Name: creating two-dimensional patterned substrates for protein and cell confinement
Uploaded: 2011-09-06T13:00:00
Description: Watch this Scientific Journal Video about Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement at JoVE.com

हम वाशिंगटन विश्वविद्यालय जिसका सामूहिक ज्ञान के इस प्रोटोकॉल संभव बनाया है में पूरे Maurer समूह स्वीकार करना चाहते हैं. मानसिक स्वास्थ्य के राष्ट्रीय संस्थान (1R01MH085495) द्वारा इस कार्य के लिए अनुदान उपलब्ध कराया जाता है.

1. नमूनों की तैयारी मास्टर (चित्र 1) <ol><li> केंद्र स्पिन coater पर सिलिकॉन वफ़र और एसीटोन के साथ तालिका 1 में दो चक्र स्पिन कार्यक्रम के प्रारंभिक चरण के दौरान वफ़र कुल्ला. एसीटोन स्पिन एक साफ, सूखे वफ़र छोड़ने का...

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Am. Chem. Soc. , 9576-9577 (1995).</li><li>Mrksich, M., Dike, L., Tien, J., Ingber, D., Whitesides, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+microcontact+printing+to+pattern+the+attachment+of+mammalian+cells+to+self-assembled+monolayers+of+alkanethiolates+on+transparent+films+of+gold+and+silver.">Using microcontact printing to pattern the attachment of mammalian cells to self-assembled monolayers of alkanethiolates on transparent films of gold and silver.</a> Experimental Cell Research. , 305-313 (1997).</li><li>Prime, K. L., Whitesides, G. 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R., Kuck, V., Katz, H., Amundson, K., Ewing, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paper-like+electronic+displays:+Large-area+rubber-stamped+plastic+sheets+of+electronics+and+microencapsulated+electrophoretic+inks.">Paper-like electronic displays: Large-area rubber-stamped plastic sheets of electronics and microencapsulated electrophoretic inks.</a> Proc Natl Acad Sci U S A. 98, 4835-4840 (2001).</li><li>Yanker, D., Maurer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+printing+of+trichlorosilanes+on+glass+for+selective+protein+adsorption+and+cell+growth.">Direct printing of trichlorosilanes on glass for selective protein adsorption and cell growth.</a> Molecular Biosystems. 4, 502-504 (2008).</li><li>Johnson, D., Maurer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recycling+and+reusing+patterned+self-assembled+monolayers+for+cell+culture.">Recycling and reusing patterned self-assembled monolayers for cell culture.</a> Chemical Communications. , 520-522 (2011).</li><li>Herne, T., Tarlov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+DNA+probes+immobilized+on+gold+surfaces.">Characterization of DNA probes immobilized on gold surfaces.</a> J. Am. Chem. Soc. , 8916-8920 (1997).</li><li>Hanson, E., Schwartz, J., Nickel, B., Koch, N., Danisman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bonding+self-assembled,+compact+organophosphonate+monolayers+to+the+native+oxide+surface+of+silicon.">Bonding self-assembled, compact organophosphonate monolayers to the native oxide surface of silicon.</a> J. Am. Chem. Soc. , 16074-16080 (2003).</li><li>Johannes, M., Cole, D., Clark, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscope+based+nanofabrication+of+master+pattern+molds+for+use+in+soft+lithography.">Atomic force microscope based nanofabrication of master pattern molds for use in soft lithography.</a> Applied Physics Letters. , (2007).</li><li>Bessueille, F., Pla-Roca, M., Mills, C. A., Martinez, E., Samitier, J., Errachid, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submerged+microcontact+printing+(S&#x03BC;CP):+An+unconventional+printing+technique+of+thiols+using+high+aspect+ratio,+elastomeric+stamps.">Submerged microcontact printing (S&#x03BC;CP): An unconventional printing technique of thiols using high aspect ratio, elastomeric stamps.</a> Langmuir. , 12060-12063 (2005).</li><li>Xia, Y., Whitesides, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extending+microcontact+printing+as+a+microlithographic+technique.">Extending microcontact printing as a microlithographic technique.</a> Langmuir. , 2059-2067 (1997).</li><li>Biasco, A., Pisignano, D., Krebs, B., Pompa, P. P., Persano, L., Cingolani, R., Rinaldi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformation+of+microcontact-printed+proteins+by+atomic+force+miroscopy+molecular+sizing.">Conformation of microcontact-printed proteins by atomic force miroscopy molecular sizing.</a> Langmuir. , 5154-5158 (2005).</li><li>Shen, K., Qi, J., Kam, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcontact+printing+of+proteins+for+cell+biology.">Microcontact printing of proteins for cell biology.</a> J Vis Exp. (22), e1065-e1065 (2008).</li><li>Piner, R., Zhu, J., Xu, F., Hong, S., Mirkin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Dip-pen"+nanolithography.">"Dip-pen" nanolithography.</a> Science. 283, 661-663 (1999).</li><li>Ryan, D., Parviz, B. A., Linder, V., Semetey, V., Sia, S. K., Su, J., Mrksich, M., 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=Patterning+multiple+aligned+self-assembled+monolayers+using+light.">Patterning multiple aligned self-assembled monolayers using light.</a> Langmuir. , 9080-9088 (2004).</li></ol>

