Name: engineering three-dimensional epithelial tissues embedded within extracellular matrix
Uploaded: 2016-07-10T14:00:00
Description: Watch this Scientific Journal Video about Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix at JoVE.com

Dette arbejde blev støttet delvist af tilskud fra NIH (HL118532, HL120142, CA187692), David &amp; Lucile Packard Foundation, Camille &amp; Henry Dreyfus Foundation, og Burroughs Velkommen fonden. ASP blev delvist understøttet af en Charlotte Elizabeth Procter hædrende Fellowship.

1. Fremstilling af opløsninger <ol><li> Til fremstilling af en 5 mg / ml opløsning af insulin, fortyndes den pulveriserede insulin lager med 5 mM saltsyre (HCI) i dH2O (500 mg insulin i 100 ml opløsningsmiddel). Fremstilling af 100 ml opløsningsmiddel ved tilsætning af 50 pi koncentreret HCI til 100 ml destilleret vand (dH 2 O). </li><li> For at gøre en 1x opløsning af PBS, fortyndes 10x phosphatbufret saltvand (PBS) stamopløsning til 1x med dH2O under sterile betingelser. </li...

<ol>
	<li>Affolter, 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=Tube+or+not+tube:+remodeling+epithelial+tissues+by+branching+morphogenesis.">Tube or not tube: remodeling epithelial tissues by branching morphogenesis.</a> Dev Cell. 4 (1), 11-18 (2003).</li><li>Zhu, W., Nelson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+signaling+in+the+regulation+of+branching+morphogenesis.">PI3K signaling in the regulation of branching morphogenesis.</a> Biosystems. 109 (3), 403-411 (2012).</li><li>Sternlicht, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+stages+in+mammary+gland+development:+the+cues+that+regulate+ductal+branching+morphogenesis.">Key stages in mammary gland development: the cues that regulate ductal branching morphogenesis.</a> Breast Cancer Res. 8 (1), 201 (2006).</li><li>Fata, J. E., 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=The+MAPK(ERK-1,2)+pathway+integrates+distinct+and+antagonistic+signals+from+TGFalpha+and+FGF7+in+morphogenesis+of+mouse+mammary+epithelium.">The MAPK(ERK-1,2) pathway integrates distinct and antagonistic signals from TGFalpha and FGF7 in morphogenesis of mouse mammary epithelium.</a> Dev Biol. 306 (1), 193-207 (2007).</li><li>Ip, M. M., Darcy, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+mammary+primary+culture+model+systems.">Three-dimensional mammary primary culture model systems.</a> J Mammary Gland Biol Neoplasia. 1 (1), 91-110 (1996).</li><li>Lo, A. T., Mori, H., Mott, J., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constructing+three-dimensional+models+to+study+mammary+gland+branching+morphogenesis+and+functional+differentiation.">Constructing three-dimensional models to study mammary gland branching morphogenesis and functional differentiation.</a> J Mammary Gland Biol Neoplasia. 17 (2), 103-110 (2012).</li><li>Nelson, C. M., Vanduijn, M. M., Inman, J. L., Fletcher, D. A., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+geometry+determines+sites+of+mammary+branching+morphogenesis+in+organotypic+cultures.">Tissue geometry determines sites of mammary branching morphogenesis in organotypic cultures.</a> Science. 314 (5797), 298-300 (2006).</li><li>Gjorevski, N., Nelson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+patterns+of+mechanical+stress+are+required+for+branching+morphogenesis.">Endogenous patterns of mechanical stress are required for branching morphogenesis.</a> Integr Biol (Camb). 2 (9), 424-434 (2010).</li><li>Gjorevski, N., Nelson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+mechanical+strains+and+stresses+around+quiescent+engineered+three-dimensional+epithelial+tissues.">Mapping of mechanical strains and stresses around quiescent engineered three-dimensional epithelial tissues.</a> Biophys J. 103 (1), 152-162 (2012).</li><li>Gjorevski, N., Piotrowski, A. S., Varner, V. D., Nelson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+tensile+forces+drive+collective+cell+migration+through+three-dimensional+extracellular+matrices.">Dynamic tensile forces drive collective cell migration through three-dimensional extracellular matrices.</a> Sci Rep. 5, 11458 (2015).</li><li>Zhu, W., Nelson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+regulates+branch+initiation+and+extension+of+cultured+mammary+epithelia+via+Akt+and+Rac1+respectively.">PI3K regulates branch initiation and extension of cultured mammary epithelia via Akt and Rac1 respectively.</a> Dev Biol. 379 (2), 235-245 (2013).</li><li>Barcellos-Hoff, M. H., Aggeler, J., Ram, T. G., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+differentiation+and+alveolar+morphogenesis+of+primary+mammary+cultures+on+reconstituted+basement+membrane.">Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane.</a> Development. 105 (2), 223-235 (1989).</li><li>Hirai, Y., 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=Epimorphin+functions+as+a+key+morphoregulator+for+mammary+epithelial+cells.">Epimorphin functions as a key morphoregulator for mammary epithelial cells.</a> J Cell Biol. 140 (1), 159-169 (1998).</li><li>Pavlovich, A. L., Manivannan, S., Nelson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+stroma+induces+branching+morphogenesis+of+engineered+epithelial+tubules.">Adipose stroma induces branching morphogenesis of engineered epithelial tubules.</a> Tissue Eng Part A. 16 (12), 3719-3726 (2010).</li></ol>

