Name: Una piattaforma innovativa per lo stretching e limaging cellulare in vitro (Video) | JoVE Journal
Uploaded: 2026-03-10T13:45:00.000+00:00
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Description: Scopri una piattaforma di stretching cellulare compatibile con microscopio per studiare la risposta meccanica di cellule muscolari lisce dell'aria umane. Questo metodo consente imaging in vivo e analisi quantitativa delle forze di trazione.

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<li>Arold, S. P., Bartolák-Suki, E., Suki, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Variable+stretch+pattern+enhances+surfactant+secretion+in+alveolar+type+II+cells+in+culture%5BTitle%5D)+AND+%22Am+J+Physiol+Lung+Cell+Mol+Physiol%22%5BJournal%5D)">Variable stretch pattern enhances surfactant secretion in alveolar type II cells in culture.</a> Am J Physiol Lung Cell Mol Physiol. 296, L574-L581 (2009).</li>
<li>Trepat, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Universal+physical+responses+to+stretch+in+the+living+cell%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Universal physical responses to stretch in the living cell.</a> Nature. 447, 592-595 (2007).</li>
<li>Benavides Damm, T., Egli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+%27s+role+in+mechanotransduction+during+muscle+development%5BTitle%5D)+AND+%22Cell+Physiol+Biochem%22%5BJournal%5D)">Calcium 's role in mechanotransduction during muscle development.</a> Cell Physiol Biochem. 33, 249-272 (2014).</li>
<li>Ito, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+Ca2%2B+influx+pathway+activated+by+mechanical+stretch+in+human+airway+smooth+muscle+cells%5BTitle%5D)+AND+%22Am+J+Respir+Cell+Mol+Biol%22%5BJournal%5D)">A novel Ca2+ influx pathway activated by mechanical stretch in human airway smooth muscle cells.</a> Am J Respir Cell Mol Biol. 38, 407-413 (2008).</li>
<li>Ito, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stretch-activated+calcium+mobilization+in+airway+smooth+muscle+and+pathophysiology+of+asthma%5BTitle%5D)+AND+%22Curr+Opin+Physiol%22%5BJournal%5D)">Stretch-activated calcium mobilization in airway smooth muscle and pathophysiology of asthma.</a> Curr Opin Physiol. 21, 65-70 (2021).</li>
<li>Ito, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Actin+cytoskeleton+regulates+stretch-activated+Ca2%2B+influx+in+human+pulmonary+microvascular+endothelial+cells%5BTitle%5D)+AND+%22Am+J+Respir+Cell+Mol+Biol%22%5BJournal%5D)">Actin cytoskeleton regulates stretch-activated Ca2+ influx in human pulmonary microvascular endothelial cells.</a> Am J Respir Cell Mol Biol. 43, 26-34 (2010).</li>
<li>Tanaka, Y., Hata, S., Ishiro, H., Ishii, K., Nakayama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quick+stretch+increases+the+production+of+inositol+1%2C4%2C5-trisphosphate+%28IP3%29+in+porcine+coronary+artery%5BTitle%5D)+AND+%22Life+Sci%22%5BJournal%5D)">Quick stretch increases the production of inositol 1,4,5-trisphosphate (IP3) in porcine coronary artery.</a> Life Sci. 55, 227-235 (1994).</li>
<li>Brown, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Techniques+for+mechanical+stimulation+of+cells+in+vitro%3A+a+review%5BTitle%5D)+AND+%22J+Biomech%22%5BJournal%5D)">Techniques for mechanical stimulation of cells in vitro: a review.</a> J Biomech. 33, 3-14 (2000).</li>
