Name: characterizing cell migration within three-dimensional in vitro wound environments
Uploaded: 2017-08-16T13:00:00
Description: Watch this Scientific Journal Video about Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments at JoVE.com

Finansiering til dette arbejde blev leveret gennem American Heart Association (16SDG29870005) og North Carolina State universitetsforskning og Innovation frø finansiering.

1. dag 1: celle og reagens Bemærk: alle celle og reagens forberedelse bør udføres i en biologisk sikkerhed kabinet til at forhindre forurening af prøver.
<ol><li>Varmt filtreret Dulbecco &#39; s ændret Eagle &#39; s Medium (DMEM), føtal bovint serum (FBS), L-glutamin, og penicillin/streptomycin i et 37 ° C vandbad.</li>
	<li>Forbered neonatal human dermal fibroblast (HDFn) medier ved at supplere varmede DMEM med 10% FBS, 1% penicillin/streptomycin og 1% L-glutamin.</li>
	<li>Tø et ...

<ol>
	<li>Chester, D., Brown, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+biophysical+properties+of+provisional+matrix+proteins+in+wound+repair.">The role of biophysical properties of provisional matrix proteins in wound repair.</a> Matrix Biol. , (2016).</li><li>Martin, P., Leibovich, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+cells+during+wound+repair:+the+good,+the+bad+and+the+ugly.">Inflammatory cells during wound repair: the good, the bad and the ugly.</a> Trends in Cell Biology. 15 (11), 599-607 (2005).</li><li>Lin, B., Yin, T., Wu, Y. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-dimensional+topography+underlies+three-dimensional+fibrillar+cell+migration.">One-dimensional topography underlies three-dimensional fibrillar cell migration.</a> J Cell Bio. 184 (4), 481-490 (2009).</li><li>Fraley, S. I., 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=A+distinctive+role+for+focal+adhesion+proteins+in+three-dimensional+cell+motility.">A distinctive role for focal adhesion proteins in three-dimensional cell motility.</a> Nat Cell Biol. 12 (6), 598-604 (2010).</li><li>Petrie, R. J., Doyle, A. D., Yamada, 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=Random+versus+directionally+persistent+cell+migration.">Random versus directionally persistent cell migration.</a> Nat Rev. Mol Cell Biol. 10 (8), 538-549 (2009).</li><li>Rommerswinkel, N., Niggemann, B., Keil, S., Z&#228;nker, K. S., Dittmar, T. <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+within+a+Three-dimensional+Collagen+Matrix.">Analysis of Cell Migration within a Three-dimensional Collagen Matrix.</a> J Vis Exp. (92), (2014).</li><li>Jonkman, J. E. N., 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=An+introduction+to+the+wound+healing+assay+using+live-cell+microscopy.">An introduction to the wound healing assay using live-cell microscopy.</a> Cell Adh Migr. 8 (5), 440-451 (2014).</li><li>L&#228;mmermann, T., 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=Rapid+leukocyte+migration+by+integrin-independent+flowing+and+squeezing.">Rapid leukocyte migration by integrin-independent flowing and squeezing.</a> Nature. 453 (7191), 51-55 (2008).</li><li>Chen, Z., Yang, J., Wu, B., Tawil, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Three-Dimensional+Wound+Healing+Model.">A Novel Three-Dimensional Wound Healing Model.</a> J Dev Biol. 2 (4), 198-209 (2014).</li><li>Ross, R., Benditt, E. P. <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+and+collagen+formation.">Wound healing and collagen formation.</a> The J Cell Biol. 12 (3), 533-551 (1962).</li><li>Sproul, E. P., Argraves, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cytokine+axis+regulates+elastin+formation+and+degradation.">A cytokine axis regulates elastin formation and degradation.</a> Matrix Biol. 32 (2), 86-94 (2013).</li><li>Almine, J. F., Wise, S. G., Weiss, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastin+signaling+in+wound+repair.">Elastin signaling in wound repair.</a> Birth Defects REs C Embryo Today. 96 (3), 248-257 (2012).</li><li>Tracy, L. E., Minasian, R. A., Caterson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Matrix+and+Dermal+Fibroblast+Function+in+the+Healing+Wound.">Extracellular Matrix and Dermal Fibroblast Function in the Healing Wound.</a> Adv Wound Care. 5 (3), 119-136 (2016).</li><li>Cox, S., Cole, M., Tawil, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+of+Human+Dermal+Fibroblasts+in+Three-Dimensional+Fibrin+Clots:+Dependence+on+Fibrinogen+and+Thrombin+Concentration.">Behavior of Human Dermal Fibroblasts in Three-Dimensional Fibrin Clots: Dependence on Fibrinogen and Thrombin Concentration.</a> Tissue Eng. 10 (5-6), 942-954 (2004).</li><li>Brown, A. C., Barker, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin-based+biomaterials:+Modulation+of+macroscopic+properties+through+rational+design+at+the+molecular+level.">Fibrin-based biomaterials: Modulation of macroscopic properties through rational design at the molecular level.</a> Acta Biomaterialia. 10 (4), 1502-1514 (2014).</li><li>Bartosh, T. J., Ylostalo, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+anti-inflammatory+mesenchymal+stem/precursor+cells+(MSCs)+through+sphere+formation+using+hanging+drop+culture+technique.">Preparation of anti-inflammatory mesenchymal stem/precursor cells (MSCs) through sphere formation using hanging drop culture technique.</a> Curr Protoc Stem Cell Biol. 28, (2014).</li><li>Bainbridge, P. <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+and+the+role+of+fibroblasts.">Wound healing and the role of fibroblasts.</a> J Wound Care. 22 (8), 410-412 (2013).</li></ol>