The protocol described above outlines a method to produce identical epithelial tissues of pre-defined shape, enabling spatial control of the mechanical stress experienced by cells in the tissue. An elastomeric mold is used to create cavities in type I collagen that are then filled with epithelial cells and covered with an additional collagen layer such that cells are completely encapsulated in a 3D collagen matrix environment. Further culture of these tissues and treatment with growth factors to induce branching from the...

Udviklingen af ​​forgrenede epitelvæv, kendt som forgrening morfogenese, reguleres af celleafledte, fysiske og miljømæssige faktorer. I mælkekirtlen, forgrening morfogenese er en iterativ proces, hvorigennem styret kollektiv cellemigrering skaber en trælignende arkitektur. Det første skridt er primære knop dannelse fra mælkekanalerne, efterfulgt af gren initiering og forlængelse 1,2. Invasion af grene i det omgivende stroma induceres af den systemiske afgivelse af steroidhormoner ved puberteten. Nye primære knopper derefter starter fra enderne af eksisterende filialer, og denne proces fortsætter med at skabe en epitelial træ 3. Selv om der er identificeret mange vigtige biokemiske signaler, en omfattende forståelse af cellens biologiske mekanismer, der styrer denne komplekse udviklingsproces øjeblikket mangler. Desuden mekanistiske undersøgelser af påvirkninger af specifikke signaler er vanskelige at dekonstruere fra erfamenter in vivo, så præcise spatiotemporale forstyrrelser og målinger er ofte ikke muligt. Tre-dimensionelle (3D) kultur teknikker, såsom hele orgel kultur, primære organoids, og cellekultur modeller, er nyttige værktøjer til systematisk at undersøge mekanismerne bag vævsmorfogenese 4-6. Disse kan være særligt nyttige til bestemmelse af påvirkninger af specifikke faktorer enkeltvis, såsom mekaniske kræfter og biokemiske signaler, på en række celle- adfærd, herunder migration, proliferation og differentiering. 6 Engineered cellekulturmodeller, navnlig let aktivere den forstyrrelse af individuelle celler og deres mikromiljø. Én sådan kultur model bruger en mikrofabrikation tilgang til ingeniør model brystepitelceller væv med kontrolleret 3D-struktur, der konsekvent og reproducerbart danner grene, der vandrer kollektivt ved induktion med enppropriate vækstfaktorer. Den største fordel ved modellen er evnen til præcist at manipulere og måle effekterne af fysiske og biokemiske faktorer, såsom mønstre af mekanisk belastning, med høj statistisk konfidens. Denne teknik, sammen med datamodellering, er allerede blevet anvendt til at bestemme de relative bidrag fra fysiske og biokemiske signaler i vejledning af den normale udvikling af brystepitelceller væv og andre forgrenede epitel 7-11. Præsenteret her er en detaljeret protokol til opbygning disse model væv, som let kan udvides til andre typer af celler og ekstracellulær matrix (ECM) geler, og som tjener som et potentielt værktøj til test af terapeutiske midler.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Ellsworth Adhesives</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS curing agent</td>
			<td>Ellsworth Adhesives</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithographically patterned silicon master</td>
			<td>self-made</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic weigh boat</td>
			<td>Fisher Scientific</td>
			<td>08-732-115</td>
			<td></td>
		</tr>
		<tr>
			<td>100-mm-diameter Petri dishes</td>
			<td>BioExpress</td>
			<td>D-2550-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Alcohol 200 Proof</td>
			<td>Pharmco-Aaper</td>
			<td>111000200</td>
			<td>Make a 70% EtOH (v:v) solution by mixing with dH2O</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>American Safety Razor</td>
			<td>620179</td>
			<td></td>
		</tr>
		<tr>
