Name: a device for performing cell migration/wound healing in a 96-well plate
Uploaded: 2017-03-07T15:00:00.000+00:00
Duration: 5 min 46 s
Description: Watch this Scientific Journal Video about A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate at JoVE.com

Gli autori desiderano ringraziare il Sig Tam Po Leung, Mr. Wong Chi Kin e lo staff tecnico della Facoltà di Scienze Workshop, Hong Kong Baptist University, per le loro competenze tecniche e consigli per rendere il prototipo di questo feritore.

1. Introduzione alle parti del feritore (Figura 1) <ol><li> Preparare il supporto del perno fissando i perni ferimento a distanze uguali. Tenere i puntali e regolare l&#39;altezza della punta spostando i perni ferimento regolabili su e giù. </li><li> Montare la guida barra regolabile su diverse marche di piastre di coltura a 96 pozzetti e assicurarsi che l&#39;area ferimento è in una posizione fissa. </li><li> Fissare il perno ferimento regolabile stringendo la vite esagonale. Fissare la guida bar in posizione stringendo i tappi a testa esagonale incassata su entrambe le estremità. </li></ol> 2. Impostare la larghezza del supporto del perno (Figura 2) <ol><li> Allentare i tappi a testa esagonale incassata con la chiave esagonale M5. </li><li> Inserire un numero adeguato di regolazione anelli ad entrambi i lati del supporto del perno in modo che le barre di guida si adattano perfettamente con la larghezza della piastra di coltura a 96 pozzetti. NOTA: Corning e piastre Iwaki richiede 1 paio di grandi anelli e 1 paio di piccoli anelli. Falcon e Nunc plAtes bisogno 1 paio di grandi anelli. </li><li> Serrare la brugola esagonale tappi per fissare la barra di guida. </li></ol> 3. Regolare le barre di guida (Figura 3) <ol><li> Montare i perni ferendo con sterili punte di pipette di plastica da 10 microlitri. </li><li> Allentare i tappi a testa esagonale incassata con la chiave esagonale M5. </li><li> Tenere il feritore e immergere i perni ferimento in una colonna di una piastra di coltura a 96 pozzetti. </li><li> Regolare l&#39;altezza delle barre di guida fino a quando tutti i suggerimenti toccare appena il fondo dei pozzetti. </li><li> Serrare i tappi a brugola esagonale. Assicurarsi che il feritore ora si adatta perfettamente al piastra di coltura e che i perni sono ben posizionate nel mezzo dei pozzi. </li></ol> 4. La calibrazione dei perni <ol><li> Tenere il feritore perpendicolare ad una superficie sterile piatta (cioè Petri) e allentare tutte le viti a testa esagonale con la chiave esagonale M3. </li><li> Toccare il feritore fino a quando tutti i suggerimenti in modo uniforme toccare la superficie sterile piatta. &lt;/ Li&gt; <li> Serrare di nuovo le viti esagonali per bloccare i perni in posizione. </li><li> Controllare la regolarità di ogni punta toccando il feritore delicatamente sulla superficie sterile piatta. </li></ol> 5. Scratching cellulare monostrato (figura 4) <ol><li> Seme umano ombelicale cellule endoteliali vascolari su un 96-pozzetti di coltura cellulare di gelatina rivestite 0,1%. cellule di coltura in media 199 integrate con il 20% di siero di calore-inattivato fetale bovino, 1% di penicillina / streptomicina e 0,09 g / L di eparina. Mantenere le cellule in un incubatore umidificato con 5% di anidride carbonica a 37 ° C durante la notte prima di essere utilizzato. Le cellule devono raggiungere il 100% di confluenza prima ferimento. </li><li> Collocare le punte ferimento alla sinistra (o destra) lato di ciascun pozzetto nella stessa colonna della piastra di coltura. Far scorrere il feritore verso l&#39;altro lato del pozzo; assicurarsi che le punte ferimento tocchino il fondo del pozzo. </li><li> Ripetere la graffiare (Passo 5.2) per tutte le colonne. </li><li> Dopo il ferimento, scarti tegli media in ciascun pozzetto e sostituirlo con terreno contenente composti di test freschi. </li></ol> 6. Acquisizione dati e Image Analysis <ol><li> Immagine tutto bene con la ferita meccanica sul monostrato cellulare utilizzando un microscopio a bassa potenza con fotocamera digitale (es obiettivo 10X) immediatamente dopo ferimento (t = 0 h). </li><li> Aggiungere composti di prova se desiderato e incubare per il tempo desiderato. Immagine tutto bene con l&#39;area della ferita dopo il tempo di incubazione desiderato (t = Dh). </li><li> L&#39;utilizzo di immagini software di analisi come ad esempio ImageJ ( <a href="https://imagej.nih.gov/ij/">https://imagej.nih.gov/ij/</a> ), misurare l&#39;area ferito l&#39;immagine utilizzando lo strumento a mano libera. </li><li> Calcolare la percentuale di chiusura della ferita: <img alt="Equazione" src="/files/ftp_upload/55411/55411eq1.jpg" /></li></ol>