<li>Kamble, H., Barton, M. J., Jun, M., Park, S., Nguyen, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+stretching+devices+as+research+tools%3A+engineering+and+biological+considerations%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Cell stretching devices as research tools: engineering and biological considerations.</a> Lab Chip. 16, 3193-3203 (2016).</li>
<li>Fredberg, J. J., Bunk, D., Ingenito, E., Shore, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+resistance+and+the+contractile+state+of+lung+parenchyma%5BTitle%5D)+AND+%22J+Appl+Physiol%22%5BJournal%5D)">Tissue resistance and the contractile state of lung parenchyma.</a> J Appl Physiol. 74, 1387-1397 (1993).</li>
<li>Shen, Z. L., Dodge, M. R., Kahn, H., Ballarini, R., Eppell, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stress-strain+experiments+on+individual+collagen+fibrils%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Stress-strain experiments on individual collagen fibrils.</a> Biophys J. 95, 3956-3963 (2008).</li>
<li>Atcha, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+low-cost+mechanical+stretching+device+for+uniaxial+strain+of+cells%3A+a+platform+for+pedagogy+in+mechanobiology%5BTitle%5D)+AND+%22J+Biomech+Eng%22%5BJournal%5D)">A low-cost mechanical stretching device for uniaxial strain of cells: a platform for pedagogy in mechanobiology.</a> J Biomech Eng. 140, 0810051-0810059 (2018).</li>
<li>Tschumperlin, D. J., Margulies, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Equibiaxial+deformation-induced+injury+of+alveolar+epithelial+cells+in+vitro%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Equibiaxial deformation-induced injury of alveolar epithelial cells in vitro.</a> Am J Physiol. 275, L1173-L1183 (1998).</li>
<li>Meza, D., Abejar, L., Rubenstein, D. A., Yin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+shearing-stretching+device+that+can+apply+physiological+fluid+shear+stress+and+cyclic+stretch+concurrently+to+endothelial+cells%5BTitle%5D)+AND+%22J+Biomech+Eng%22%5BJournal%5D)">A shearing-stretching device that can apply physiological fluid shear stress and cyclic stretch concurrently to endothelial cells.</a> J Biomech Eng. 138, 4032550(2016).</li>
<li>Ursekar, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design+and+construction+of+an+equibiaxial+cell+stretching+system+that+is+improved+for+biochemical+analysis%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Design and construction of an equibiaxial cell stretching system that is improved for biochemical analysis.</a> PLoS One. 9, e90665(2014).</li>
<li>Pelham, R. J., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+locomotion+and+focal+adhesions+are+regulated+by+substrate+flexibility%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Cell locomotion and focal adhesions are regulated by substrate flexibility.</a> Proc Natl Acad Sci U S A. 94, 13661-13665 (1997).</li>
<li>Cell stretching bioreactor FAQs. , Flexcell International. 
 <a target="_blank" href="https://www.flexcellint.com/resources/faq">https://www.flexcellint.com/resources/faq</a> (2024).</li>
<li>Bossé, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+strain+on+airway+smooth+muscle+during+a+deep+inspiration+to+total+lung+capacity%5BTitle%5D)+AND+%22J+Eng+Sci+Med+Diagn+Ther%22%5BJournal%5D)">The strain on airway smooth muscle during a deep inspiration to total lung capacity.</a> J Eng Sci Med Diagn Ther. 2, 010802(2019).</li>