			<td>1:1 Dulbecco&#8217;s Modified Eagle&#8217;s Medium : Ham&#8217;s F12 Nutrient Mixture (DMEM/F12) (1:1)</td>
			<td>Hyclone</td>
			<td>SH30023FS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150H</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Hank&#8217;s balanced salt solution (HBSS)</td>
			<td>Life Technologies</td>
			<td>14185-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma Aldrich</td>
			<td>I6634-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Life Technologies</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Phosphate-buffered saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BP399-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma Aldrich</td>
			<td>221465-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine type I collagen (non-pepsinized)</td>
			<td>Koken</td>
			<td>IAC-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin from bovine serum (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>A-7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved stainless steel tweezers</td>
			<td>Dumont</td>
			<td>7</td>
			<td></td>
		</tr>
		<tr>
			<td>35-mm-diameter tissue culture dishes</td>
			<td>BioExpress</td>
			<td>T-2881-6</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>BioExpress</td>
			<td>C-3394-2</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf Safe-Lock Tube</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Circular #1 glass coverslips, 15-mm in diameter</td>
			<td>Bellco Glass Inc.</td>
			<td>Special order</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% 1X Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>VWR</td>
			<td>100503-916</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Perkin Elmer</td>
			<td>N9300260</td>
			<td>Detergent</td>
		</tr>
		<tr>
			<td>HGF</td>
			<td>Sigma Aldrich</td>
			<td>H 9661</td>
			<td>Resuspended in dH2O at 50 mg/mL</td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse FAK antibody</td>
			<td>Life Technologies</td>
			<td>AMO0672</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa 488 antibody</td>
			<td>Life Technologies</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Adobe Photoshop</td>
			<td>Adobe</td>
			<td>N/A</td>
			<td>Used for color-coding pixel frequency maps.</td>
		</tr>
		<tr>
			<td>FIJI (ImageJ)</td>
			<td>NIH</td>
			<td>N/A</td>
			<td>Free image analysis software used for thresholding, registering, and overlaying images to create a pixel frequency map. The StackReg plugin was used for registering binary images.</td>
		</tr>
	</tbody>
</table>

engineering three-dimensional epithelial tissues embedded within extracellular matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

Oversigtsdiagram af mamma-epitelvæv mikrofabrikation En generel skematisk af mikrofabrikation procedure skitserer det eksperimentelle arbejde flow er vist i figur 1. Slutresultatet er en vifte af epitelvæv med identisk geometri og afstand, der er fuldstændig indlejret i en ECM gel. Et repræsentativt eksperiment anvender EpH4 musebryst epitelceller dyrket i en gel af bovin type I collagen i en koncentration på 4 mg / ml. ...

Watch this Scientific Journal Video about Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix at JoVE.com

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

Dette håndskrift beskriver en blød litografi-baseret teknik til at konstruere ensartede arrays af tredimensionale (3D) epitelvæv med defineret geometri omgivet af ekstracellulær matrix. Denne metode kan underkastes en lang række celletyper og eksperimentelle sammenhænge og giver mulighed for high-throughput screening af identiske gentagelser.

Engineering Tredimensionale epitelvæv indlejret i ekstracellulær matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

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