Name: observing and quantifying fibroblast-mediated fibrin gel compaction
Uploaded: 2014-01-16T13:45:00
Description: Watch this Scientific Journal Video about Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction at JoVE.com

Ringraziamo George Giudice e Steven Eliason per aver donato fibroblasti dermici umani e Ramesh Raghupathy per l'aiuto con l'algoritmo di tracciamento del ceppo. Il sostegno a questo lavoro è stato fornito da un Dipartimento dell'Assistenza ai laureati del Dipartimento dell'Istruzione degli Stati Uniti nelle aree della National Need Fellowship (GAANN P200A120071).

1. Preparazione stencil
Preparare uno stencil su Parafilm per disartillare la posizione di ogni espianto, seguendo lageometria desiderata (figure 1A e 1B). Distanziare ciascuno di circa 1-2 mm di distanza. Questa distanza corrisponde a una spaziatura ideale per generare l'allineamento delle fibre tra le espianto. Attaccare lo stencil sotto l'area del vetro di copertura in cui il campione verrà preparato con nastro adesivo.
<p class="jove_ti...

<ol>
	<li>Grinnell, F., Petroll WM, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+motility+and+mechanics+in+three-dimensional+collagen+matrices.">Cell motility and mechanics in three-dimensional collagen matrices.</a> Annu. Rev. Cell. Dev. Biol. 26, 335-361 (2001).</li><li>Sander, E. A., Barocas, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomimetic collagen tissues: collagenous tissue engineering and other applications. In: Collagen: Structure and Mechanics. , (2008).</li><li>Pedersen JA, ., Swartz MA, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanobiology+in+the+third+dimension.">Mechanobiology in the third dimension.</a> Ann. Biomed. Eng. 33 (11), 1469-1490 (2005).</li><li>Bell, E., Ivarsson, B., Merrill, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+tissue-like+structure+by+contraction+of+collagen+lattices+by+human+fibroblasts+of+different+proliferative+potential+in+vitro.">Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro.</a> Proc. Natl. Acad. Sci. U.S.A. 76 (3), 1274 (1979).</li><li>Guidry, C., Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+mechanism+of+hydrated+collagen+gel+reorganization+by+human+skin+fibroblasts.">Studies on the mechanism of hydrated collagen gel reorganization by human skin fibroblasts.</a> J. Cell. Sci. 79 (1), 67-81 (1985).</li><li>Fluck, J., Querfeld, C., Cremer, A., Niland, S., Krieg, T., Sollberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+human+primary+fibroblasts+undergo+apoptosis+in+three-dimensional+contractile+collagen+gels.">Normal human primary fibroblasts undergo apoptosis in three-dimensional contractile collagen gels.</a> J. Invest. Dermatol. 110 (2), 153-157 (1998).</li><li>Rosenfeldt, H., Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+quiescence+and+the+disruption+of+ERK+signaling+in+mechanically+unloaded+collagen+matrices.">Fibroblast quiescence and the disruption of ERK signaling in mechanically unloaded collagen matrices.</a> J. Biol. Chem. 275 (5), 3088-3092 (2000).</li><li>Nakagawa, S., Pawelek, P., Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+of+fibroblasts+in+contracted+collagen+gels:+Effects+on+cell+growth+and+biosynthetic+activity.">Long-term culture of fibroblasts in contracted collagen gels: Effects on cell growth and biosynthetic activity.</a> J. Invest. Dermatol. 93 (6), 792-798 (1989).</li><li>Harris, A. K., Stopak, D., Wild, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+traction+as+a+mechanism+for+collagen+morphogenesis.">Fibroblast traction as a mechanism for collagen morphogenesis.</a> Nature. 290, 249-251 (1981).</li><li>Stopak, D., Harris AK, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connective+tissue+morphogenesis+by+fibroblast+traction:+I.+tissue+culture+observations.">Connective tissue morphogenesis by fibroblast traction: I. tissue culture observations.</a> Dev. Biol. 90 (2), 383-398 (1982).</li><li>Sawhney, R. K., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+local+movements+of+collagen+fibers+by+fibroblasts+drive+the+rapid+global+self-organization+of+collagen+gels.">Slow local movements of collagen fibers by fibroblasts drive the rapid global self-organization of collagen gels.</a> J. Cell. Biol. 157 (6), 1083-1092 (2002).</li><li>Provenzano, P. P., Inman, D. R., Eliceiri, K. W., Trier, S. M., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+guidance+mediated+three-dimensional+cell+migration+is+regulated+by+rho/ROCK-dependent+matrix+reorganization.">Contact guidance mediated three-dimensional cell migration is regulated by rho/ROCK-dependent matrix reorganization.</a> Biophys. J. 95 (11), 5374 (2008).</li><li>Sander, E., Barocas, V., Tranquillo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+fiber+alignment+pattern+alters+extracellular+matrix+synthesis+in+fibroblast-populated+fibrin+gel+cruciforms+and+correlates+with+predicted+tension.">Initial fiber alignment pattern alters extracellular matrix synthesis in fibroblast-populated fibrin gel cruciforms and correlates with predicted tension.