<li>Yoshie, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Traction+force+screening+enabled+by+compliant+PDMS+elastomers%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Traction force screening enabled by compliant PDMS elastomers.</a> Biophys J. 114, 2194-2199 (2018).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+an+open+source+platform+for+biological+image+analysis%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Fiji: an open source platform for biological image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li>
<li>Butler, J. P., Toli ć-Nørrelykke, I. M., Fabry, B., Fredberg, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Traction+fields%2C+moments%2C+and+strain+energy+that+cells+exert+on+their+surroundings%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">Traction fields, moments, and strain energy that cells exert on their surroundings.</a> Am J Physiol Cell Physiol. 282, C595-C605 (2002).</li>
<li>Dessalles, C. A., Leclech, C., Castagnino, A., Barakat, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integration+of+substrate-+and+flow-derived+stresses+in+endothelial+cell+mechanobiology%5BTitle%5D)+AND+%22Commun+Biol%22%5BJournal%5D)">Integration of substrate- and flow-derived stresses in endothelial cell mechanobiology.</a> Commun Biol. 4, 764(2021).</li>
<li>Ji, G., Barsotti, R. J., Feldman, M. E., Kotlikoff, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stretch-induced+calcium+release+in+smooth+muscle%5BTitle%5D)+AND+%22J+Gen+Physiol%22%5BJournal%5D)">Stretch-induced calcium release in smooth muscle.</a> J Gen Physiol. 119, 533-543 (2002).</li>
<li>Davis, M. J., Meininger, G. A., Zawieja, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stretch-induced+increases+in+intracellular+calcium+of+isolated+vascular+smooth+muscle+cells%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Stretch-induced increases in intracellular calcium of isolated vascular smooth muscle cells.</a> Am J Physiol. 263, H1292-H1299 (1992).</li>
<li>Naruse, K., Sokabe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Involvement+of+stretch-activated+ion+channels+in+Ca2%2B+mobilization+to+mechanical+stretch+in+endothelial+cells%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Involvement of stretch-activated ion channels in Ca2+ mobilization to mechanical stretch in endothelial cells.</a> Am J Physiol. 264, C1037-C1044 (1993).</li>
<li>Arold, S. P., Bartol ák-Suki, E., Suki, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Variable+stretch+pattern+enhances+surfactant+secretion+in+alveolar+type+II+cells+in+culture%5BTitle%5D)+AND+%22Am+J+Physiol+Lung+Cell+Mol+Physiol%22%5BJournal%5D)">Variable stretch pattern enhances surfactant secretion in alveolar type II cells in culture.</a> Am J Physiol Lung Cell Mol Physiol. 296, L574-L581 (2009).</li>
<li>Cell stretching bioreactors. , Flexcell International. 
 <a target="_blank" href="https://www.flexcellint.com">https://www.flexcellint.com</a> (2024).</li>
<li>Mitrossilis, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+response+to+stiffness+exhibits+muscle-like+behavior%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Single-cell response to stiffness exhibits muscle-like behavior.</a> Proc Natl Acad Sci U S A. 106, 18243-18248 (2009).</li>
<li>Parameswaran, H., Lutchen, K. R., Suki, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+computational+model+of+the+response+of+adherent+cells+to+stretch+and+changes+in+substrate+stiffness%5BTitle%5D)+AND+%22J+Appl+Physiol%22%5BJournal%5D)">A computational model of the response of adherent cells to stretch and changes in substrate stiffness.</a> J Appl Physiol. 116, 825-834 (2014).</li>
</ol>