Denne protokol giver mulighed for undersøgelse af celle migration i sårheling forbundet ECMs gennem en in vitro- 3D model. Et afgørende skridt i korrekt udførelse af fremgangsmåden er en hensigtsmæssig udvikling af fibroblast spheroids; celle koncentration under forberedelse var optimeret til at give en begyndelseskoncentration af 2.500 celler/slip, så spheroids form pålideligt over en inkubationstid på 72 h. Hvis et tilstrækkeligt antal celler anvendes, spheroids kan ikke sammenregnet effektivt og kan...

Sårheling er en kompleks proces, hvilket resulterer i genoprettelse af vævet intakt efter skade. Denne proces er opdelt i fire overlappende faser omfattet af hæmostase, betændelse, spredning og remodellering, som reguleres hver af en kompleks kombination af opløselige stikord, celle-matrix interaktioner og cell-celle kommunikation. 1 , 2 orkestrering af disse stikord styrer et væld af cellulære svar i sårheling herunder vigtigere, celle migration. 3 , 4 celle migration er en yderst dynamisk proces afhængig af de biokemiske og biofysiske egenskaber af ekstracellulære matrix milieu og cellulære fænotyper. 5 celler løbende sonde deres ekstracellulære matrix miljø gennem integrin-medieret veje; denne proces gør det muligt for cellerne til at fornemme en bred vifte af matrix egenskaber herunder topografi, stivhed og indespærring. Todimensionale (2D) analyse af celle migration viser, at migration er drevet af lamellipodia dannelse på forkant gennem generation af actin glødetrådens bundter. Efterfølgende dannelse af fokale sammenvoksninger på forkant i kombination med actomyosin drevet sammentrækning og retraktion på bagkanten, drev cellen frem. 6 tredimensionalt (3D) cellulære migration er i øjeblikket ikke godt forstået, men nyere undersøgelser viser, at 3D migration er markant anderledes end de før beskrevne mekanisme i 2D og er sandsynligvis drevet af alterative mekanismer. For eksempel, de seneste undersøgelser af Petrie mfl. påvist, at afhængigt af egenskaberne for ECM, celler i 3D matricer kan skifte mellem lamellipodium og lobopodium drevet mekanismer af migration. 7 fordi celle migration er et afgørende element i sårheling svar, 3D-modeller af celle migration er afgørende for at forstå fysiologiske reaktioner på forskellige stimuli under sårheling. Selv om celle vandrende adfærd på 2D overflader har vist sig at variere meget fra migration i 3D matricer, udnytte de fleste nuværende in vitro- modeller af celle migration under sårheling proces, herunder de veletablerede scratch assay, 2D éncellelag kulturer. 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 mere nylig udviklet assays har udnyttet en 3D matrix, men stadig kultur celler i en éncellelag på toppen af matricen, hvilket begrænser evnen til at virkelig sammenfatte tredimensionalitet af celle miljø in vivo. 15
Formålet med metoden beskrevet heri er at undersøge celle migration i en fysiologisk relevante 3D model i stedet for på en 2D overflade, for at opnå en mere robust forståelse af migration svar forbundet med de anvendte stimuli. Denne metode giver mulighed for evaluering af celle migration inden for en 3D fibrin blodprop matrix i overværelse af faktorer, der aktivere eller hæmme veje tilknyttet celle migration. En fibrin matrix blev valgt for denne metode på grund af den kritiske rolle fibrin spiller i de tidlige stadier af sårheling; følgende skade, en fibrin blodprop er dannet inden for sår miljøer at dæmme op for blodtab og fungerer som et stillads til første celle infiltration under væv reparation og ombygning. 2 , 16 , 17 , 18 , 19 , 20 desuden fibrin bruges klinisk som en kirurgisk fugemasse og sammensætningen af fugemasser har været bundet til celle migration og ultimative healing resultater. En tidligere undersøgelse af Cox mfl. undersøgt fibroblast migration med fibrin blodpropper består af varierende fibrin og thrombin koncentrationer med henblik på at optimere en fibrin fugemasse. I disse undersøgelser, var migration af celler ud af fibrin blodpropper på plastic retter kvantificeret. 20 her præsenterer vi en metode, der giver mulighed for kvantificering af migration af celler i 3D fibrin miljø. Mens metoden beskrevet i denne protokol specifikt bruger fibrin, kan denne model let modificeret til at bruge alternative matrix materialer som ønsket, såsom kollagen eller andre 3D matricer. Derudover præsenterer vi brugen af fibroblast spheroids i denne analyse, fordi fibroblast migration i såret seng er af afgørende betydning for ekstracellulære matrix syntese og væv reparation/ombygning. Men under reparation processen neutrofile er den første celletype skal overflyttes til sår bed, efterfulgt af makrofager. Fibroblaster derefter begynde at infiltrere sår miljø ca 48 timer efter første skade, i begyndelsen af den proliferative fase af sårheling proces. 19 denne analyse og kan let modificeret til at omfatte neutrofiler, makrofager eller andre celletyper til at vurdere, hvordan ændringer i fibrin blodprop struktur påvirker migration af disse celletyper. 21