<ol>
	<li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3 (5), 362-374 (2003).</li><li>Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prespecification+and+plasticity:+shifting+mechanisms+of+cell+migration.">Prespecification and plasticity: shifting mechanisms of cell migration.</a> Curr Opin Cell Biol. 16 (1), 14-23 (2004).</li><li>Lampugnani, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+into+a+wounded+area+in+vitro.">Cell migration into a wounded area in vitro.</a> Methods Mol Biol. 96, 177-182 (1999).</li><li>Sholley, M. M., Gimbrone, M. A., Cotran, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+migration+and+replication+in+endothelial+regeneration:+a+study+using+irradiated+endothelial+cultures.">Cellular migration and replication in endothelial regeneration: a study using irradiated endothelial cultures.</a> Lab Invest. 36 (1), 18-25 (1977).</li><li>Gotlieb, A. I., Spector, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+into+an+in+vitro+experimental+wound:+a+comparison+of+porcine+aortic+endothelial+and+smooth+muscle+cells+and+the+effect+of+culture+irradiation.">Migration into an in vitro experimental wound: a comparison of porcine aortic endothelial and smooth muscle cells and the effect of culture irradiation.</a> Am J Pathol. 103 (2), 271-282 (1981).</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. 18 (5), 9980 (2015).</li><li>Yarrow, J. C., Periman, Z. E., Westwood, N. J., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+cell+migration+assay+using+scratch+wound+healing,+a+comparsion+of+image-based+readout+methods.">A high-throughput cell migration assay using scratch wound healing, a comparsion of image-based readout methods.</a> BMC Biotechnol. 4, 21 (2004).</li><li>Lauder, H., Frost, E. E., Hiley, C. R., Fan, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+repair+process+involved+in+the+repair+of+a+cell+monolayer+using+an+in+vitro+model+of+mechanical+injury.">Quantification of the repair process involved in the repair of a cell monolayer using an in vitro model of mechanical injury.</a> Angiogenesis. 2 (1), 67-80 (1998).</li><li>Yue, P. Y. K., Leung, E. P. Y., Mak, N. K., Wong, R. N. 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+simplified+method+for+quantifying+cell+migration/+wound+healing+in+96-well+plates.">A simplified method for quantifying cell migration/ wound healing in 96-well plates.</a> J. Biomol Screen. 15 (4), 427-433 (2010).</li><li>Yue, P. 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=Elucidation+of+the+mechanisms+underlying+the+angiogenic+effects+of+ginsenoside+Rg(1)+in+vivo+and+in+vitro.">Elucidation of the mechanisms underlying the angiogenic effects of ginsenoside Rg(1) in vivo and in vitro.</a> Angiogenesis. 8 (3), 205-216 (2005).</li><li>Kwok, H. H., Chan, L. S., Poon, P. Y., Yue, P. Y., Wong, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ginsenoside-Rg1+induces+angiogenesis+by+the+inverse+regulation+of+MET+tyrosine+kinase+receptor+expression+through+miR-23a.">Ginsenoside-Rg1 induces angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a.</a> Toxicol Appl Pharmacol. 287 (3), 276-283 (2015).</li></ol>

Gli autori dichiarano di non avere interessi finanziari concorrenti.