<table><tbody><tr><td>&nbsp; Dulbecco' s Eagle modificata s medium/F12</td><td>Fisher Scientific</td><td>12634010</td><td>500 mL&nbsp;</td></tr><tr><td>(4-benzoilbenzil) cloruro trimetilammonio (PLPP)</td><td>Alv&eacute; Ole</td><td>N/A</td><td>14,5mg/mL</td></tr><tr><td>0,1% di gelatina coniugata con fluoresceina &nbsp;</td><td>Invitrogen</td><td>G13187</td><td></td></tr><tr><td>Soluzione di gelatina allo 0,1%</td><td>Sigma-Aldrich</td><td>ES-006</td><td>500 mL&nbsp;</td></tr><tr><td>1 & mu; Microsfere fluorescenti rosse modificate con carbossilato con diametro m</td><td>FluoSpheres</td><td>F8816</td><td></td></tr><tr><td>1&times; Soluzione di amminoacidi non essenziali MEM</td><td>Sigma-Aldrich</td><td>M7145</td><td>100 mL</td></tr><tr><td>1&times; Penicillina/streptomicina</td><td>Fisher Scientific</td><td>15-140-122</td><td>100 mL</td></tr><tr><td>Siero fetale bovino al 10%</td><td>Termo-Scientifico</td><td>A5256701</td><td>500 mL&nbsp;</td></tr><tr><td>Tubo da 15 mL</td><td>Fisher Scientific</td><td>14-959-53A</td><td></td></tr><tr><td>1x glutamine &nbsp;</td><td>Fisher Scientific</td><td>MT10010CM</td><td></td></tr><tr><td>1x Integratore di Crescita di Insulina-Transferrina-Selenio</td><td>Scienze della Vita di Corning</td><td>25-800-CR</td><td></td></tr><tr><td>Amfotericina B</td><td>Sigma-Aldrich</td><td>A2942</td><td>25 & mu; g/litro</td></tr><tr><td>Cuscinetti a sfere</td><td>McMaster-Carr&nbsp;</td><td>N/A</td><td></td></tr><tr><td>Diidrato di cloruro di calcio</td><td>Sigma-Aldrich</td><td>223506</td><td></td></tr><tr><td>Ferrovia per carrozze</td><td>McMaster-Carr&nbsp;</td><td>N/A</td><td></td></tr><tr><td>FLIPR Ca2+ 6</td><td>Dispositivi molecolari</td><td>R8190</td><td></td></tr><tr><td>GraphPad Prism</td><td>Dotmatics</td><td>N/A</td><td></td></tr><tr><td>Prosciutto' s F-12 medium & nbsp;</td><td>Sigma-Aldrich</td><td>N8641</td><td>500 mL&nbsp;</td></tr><tr><td>La soluzione salina bilanciata di Hanks&nbsp;</td><td>Termo-Scientifico</td><td>14025092</td><td>500 mL&nbsp;</td></tr><tr><td>BUFFER HEPES&NBSP;</td><td>Termo-Scientifico</td><td>15630080</td><td>100 mL</td></tr><tr><td>Leonardo Software</td><td>Alv&eacute; Ole</td><td>N/A</td><td></td></tr><tr><td>Matlab</td><td>Mathworks</td><td>N/A</td><td></td></tr><tr><td>metassil polietilenico glicole e linea; Succinimidile Valerato (mPEG-SVA)</td><td>Laysan Bio Inc</td><td>N/A</td><td>50mg/mL</td></tr><tr><td>Nusil Gel-8100</td><td>Nusil&nbsp;</td><td>N/A</td><td></td></tr><tr><td>Piatti di Petri</td><td>Termo-Scientifico</td><td>263991</td><td>90 mm&nbsp;</td></tr><tr><td>soluzione salina tamponata fosfato &nbsp;</td><td>Sigma-Aldrich</td><td>10010023</td><td>500 mL&nbsp;</td></tr><tr><td>poli-L-lisina (PLL)</td><td>Sigma-Aldrich</td><td>P8920</td><td>500&mu; g/mL</td></tr><tr><td>Cellule muscolari lisce delle vie aeree umane primarie &nbsp;</td><td>ATCC</td><td>PCS-130-011</td><td></td></tr><tr><td>Modulo ottico PRIMO &nbsp;</td><td>Alv&eacute; Ole</td><td>N/A</td><td></td></tr><tr><td>Viti</td><td>McMaster-Carr&nbsp;</td><td>N/A</td><td></td></tr><tr><td>SigmaStat</td><td>Systat Software</td><td>N/A</td><td></td></tr><tr><td>Rivestimento a filatura</td><td>Laurell</td><td>Modello WS-650MZ-23NPPB</td><td></td></tr><tr><td>Componenti di attacco per barella e palco&nbsp;</td><td>Struttura per strumenti scientifici dell'Università di Boston</td><td>N/A</td><td>Acciaio inox</td></tr><tr><td>Kit base Elastomer e Agente di Polimerizzazione Sylgard 184</td><td>Dow</td><td>H047J8Q026</td><td></td></tr><tr><td>Fiaschetta per coltura tessutale T-25 - Tappo di ventilazione, sterile</td><td>CellTreat</td><td>229331</td><td></td></tr><tr><td>Depressore linguale&nbsp;</td><td>Termo-Scientifico</td><td>22-363-154</td><td></td></tr></tbody></table>