For at gennemføre denne analyse, er en fibroblast klumpformet først dannet, ved hjælp af en ændring af teknikker tidligere beskrevet for stamcelleforskning kultur. 22 fibroblast klumpformet overføres efterfølgende til en 3D fibrin matrix, og udvækst kan derefter nemt overvåges og kvantificeres over flere dage. Denne protokol tager hensyn til 3D af fysiologiske systemer ved at integrere 3D celle spheroids inden for en fibrin matrix, således at man undgår begrænsningerne i relevans som følge af en ved hjælp af en 2D vækst overflade med en celle éncellelag til vandrende adfærd, mens stadig mulighed for evaluering af celle adfærd i et stærkt kontrolleret in vitro- miljø.

<table border="0" cellpadding="0" cellspacing="0" width="692">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Materials</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Dulbecco&#39;s Modified Eagle&#39;s Medium, 4.5 g/L Glucose, w/ Sodium Pyruvate, w/out L-Glutamine</td>
			<td style="width:91px;">VWR</td>
			<td>VWRL0148-0500</td>
			<td style="width:217px;">DMEM already containing L-glutamine can also be used</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">100mm Tissue Culture Dish, Non-Treated, Sterilized, Non-Pyrogenic</td>
			<td style="width:91px;">VWR</td>
			<td>10861-594</td>
			<td style="width:217px;">Dishes of any size can be used for hanging drop culture&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Fetal Bovine Serum from USDA approved countries, heat inactivated, sterile-filtered, cell culture tested</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td>12306C-500ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">L-glutamine</td>
			<td style="width:91px;">Fisher Scientific</td>
			<td>ICN1680149</td>
			<td style="width:217px;">Not needed if using DMEM that already contains L-glutamine&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:267px;">Penicillin-Streptomycin Solution stabilized, sterile-filtered, with 10,000 units penicillin and 10 mg streptomycin/mL</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td>P4333-100ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Human Dermal Fibroblasts, neonatal</td>
			<td style="width:91px;">Thermo Fisher</td>
			<td>C0045C</td>
			<td style="width:217px;">Can be replaced with other cell type of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">21 G x 1 1/2&#39; needle</td>
			<td style="width:91px;">BD&#160;</td>
			<td>305167</td>
			<td>22 G x 1 1/2 needles will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">1 mL syringe</td>
			<td style="width:91px;">VWR</td>
			<td>89174-491</td>
			<td>Syringes of any volume can be used</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Cell Culture Multiwell Plates, Polystyrene, Greiner Bio-One (Individially Wrapped) (48 wells)</td>
			<td style="width:91px;">VWR</td>
			<td>82051-004&#160;</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Human Fibrinogen - Plasminogen, von Willebrand Factor and Fibronectin Depleted</td>
			<td style="width:91px;">Enzyme Research Laboratories</td>
			<td>FIB 3</td>
			<td style="width:217px;">Can be replaced with matrix protein of interest</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Human Alpha Thrombin</td>
			<td style="width:91px;">Enzyme Research Laboratories</td>
			<td>HT 1002a</td>
			<td style="width:217px;">May not be necessary depending on matrix protein of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Equipment</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">BSL2 cell culture hood</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cell culture incubator</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Inverted microscope with 10X objective</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Centrifuge compatibile with 15 mL tubes</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing cell migration within three-dimensional in vitro wound environments