Il nostro feritore cella ha diverse caratteristiche uniche nel risolvere i problemi dei test di migrazione delle cellule tradizionali. Il feritore cellule meccanico a 8 canali è realizzato in acciaio inox di alta qualità (acciaio 304) con una lunga durata di vita che può essere sterilizzato in autoclave. Quasi tutti marchi commerciali di piastre di coltura a 96 pozzetti disponibili sul mercato possono essere utilizzati con questo wounder cellule meccanica causa della barra guida regolabile. Il disegno della barra gui...

La migrazione cellulare gioca un ruolo importante nei processi cellulari, come ad esempio l&#39;embriogenesi, la neurogenesi, l&#39;angiogenesi, la guarigione delle ferite, delle mucose riparazione, epiteliali-mesenchimali-transizioni in fase di sviluppo normale e la situazione della malattia 1. Si tratta di un processo complicato, che richiede un coordinamento delle numerose manifestazioni inter- e intra-cellulari, compresi segnalazione interazioni molecola, la polarizzazione delle cellule, la riorganizzazione del citoscheletro, rimodellamento della matrice, protrusione della membrana e dinamica cellula-cellula adesione modulazione 2. Studiando la migrazione delle cellule, la scoperta e la convalida delle sostanze chimiche o biomolecole che conducono al movimento delle cellule e le relative vie biochimiche possono essere determinati. Questo processo fondamentale può essere utilizzato vantaggiosamente come proxy per varie applicazioni, come il trattamento di malattia e drug targeting. Il saggio ferendo è uno dei saggi di migrazione cellulare 3. Qui, unsingolo campione di cellule viene parzialmente rimosso dai mezzi meccanici per produrre una zona denudata cui le cellule migreranno per coprire l&#39;area. La percentuale di recupero in un particolare momento sarà monitorato. Quando si maneggiano gran numero di campioni, questioni come il costo sperimentale e la contaminazione incrociata all&#39;interno di campioni possono essere problematico. Anche se molti strumenti commerciali e saggi sono disponibili per lo studio migrazione delle cellule 4, 5, 6, molti di loro richiesto attrezzature costose e sofisticate e miglioramenti sono ancora necessari. Per queste ragioni, un wounder cellule meccanico 8 canali (Figura 1) è sviluppato. Il nostro wounder cellule meccanica 8 canali ha incorporato diverse caratteristiche uniche nel risolvere i suddetti problemi. Esso fornisce la flessibilità necessaria per eseguire la migrazione delle cellule / ferendo saggi nel formato piastra di coltura a 96 pozzetti. La regolazione guprogettazione bar Iding assicura che la zona zero è in posizione centrale di ogni bene. Inoltre, l&#39;altezza delle barre di guida può essere regolato in modo che il feritore è applicabile per varie marche di piastre di coltura. Infine, il design pin ferimento regolabile consente un ancora di contatto delle punte delle pipette con la superficie della piastra di coltura per ottenere simultaneamente e riproducibile ferimento 7, 8.

<table><tbody><tr><td>96-well cell culture plate</td><td>Nunc</td><td>167008</td><td>Other brands of 96-well cell culture plate can also be used</td></tr><tr><td>P10 pipette tips</td><td>Axygen</td><td>301-03-051</td><td>Short P10 pipette tip is more easy to create a clear wound</td></tr><tr><td>Wounder</td><td>R&amp;amp;P Technology Limited</td><td></td><td></td></tr><tr><td>Medium 199</td><td>Sigma</td><td>M2520-1L</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>26140079</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140122</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa</td><td>Sigma</td><td>H3393</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr><tr><td>Gelatin from bovine skin</td><td>Sigma</td><td>G9391</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr></tbody></table>

