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

Dit werk werd mede ondersteund door subsidies van de NIH (HL118532, HL120142, CA187692), de David en Lucile Packard Foundation, de Camille en Henry Dreyfus Stichting, en de Burroughs Welcome Fund. ASP werd mede ondersteund door een Charlotte Elizabeth Procter eretitel Fellowship.

1. Voorbereiding van de Solutions <ol><li> Een 5 mg / ml oplossing van insuline te bereiden, verdun het gepoederde insuline leverbaar met 5 mM zoutzuur (HCl) in dH 2 O (500 mg insuline in 100 ml oplosmiddel). Bereid 100 ml oplosmiddel door toevoeging van 50 pi geconcentreerd HCl aan 100 ml gedestilleerd water (dH 2 O). </li><li> Aan een oplossing van 1x PBS te verdunnen 10x fosfaatgebufferde zoutoplossing (PBS) stockoplossing aan 1x met dH 2 O onder steriele omstandigheden. </li><li> B...

<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. 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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. 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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...

De ontwikkeling van vertakte epitheliale weefsels, zogenaamde vertakking morfogenese wordt geregeld door cel afkomstige, fysieke en omgevingsfactoren. In de melkklier, vertakkende morfogenese is een iteratief proces waarbij geleid collectieve celmigratie wordt een boom-achtige architectuur. De eerste stap is primair knopvorming uit de melkkanalen, gevolgd door tak initiatie en elongatie 1,2. Invasie van takken in het omliggende stroma wordt geïnduceerd door de systemische afgifte van steroïde hormonen in de puberteit. Nieuwe primaire knoppen vervolgens initiëren van de uiteinden van de bestaande vestigingen, en dit proces blijft een epitheliale boom 3 te creëren. Hoewel veel belangrijke biochemische signalen zijn geïdentificeerd, een uitgebreide kennis van de celbiologische mechanismen die leiden dit complex ontwikkelingsproces wordt momenteel ontbreekt. Bovendien mechanistische studies over de invloed van specifieke signalen zijn moeilijk te ontleden van experigen in vivo, zo precies spatiotemporele verstoringen en metingen zijn vaak niet mogelijk. Driedimensionale (3D) cultuur technieken, zoals hele orgelcultuur, primaire organoids en celcultuur modellen, zijn nuttige hulpmiddelen voor het systematisch onderzoek naar de mechanismen die ten grondslag liggen aan weefsel morfogenese 4-6. Deze kunnen bijzonder nuttig zijn voor het bepalen van de invloed van bepaalde factoren afzonderlijk, zoals mechanische krachten en biochemische signalen, op een verscheidenheid van cel gedrag, zoals migratie, proliferatie en differentiatie. 6 Engineered celkweekmodellen name gemakkelijk mogelijk de verstoring van afzonderlijke cellen en hun micromilieu. Een dergelijke cultuur model maakt gebruik van een microfabrication gebaseerde benadering van model borstklier epitheliale weefsels met gecontroleerde 3D-structuur die consistent en reproduceerbaar te vormen takken die samen migreren wanneer geïnduceerd met de een ingenieurppropriate groeifactoren. Het grote voordeel van het model is de mogelijkheid om precies te manipuleren en de effecten van fysische en biochemische factoren, zoals patronen van mechanische belasting, hoge statistische betrouwbaarheid te meten. Deze techniek, samen met computermodellen is al gebruikt om de relatieve bijdrage van fysische en biochemische signalen in de leiding van de normale ontwikkeling van mammaire epitheliale weefsels en andere epithelia vertakte 7-11 bepalen. Hier voorgesteld is een gedetailleerd protocol voor het bouwen van deze model weefsels, die gemakkelijk kan worden uitgebreid tot andere typen cellen en extracellulaire matrix (ECM) gels en die dient als een potentieel middel voor het testen van geneesmiddelen.

<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.

Algemeen schema van borstklieren epitheelweefsel microfabrication Een algemeen schema van de microfabrication procedure waarin het experimentele workflow wordt weergegeven in figuur 1. Het resultaat is een array van epitheliale weefsels van identieke geometrie en afstand die volledig zijn ingebed in een gel ECM. Een representatief experiment gebruikt EpH4 muizen mammaire epitheliale cellen gekweekt in een gel van bovine type ...

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

Dit manuscript beschrijft een zachte lithografie gebaseerde techniek uniforme arrays van driedimensionale (3D) epitheliale weefsels gedefinieerde geometrische midden van extracellulaire matrix ingenieur. Deze methode is vatbaar voor een grote verscheidenheid aan celtypen en experimentele contexten en zorgt voor high-throughput screening van identieke herhalingen.

Techniek Driedimensionale epitheelweefsels Embedded binnen extracellulaire matrix

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

Research

JoVE Journal

Bioengineering

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Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

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Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

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This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

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Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

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A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

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