</a> Ann. Biomed. Eng. 39 (2), 714-729 (2011).</li><li>Barocas, V. H., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+anisotropic+biphasic+theory+of+tissue-equivalent+mechanics:+the+interplay+among+cell+traction,+fibrillar+network+deformation,+fibril+alignment,+and+cell+contact+guidance.">An anisotropic biphasic theory of tissue-equivalent mechanics: the interplay among cell traction, fibrillar network deformation, fibril alignment, and cell contact guidance.</a> J. Biomech. Eng. 119, 137-146 (1997).</li><li>Neidert, M. R., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+valves+with+commissural+alignment.">Tissue-engineered valves with commissural alignment.</a> Tissue Eng. 12 (4), 891-903 (2006).</li><li>Robinson PS, ., Robinson PS, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+biaxial+behavior+of+fibrin-based+tissue-engineered+heart+valve+leaflets.">Planar biaxial behavior of fibrin-based tissue-engineered heart valve leaflets.</a> Tissue Eng. Part A. 15 (10), 2763-2772 (2009).</li><li>Grassl, E., Oegema, T., Tranquillo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+as+an+alternative+biopolymer+to+type+I+collagen+for+the+fabrication+of+a+media+equivalent.">Fibrin as an alternative biopolymer to type I collagen for the fabrication of a media equivalent.</a> J. Biomed. Mater. Res. 60 (4), 607-612 (2002).</li><li>Paten, J. A., Zareian, R., Saeidi, N., Melotti, S. A., Ruberti, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+performance+of+an+optically+accessible,+low-volume,+mechanobioreactor+for+long-term+study+of+living+constructs.">Design and performance of an optically accessible, low-volume, mechanobioreactor for long-term study of living constructs.</a> Tissue Eng. Part C Methods. 17 (7), 775-788 (2001).</li><li>Ye KY, ., Sulli van KE, ., Black LD, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+cardiomyocytes+in+a+fibrin+hydrogel+for+cardiac+tissue+engineering.">Encapsulation of cardiomyocytes in a fibrin hydrogel for cardiac tissue engineering.</a> J. Vis. Exp. (55), (2011).</li><li>Ahmann, K. A., Weinbaum, J. S., Johnson, S. L., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+degradation+enhances+vascular+smooth+muscle+cell+proliferation+and+matrix+deposition+in+fibrin-based+tissue+constructs+fabricated+in+vitro.">Fibrin degradation enhances vascular smooth muscle cell proliferation and matrix deposition in fibrin-based tissue constructs fabricated in vitro.</a> Tissue Eng. Part A. 16 (10), 3261-3270 (2010).</li><li>Raghupathy, R., Witzenburg, C., Lake, S. P., Sander, E. A., Barocas, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+regional+mechanical+anisotropy+in+soft+tissue+analogs.">Identification of regional mechanical anisotropy in soft tissue analogs.</a> J. Biomech. Eng. 133, (2011).</li><li>Robinson, P. S., Johnson, S. L., Evans, M. C., Barocas, V. H., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+tissue-engineered+valves+from+cell-remodeled+fibrin+with+commissural+alignment+of+cell-produced+collagen.">Functional tissue-engineered valves from cell-remodeled fibrin with commissural alignment of cell-produced collagen.</a> Tissue Eng. Part A. 14 (1), 83-95 (2008).</li><li>Gabbiani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myofibroblast+in+wound+healing+and+fibrocontractive+diseases.">The myofibroblast in wound healing and fibrocontractive diseases.</a> J. Pathol. 200 (4), 500-503 (2003).</li><li>PaszeK, M. J., 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=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8 (3), 241-254 (2005).</li><li>Grinnell, F., Rocha L, B., Iucu, C., Rhee, S., Jiang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nested+collagen+matrices:+A+new+model+to+study+migration+of+human+fibroblast+populations+in+three+dimensions.">Nested collagen matrices: A new model to study migration of human fibroblast populations in three dimensions.</a> Exp. Cell. Res. 312 (1), 86-94 (2006).</li><li>Miron-Mendoza, M., Seemann, J., Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+differential+regulation+of+cell+motile+activity+through+matrix+stiffness+and+porosity+in+three+dimensional+collagen+matrices.">The differential regulation of cell motile activity through matrix stiffness and porosity in three dimensional collagen matrices.</a> Biomaterials. 31 (25), 6425-6435 (2010).</li><li>Wakatsuki, T., Kolodney MS, ., Zahalak GI, ., Elson EL, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+mechanics+studied+by+a+reconstituted+model+tissue.">Cell mechanics studied by a reconstituted model tissue.</a> Biophys. J. 79 (5), 2353-2368 (2000).</li><li>De Jesus, A., Aghvami, M., Sander, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast-mediated+fiber+realignment+in+fibrin+gels.">Fibroblast-mediated fiber realignment in fibrin gels.</a> , (2013).</li><li>Aghvami, M., Barocas, V. H., Sander, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+mechanical+simulations+of+cell+compacted+collagen+gels.">Multiscale mechanical simulations of cell compacted collagen gels.</a> J. Biomech. Eng. 135, (2013).</li></ol>