I øjeblikket de fleste in vitro- modeller af sårheling, såsom veletablerede scratch assays, indebærer at studere celle migration og sår lukning på todimensionelle flader. Den fysiologiske miljø i hvilke i vivo sår healing finder sted er imidlertid tre-dimensionelle snarere end to-dimensionelle. Det bliver stadig tydeligere, at celle adfærd varierer stærkt i to-dimensionelle vs tre-dimensionelle miljøer; Derfor er der behov for mere fysiologisk relevante in vitro- modeller for at studere celle migration adfærd i såret lukning. Den metode beskrevet heri giver mulighed for undersøgelse af celle migration i en tre-dimensionelle model, der bedre afspejler fysiologiske tilstande end tidligere etablerede to-dimensionelle scratch assays. Formålet med denne model er at vurdere celle udvækst via gennemgangen af celle migration fra en klumpformet krop er indlejret i en fibrin matrix i overværelse af pro eller anti migratory faktorer. Du bruger denne metode, celle udvækst fra klumpformet kroppen i en tredimensional matrix kan observeres og er let kvantificerbare over tid via brightfield mikroskopi og analyse af klumpformet krop område. Effekten af pro-vandrende og/eller hæmmende faktorer på celle migration kan også blive evalueret i dette system. Denne metode giver forskere med en enkel metode til at analysere celle migration i tre-dimensionelle sår forbundet matricer i vitro, hvilket øger relevansen af in vitro celle undersøgelser forud for anvendelsen af i vivo dyr modeller.

Klumpformet kultur kan udnyttes til korrekt vurdering af effekten af pro- og anti-trækfugle agenter på fibroblast migration in vitro- 3D fibroblast spheroids kan dannes via hængende drop kultur over en periode på 72 h (figur 1). Efter perioden kultur spheroids er indlejret i matrixen blodprop og afbildet ved hjælp af brightfield mikroskopi (figur 2). De første områder i spheroids (afbildet i figur som d...

Watch this Scientific Journal Video about Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments at JoVE.com

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Målet med denne protokol er at vurdere effekten af pro - og anti - migratory faktorer på celle migration inden for en tre-dimensionel fibrin matrix.

Kendetegner celle Migration inden for tre-dimensionelle In Vitro sår miljøer

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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Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

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Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

Mikroinjektion Wound Assay og<em&gt; In vivo</em&gt; Lokalisering af epidermale Wound Respons Journalister i<em&gt; Drosophila</em&gt; Embryoner.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

Analyse af Cell Migration inden for en tre-dimensionel kollagenmatrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

Generation af en tre-dimensionel fuld tykkelse Skin Equivalent og Automated Anskydning

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

Øjenirritation Test (EIT) for Hazard Identifikation af Eye Irriterer Kemikalier hjælp rekonstrueret human Cornea-lignende Epithelial (RHCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Udarbejdelse og analyse af<em&gt; In vitro</em&gt; Tre Dimensional brystcarcinoma surrogater

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

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, ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

Dyrkning pattedyrsceller i tre-dimensionelle peptid stilladser

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