PDMS स्टांप निर्माण के लिए इस्तेमाल किया मास्टर के lithographic उत्पादन में मुद्दों का एक नंबर पैदा कर सकते हैं. अपूर्ण फोटो अस्पष्ट और अस्पष्ट पैटर्न और विरोध में लिपटे बढ़े या लापता सुविधाओं में वफ़र परिणामों ...

<table border="1"><tbody><tr><th> अभिकर्मक के नाम </th><th> कंपनी </th><th> सूचीपत्र संख्या </th><th> टिप्पणियां (वैकल्पिक) </th></tr><tr><td> सिलिकॉन वफ़र </td><td> वेफर सेवा को पुनः प्राप्त </td><td></td><td> 2 इंच </td></tr><tr><td> स्पिन coater / हॉट प्लेट </td><td> शराब बनानेवाला विज्ञान </td><td> CEE 200CB स्पिन बनाओ सिस्टम </td><td></td></tr><tr><td> AZ9245 photoresist </td><td> Mays केमिकल कंपनी </td><td> 105880034-1160 </td><td></td></tr><tr><td> प्रत्यक्ष लिखना photolithography प्रणाली </td><td> Microtech srl </td><td> LW325 LaserWriter प्रणाली </td><td></td></tr><tr><td> मुखौटा aligner </td><td> HTG </td><td> 3HR </td><td></td></tr><tr><td> Az 400K डेवलपर </td><td> Mays केमिकल कंपनी </td><td> 105880018-1160 </td><td></td></tr><tr><td> Sylgard 182 सिलिकॉन Elastomer किट </td><td> डॉव Corning </td><td></td><td></td></tr><tr><td> 25 कोई मिमी. 1 गोल कांच coverslips </td><td> VWR </td><td> 16004-310 </td><td></td></tr><tr><td> प्लाज्मा आक्सीकारक </td><td> Diener </td><td> Femto </td><td></td></tr><tr><td> टाइटेनियम टुकड़े Kamis </td><td> निगमित </td><td></td><td> 99.95 शुद्ध% </td></tr><tr><td> गोल्ड छर्रों </td><td> Kamis शामिल </td><td></td><td> 99.999 शुद्ध% </td></tr><tr><td> इलेक्ट्रॉन बीम बाष्पीकरण </td><td> कर्ट जे Lesker </td><td> PVD 75 तनु फिल्म बयान प्रणाली </td><td> इलेक्ट्रॉन बीम गौण के साथ </td></tr><tr><td> Hexadecanethiol </td><td> अल्फा Aesar </td><td> A11362 </td><td></td></tr><tr><td> 1 - mercaptoundec-11-YL) tetra (ethyleneglycol) </td><td> सिग्मा Aldrich </td><td> 674508 </td><td></td></tr><tr><td> इथेनॉल </td><td> Pharmco - aaper </td><td> 111000200 </td><td> 200 सबूत निरपेक्ष, </td></tr><tr><td> Parafilm </td><td> VWR </td><td> 52858-000 </td><td></td></tr><tr><td> DPBS </td><td> VWR </td><td> 4500-434 </td><td> कैल्शियम और मैग्नीशियम के बिना </td></tr><tr><td> माउस laminin मैं </td><td> VWR </td><td> 95036-762 </td><td></td></tr><tr><td> मानव प्लाज्मा fibronectin </td><td> Invitrogen </td><td> 33016-015 </td><td></td></tr><tr><td> AlexaFluor ® 647 carboxylic एसिड, succinimidyl एस्टर </td><td> Invitrogen </td><td> एक - 20,006 </td><td></td></tr><tr><td> MitoTracker 580 लाल </td><td> Invitrogen </td><td> M22425 </td><td></td></tr><tr><td> ® 350 carboxylic एसिड, succinimidyl एस्टर AlexaFluor </td><td> Invitrogen </td><td> A-+१०१६८ </td><td></td></tr><tr><td> एंटी - laminin एंटीबॉडी </td><td> फिशर साइंटिफिक </td><td> AB2034MI </td><td></td></tr></tbody></table>

