fibrogenesis and wound contraction, where the cells are recruited to the site of injury to synthesize collagen in order to form the extracellular matrix3. Numerous mechanisms behind the migration of fibroblasts to a wound site have been studied, and they include different mechanical, physical, electrical, and chemotactic factors4. Fibroblasts respond especially well to different concentration gradients of growth factors. These different growth factors work together to optimize tissue regeneration5. While observing the chemotactic response of fibroblasts to growth factor concentrations, one can study the pattern of directional migration of fibroblasts and how they orient themselves around physical obstacles in order to reach their destination. Therefore, the goals of this study were to first develop a system in which fibroblast growth could be tracked under guidance by physical barriers and to secondly model the growth of fibroblasts as they navigate through the system.
Currently, the Boyden chamber assay is the most widely used system to measure the migration of cells6. The Boyden chamber consists of a two-chamber multi-well plate where each well may contain medium with or without chemoattractants7. A filter membrane provides a porous interface between the two chambers in each well; this creates a barrier so that cells cannot pass through unless it is by active migration. Typically, for the Boyden chamber, a chemoattractant is added to the lower chamber, and the system is allowed to equilibrate to form a gradient between the upper and lower wells4. One problem with the Boyden chamber assay is that steep gradients end up forming along a single axis perpendicular with the surface of the membrane. This causes the difference in the chemoattractant concentration between the upper and lower wells to be a lower than what was originally expected. Due to this constraint, the Boyden chamber assay makes it hard to correlate specific cell responses with particular gradient characteristics, such as the slope and the concentration difference. Without these measurements, it is hard to study multi-gradient signal integration.
To address some of the constraints of the traditional Boyden chamber assay, microfluidic assays have been developed to form customizable concentration gradients1. Standard methods for creating microfluidic systems require clean rooms for lithographic techniques. These techniques can be difficult to learn especially in a standard classroom setting. Thus, we have designed a chamber system for measuring cell migration that can be made without using a clean room. Using our system, the wells of the assay can be adjusted to a preferred size, and a linear concentration gradient of customized slope can be produced. This allows for accurate measurement of chemotaxis from random movement. The design is an inexpensive and easy-to-use system to model cell growth in response to different chemical stimuli.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Sigma-Aldrich</td>
			<td>761036</td>
			<td>poly(dimethylsiloxane) 2-part kit including silicone elastomer base and silicone elastomer curing agent</td>
		</tr>
		<tr>
			<td>Acrylic Sheets</td>
			<td>US Plastic</td>
			<td>44200</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes&#160;</td>
			<td>Falcon&#160;</td>
			<td>25373-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate&#8211;dextran</td>
			<td>Sigma-Aldrich</td>
			<td>FD20s-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose, Type I, Low EEO</td>
			<td>Sigma-Aldrich</td>
			<td>A6013-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Fisher Scientific</td>
			<td>11965092</td>
			<td>Cell media components</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serium</td>
			<td>Fisher Scientific</td>
			<td>16000036</td>
			<td>Cell media components</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Fisher Scientific</td>
			<td>15140148</td>
			<td>Cell media components</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BP24384</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS XL Cell Imaging System</td>
			<td>Thermo Fisher Scientific</td>
			<td>AME3300</td>
			<td>Instrument used for taking time-lapse images</td>
		</tr>
		<tr>
			<td>Versa LASER</td>
			<td>Universal Laser Systems, Inc.&#160;</td>
			<td>Model number VLS2.30</td>
			<td>Laser cutter used for cutting plastic</td>
		</tr>
	</tbody>
</table>