a device for performing cell migration/wound healing in a 96-well plate

La migrazione delle cellule / ferendo test è un metodo comunemente utilizzato per studiare la migrazione delle cellule e altri processi biologici, come l&#39;angiogenesi e metastasi tumorali. In questo saggio, le cellule sono coltivate per formare un monostrato confluente e una ferita meccanica viene creato graffiando con un dispositivo. Allora il tasso di migrazione delle cellule verso la zona denudato può essere monitorato mediante imaging. Il nostro wounder meccanica 8 canali è progettato per affrontare la maggior parte dei problemi associati con il test migrazione cellulare. In primo luogo, la nostra wounder può essere facilmente sterilizzato in autoclave o con comuni disinfettanti. In secondo luogo, i singoli pin regolabili permettono anche contatto con la piastra di coltura cellulare in modo che possono essere creati ferite taglienti e riproducibili. In terzo luogo, le barre di guida su entrambi i lati del wounder assicurano posizione ferimento consistente in ciascun pozzetto. L&#39;uso di puntali di plastica per il ferimento può fornire ulteriori migliore gestione del feritore e minimizzare le contaminationico. In conclusione, il nostro wounder cella può fornire ai ricercatori un dispositivo facile e riproducibile utente per l&#39;esecuzione del test migrazione cellulare utilizzando la piastra standard coltura a 96 pozzetti.

Questo wounder meccanico 8 canali è costruito graffiare un monostrato di cellule al fine di eseguire il saggio migrazione cellulare. Si tratta di un dispositivo di facile utilizzo in grado di svolgere cellulari graffiare test in piastre da 96 pozzetti in meno di un minuto senza formazione specifica. Questo wounder può introdurre aree avvolte su monostrati di cellule con una larghezza uniforme di circa 600 micron e con spigoli vivi (figure 5 e 6). La la...

Watch this Scientific Journal Video about A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate at JoVE.com

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

Qui, presentiamo un protocollo per eseguire un test di migrazione cellulare semi-high throughput su una piastra di coltura cellulare a 96 pozzetti. Questo protocollo è un metodo rapido, semplice ed economico per creare ferite scratch coerenti su un monostrato di cellule.

Un dispositivo per realizzare la migrazione delle cellule / Wound Healing in una piastra a 96 pozzetti

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

a-device-for-performing-cell-migrationwound-healing-in-a-96-well-plate

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

10.4K Views.  Hong Kong Baptist University.  Qui, presentiamo un protocollo per eseguire un test di migrazione cellulare semi-high throughput su una piastra di coltura cellulare a 96 pozzetti. Questo protocollo è un metodo rapido, semplice ed economico per creare ferite scratch coerenti su un monostrato di cellule. 

Video: A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

Valutazione del processo di migrazione e l'invasione delle cellule: un confronto dello Scratch Video basati su microscopio ferita test e le analisi della camera di Boyden

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

Ricerca

Ricerca sul cancro

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, MRP4 can regulate multiple cyclic nucleotide-dependent cellular events including cell migration. Previously, we demonstrated that in the absence of MRP4, fibroblast cells contain higher levels of intracellular cyclic nucleotides and can migrate faster. To understand the underlying mechanisms of this finding, we adopted a direct yet multifaceted approach. First, we isolated potential interacting protein complexes of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by mass-spectrometry. After identifying unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. We elucidated the potential role of the MRP4 protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Using high-content microscopy, we performed cell-migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of the actin cytoskeleton or inhibition of cAMP-dependent kinase A (PKA). To visualize signaling modulations in a migrating cell in real time, we utilized a FRET-based sensor for measuring PKA activity and found, the presence of more polarized PKA activity near the leading edge of migrating Mrp4-/- fibroblast, compared to Mrp4+/+fibroblasts. This in turn increased cortical actin formation and augmented the process of migration. Our approach enables identification of the proteins acting downstream to MRP4 and provides us with an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.

MRP4 regulates various cyclic nucleotide-dependent signaling events including a recently elucidated role in cell migration. We describe a direct, but multifaceted approach to unravel the downstream molecular targets of MRP4 resulting in identification of a unique MRP4 interactome that plays key roles in the fine-tuned regulation of fibroblast migration.