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

Watch this Scientific Journal Video about Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement at JoVE.com

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

स्व इकट्ठे monolayers (SAMs) सोने पर लंबी श्रृंखला alkane thiols गठन से अच्छी तरह से परिभाषित प्रोटीन पैटर्न और सेल कारावास के गठन के लिए substrates प्रदान करते हैं. Hexadecanethiol के microcontact मुद्रण polydimethylsiloxane (PDMS) के साथ backfilling बाद स्टांप का उपयोग कर एक glycol समाप्त alkane thiol monomer के एक पैटर्न का उत्पादन जहां प्रोटीन और कोशिकाओं को केवल मुद्रांकित hexadecanethiol क्षेत्र सोखना.

प्रोटीन और सेल बन्दिश के लिए दो आयामी नमूनों Substrates बनाना

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

Research

JoVE Journal

Bioengineering

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क्षणिक सेल झिल्ली एक मानक inkjet प्रिंटर का उपयोग Pores बनाना

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

प्रतिदीप्ति माइक्रोस्कोपी के साथ इमेजिंग सेल प्रवासन के लिए चिपकने वाला और घुलनशील Gradients बनाना

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Photolithographically परिभाषित Parylene सी पर सेल patterning: SiO<sub&gt; 2</sub&gt; Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

एकल कक्षों और 3 डी कारावास में एकल भ्रूण आदेश: उच्च सामग्री स्क्रीनिंग के लिए एक नई डिवाइस

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

एक छोटी बूंद विलय प्रतिदीप्ति वसूली के बाद एक फॉस्फोलिपिड monolayer के दो आयामी प्रसार करने के लिए उपाय

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after ...

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

सेल व्यवहार की Nanotopographical मॉड्यूलेशन के लिए nanopatterned substrates का उपयोग सिलाई तकनीक का विस्तार

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

High-resolution Patterned Biofilm Deposition Using pDawn-Ag43

pDawn-Ag43 का उपयोग करते हुए उच्च-रिज़ॉल्यूशन नमूनों वाली फिल्मी साठा

Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Keratinocyte प्रसार के दो और तीन आयामी प्रकार मैं कोलेजन Substrates पर मूल्यांकन

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

ऊतक-स्तरीय यांत्रिकी को नियंत्रित करने के लिए अनुपालन सबस्ट्राट्स पर मानव भ्रूण स्टेम सेल कालोनियों की ज्यामिति का पैटर्न

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

सेल फ्री बैक्टीरियल अर्क, लिपोसोम्स और इमल्शन ट्रांसफर का उपयोग करके प्रोटीन उत्पादक सिंथेटिक कोशिकाओं की तैयारी

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...