a customizable chamber for measuring cell migration

נדידת תאים היא חלק חיוני של מערכת חיסונית, צמיחה, וריפוי פצעים. נדידת תאים היא תהליך מורכב הכרוך אינטראקציות בין תאים, תאי מטריקס, וגורמים כימיים מסיסים ובלתי מסיס (למשל, chemoattractants). שיטות תקן למדידת ההגירה של תאים, כגון assay הקאמרית בוידן, עבודה על ידי ספירת תאים משני צדי מחיצה. טכניקות אלו קלות לשימוש; עם זאת, הם מציעים שינוי גיאומטרי קטן עבור יישומים שונים. לעומת זאת, מכשירי microfluidic יכולים לשמש כדי לצפות נדידת תאים עם מילויי ריכוז להתאמה אישיים של גורמי מסיסי 1, 2. עם זאת, שיטות להכנת מבחנים מבוססים מיקרופלואידיקה יכולות להיות קשות ללמוד. כאן אנו מתארים שיטה קלה ליצירת תאי תרבית תאים למדוד נדידת תאים בתגובה הדרגתית ריכוז כימי. המייל הסלולרי שלנושיטה קאמרית הגירה יכולה ליצור הדרגתי ריכוז ליניארי שונה על מנת ללמוד נדידת תאים עבור מגוון רחב של יישומים. שיטה זו היא יחסית קלה לשימוש והיא מבוצעת בדרך כלל על ידי סטודנטים לתואר ראשון. תא microchannel נוצר על ידי צבת הוסף אקריליק בצורה של חדר microchannel הסופי היטב לתוך צלחת פטרי. אחרי זה, פולי (dimethylsiloxane) (PDMS) נמזג על גבי להכניס את. את PDMS הורש להקשיח ואז להכניס הוסר. זה אפשר ליצירת בארות בכל צורה או גודל רצוי. תאים עשויים להיות שיצטרפו בעתיד תא microchannel, וסוכני מסיסים ניתן להוסיף אחת הבארות על ידי השריית גוש agarose ב הסוכן הרצוי. בלוק agarose מתווסף באחת הבארות, ואת הזמן לשגות תמונות שניתן לנקוט של חדר microchannel על מנת לכמת נדידת תאים. וריאציות שיטה זו יכולה להיעשות עבור יישום נתון, מה שהופך גדולות בשיטה זוhly להתאמה אישית.

איור 2 מציג את התנועה של חזית התא מעבר לתעלה בתגובת שיפוע של בסרום שור העובר להציב בקצה השני של הערוץ מהמקום שבו התאים מצופים. חזית התא מוצגת 48 שעות (איור 2 א), 72 שעות (איור 2 ב), 96 שעות (איור 2 ג), ו -120 שעות ציפוי פוסט <stron...

Watch this Scientific Journal Video about A Customizable Chamber for Measuring Cell Migration Video at JoVE.com

A Customizable Chamber for Measuring Cell Migration Video (Video) | JoVE

פרוטוקול זה מפרט שיטה להתאמה אישית למדוד נדידת תאים בתגובת chemoattractants שעשוי לשמש גם כדי לקבוע את קצב הדיפוזיה של תרופה מתוך פולימר מטריקס.

תא להתאמה אישי עבור נדידת תאים מדידות

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...

Research

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Biology

A Customizable Chamber for Measuring Cell Migration

Measuring Exocytosis in Neurons Using FM Labeling

Measuring Exocytosis in Neurons Using FM Labeling Video (Video) | JoVE

מדידת Exocytosis בנוירונים באמצעות תיוג FM

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber Video (Video) | JoVE

פורסים גירוי המוח באמצעות רשת Microfluidic ולשכת זלוף רגילה

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc Video (Video) | JoVE

הדמיה חיה של נדידת תאים גליה את הדיסק העין דמותי תסיסנית

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap Video (Video) | JoVE

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We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Measuring Peptide Translocation into Large Unilamellar Vesicles

Measuring Peptide Translocation into Large Unilamellar Vesicles Video (Video) | JoVE

מדידת טרנסלוקציה פפטיד לתוך שלפוחית ​​Unilamellar גדול

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration Video (Video) | JoVE

מיקרוסקופית Intravital של זרימת הדם בשריר העכבר Cremaster לניתוח של נדידת תאי גזע היקפי

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

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Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

In vitro Cell Migration and Invasion Assays

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Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

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The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

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Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera Video (Video) | JoVE

אופטימיזציה Assay מאפס במבחנה בדיקה של נדידת תאים עם מצלמה אופטית אוטומטית

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...