Metodi per studiare Mrp4 contenenti complessi macromolecolari nel regolamento di Fibroblasto migrazione

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

Biologia dello sviluppo

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Un flusso di lavoro semplice migrazione/invasione utilizzando un Imager di vivere-cella automatizzata

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Che caratterizzano la migrazione delle cellule all'interno del tridimensionale In Vitro ferita ambienti

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

Bioingegneria

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

Un romanzo In Vitro ferita guarigione test per valutare la migrazione cellulare

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

Immunologia e infezioni

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

La microscopia basato su metodi per la valutazione della migrazione delle cellule epiteliali durante In Vitro la guarigione arrotolata

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

Medicina

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Nella migrazione delle cellule in vitro e saggi Invasion

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

Biologia

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Scratch Migration Ssay and Dorsal Skinfold Chamber for In Vitro e In Vivo Analisi della Guarigione delle Ferite

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

Quantifying Three-Dimensional Cell Migration Within and Into Granular Hydrogel Biomaterials

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for tissue integration. This article introduces two novel approaches for quantifying cell migration within and into granular hydrogels, highlighting the distinct applications of these scaffolds. First, a cell monolayer interface assay that simulates tissue growth into granular hydrogels for integration purposes is presented. Second, a spheroid-based assay is described, designed to track cell movement within the hydrogel matrix, specifically suited for applications involving cell delivery. Both methods enable precise and controlled measurements of cell migration, providing a comprehensive toolkit for researchers utilizing granular hydrogel scaffolds. The motivation for these methods stems from the need for tailored control over cell migration within the scaffold to align with specific applications. By optimizing and standardizing these quantification techniques, researchers can iteratively refine granular hydrogel properties, ensuring their effectiveness in diverse regenerative medicine contexts. This robust set of quantitative tools offers new opportunities to enhance granular hydrogel scaffolds, advancing their use in both cell delivery and tissue integration applications.

A protocol for the quantitative evaluation of 3D cell migration within and into the interface of granular hydrogels is presented here.

Quantificazione della migrazione cellulare tridimensionale all'interno e all'interno di biomateriali granulari in idrogel

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for ...

An Innovative 3D-Printed Insert Designed to Enable Straightforward 2D and 3D Cell Cultures

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of conditioned media remains time-consuming and far from physiological and pathological conditions. Although a few models of co-culture are commercially available, they remain restricted to specific assays and are mostly for two types of cells. 
Here, 3D-printed inserts are used that are compatible with numerous functional assays. The insert allows the separation of one well of a 6-well plate into four compartments. A wide range of combinations can be set. Moreover, windows are designed in each wall of the compartments so that potential intercellular communication between every compartment is possible in the culture medium in a volume-dependent manner. For example, paracrine intercellular communication can be studied between four cell types in monolayer, in 3D (spheroids), or by combining both. In addition, a mix of different cell types can be seeded in the same compartment in 2D or 3D (organoids) format. The absence of a bottom in the 3D-printed inserts allows the usual culture conditions on the plate, possible coating on the plate containing the insert, and direct visualization by optical microscopy. The multiple compartments provide the possibility to collect different cell types independently or to use, in each compartment, different reagents for RNA or protein extraction. In this study, a detailed methodology is provided to use the new 3D-printed insert as a co-culture system. To demonstrate several capacities of this flexible and simple model, previously published functional assays of cell communication were performed in the new 3D-printed inserts and were demonstrated to be reproducible. The 3D-printed inserts and the conventional cell culture using conditioned media led to similar results. In conclusion, the 3D-printed insert is a simple device that can be adapted to numerous models of co-cultures with adherent cell types.

In this paper, a newly designed 3D-printed insert is presented as a model of co-culture and validated through the study of the paracrine intercellular communication between endothelial cells and keratinocytes.

Un innovativo inserto stampato in 3D progettato per consentire colture cellulari 2D e 3D semplici

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of ...