una piattaforma innovativa per lo stretching e l'imaging cellulare in vitro

A Novel Platform for In Vitro Cellular Stretching and Imaging

Una piattaforma innovativa per lo stretching e l'imaging cellulare in vitro

Cells respond to mechanical cues from their environment, such as changes in extracellular matrix (ECM) stiffness and cyclic strain, which regulate ...

a-novel-platform-for-in-vitro-cellular-stretching-and-imaging

Research

Ricerca

JoVE Journal

Bioengineering

Bioingegneria

Presentiamo in questo articolo un romanzo stiramento piattaforma che può essere usato per studiare le risposte delle cellule singole a complessi anisotropo deformazione meccanica biassiale e quantificare le proprietà meccaniche dei tessuti biologici.

Una piattaforma Novel Stretching per applicazioni in cellule e tessuti mechanobiology

Riportiamo un dispositivo ed un nuovo metodo per studiare le cellule ed embrioni. celle singole sono proprio ordinati in array microcavità. Il loro confinamento 3D è un passo verso ambienti 3D incontrate in condizioni fisiologiche e permette l'orientamento organelli. Controllando la forma delle cellule, questa configurazione riduce al minimo la variabilità riportata nei test standard.

Ordinazione di singole cellule e singola embrioni in 3D confinamento: Un nuovo dispositivo per l&#39;alta Screening contenuti

Un nuovo protocollo di imaging è stato sviluppato utilizzando un attuatore meccanico motorizzato personalizzato per consentire la misurazione delle risposte in tempo reale alla deformazione meccanica nelle cellule vive. Rilevante per la meccanobiologia, il sistema può applicare deformazioni fino al 20% consentendo l'imaging quasi in tempo reale con microscopia confocale o a forza atomica.

Imaging Live Cell durante meccanica Stretch

Utilizzando un dispositivo progettato in precedenza per applicare la deformazione meccanica alle celle aderenti, questo documento descrive una geometria del substrato ridisegnata e un apparato personalizzato per l'imaging a cella singola ad alta risoluzione delle cellule tese con un obiettivo di immersione in olio 100x.

Imaging ad alta risoluzione di dinamiche nucleari in cellule vive sotto tensione uniassiale

Il metodo presentato prevede lo stiramento uniassiale di idrogel morbidi 3D incorporati nella gomma siliconica, consentendo al contempo la microscopia confocale viva. Vengono dimostrate la caratterizzazione dei ceppi idrogel esterni e interni e l'allineamento delle fibre. Il dispositivo e il protocollo sviluppati possono valutare la risposta delle cellule a vari regimi di deformazione.

Ceppo controllato di idrogel 3D in imaging a microscopia dal vivo

Questo lavoro presenta un protocollo flessibile per l'utilizzo della tecnologia FLECS (fluorescently labeled elastomeric contractible surfaces) in formato microwell per una quantificazione semplificata e hands-off delle forze contrattili a singola cellula basata sugli spostamenti visualizzati di micropattern proteici fluorescenti.

Analisi semplificata e ad alta produttività della contrattilità a cella singola utilizzando elastomeri micropatterati

Qui presentiamo un versatile dispositivo di allungamento equibiassiale ad azionamento pneumatico e compatibile con la microscopia ad alta risoluzione.

Dispositivo di stretching equibiassiale per microscopia a fluorescenza confocale a cellule vive ad alto ingrandimento

In molte situazioni biologici e clinici è vantaggioso per lo studio dei processi cellulari, come si evolvono nel loro microambiente nativo. Qui si descrive il montaggio e l'uso di un basso costo in fibra ottica microscopio in grado di fornire immagini in tempo reale in coltura cellulare, studi su animali e studi clinici del paziente.

Ad alta risoluzione in fibra ottica per Microendoscopy In situ Cellular Imaging

Questo protocollo dimostrerà l'estrazione e l'analisi di acidi grassi bioattivi liberi ed esterificati dalle cellule. Gli acidi grassi sono accuratamente quantificati utilizzando la diluizione degli isotopi stabili, la cromatografia liquida chirale, la cattura di elettroni, la ionizzazione chimica atmosferica, il monitoraggio delle reazioni multiple, la spettrometria di massa (SID-LC-ECAPCI-MRM/MS).

Estrazione dei lipidi cellulari per analisi mirate Stabile diluizione isotopica cromatografia liquida-spettrometria di massa

Drosophila larve sono un sistema modello attraente per l'imaging dal vivo a causa della loro cuticola trasparente e potenti genetica. Questo protocollo descrive come utilizzare un dispositivo PDMS singolo strato, chiamato il 'chip di larva' per l'imaging dal vivo di processi cellulari all'interno dei neuroni di 3 ° instar larve di Drosophila.

Utilizzando Chips microfluidica per l&#39;imaging dal vivo e studio delle risposte lesioni in<em&gt; Drosophila</em&gt; Larve

Scopri una piattaforma di stretching cellulare compatibile con microscopio per studiare la risposta meccanica di cellule muscolari lisce dell'aria umane. Questo metodo consente imaging in vivo e analisi quantitativa delle forze di trazione.

Una piattaforma innovativa per lo stretching e l'imaging cellulare in vitro

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