Questo protocollo è stato sviluppato allo scopo di osservare e quantificare la meccanica coinvolta nel rimodellamento ECM mediato dalle cellule. Tali processi sono alla base di una serie di fenomeni biologici e hanno importanti implicazioni per i tessuti ingegneristici2,22, riducendola cicatrice 1,23e comprendendo il rimodellamento patologico deitessuti 12,24. L'uso della microscopia DIC time-lapse consente di risolvere e quantificare lo spostamento e l'allineamento delle fibre di fibrin...

Uno strumento importante per studiare le interazioni cellula-matrice è il gel di collagene popolato dallecellule 1,2. Il gel fornisce un ambiente 3D più vicino al carattere in vivo del tessuto e più adatto per comprendere il comportamento cellulare di quanto offerto dalle colture 2D tradizionali3. I primi studi in cui i fibroblasti sono stati distribuiti omogeneamente all'interno di un gel di collagene hanno scoperto che le cellule consolidano rapidamente le fibre di collagene e compattano il gel4,5. I fibroblasti contrattili in gel galleggianti liberi poi si trasformano in uno stato quiescente subito dopo che il gel ha raggiunto completamente lacompattazione 1,6,7. I fibroblasti nei gel vincolati ai confini rimangono in uno stato attivo e sintetico8 e generano l'allineamento delle fibre in modo dipendente dalla geometria del gel e dai vincoliesterni 5,9. Le differenze nell'attività cellulare sembrano essere il risultato della tensione interna (o della sua mancanza) che si sviluppa man mano che le cellule esercitano forze di trazione attraverso integrine sulle fibre di collagene nel gel.
Una variante di questa tecnica prevedeil posizionamento di espianto di fibroblasti (cioè ciuffi di cellule) a una distanza all'interno di un gel di collagene e l'osservazione delle interazioni cellula-matrice e il graduale sviluppo dell'allineamento delle fibre tra le espianto (a volte chiamate cinghie simili a legamenti)10-12. Il vantaggio principale del sistema di espianto è che consente di disporre le cellule in semplici motivi geometrici, il che rende più facile visualizzare e sondare i meccanismi alla base del riallineamento delle fibre guidato dalle cellule. Questi modelli di allineamento - che dipendono principalmente dall'interazione tra le forze di trazione cellulare, la distribuzione spaziale cellulare, la geometria del gel e i vincoli meccanici sul gel - sono importanti da capire perché svolgono un ruolo centrale nell'organizzazione globale dei tessuti, nella funzione meccanica e nell'ambiente meccanicolocale 13.
Nel campo dell'ingegneria tissutale, una strategia per la produzione di tessuti ingegnerizzati meccanicamente funzionali comporta il controllo del modello di allineamento delle fibre che si sviluppa dalla compattazione cellulare in modo che il tessuto ingegnerizzato possieda un allineamento delle fibre che imita quello del tessutonativo 14,15. Tale allineamento si ritiene necessario per i tessuti ingegnerizzati per replicare il complesso comportamento meccanico dei tessuti nativi. Una modifica di questa strategia è sostituire il gel di collagene con un gel di fibrina16. Il gel di fibrina sviluppa un modello di allineamento simile a un gel di collagene durante la compattazione. Nel corso del tempo la fibrina viene degradata e sostituita con ECM sintetizzato a cella che segue il modello iniziale di allineamento della fibra di fibrina. Il costrutto ingegnerizzato risultante ha migliorato significativamente le proprietà meccaniche rispetto ai costrutti derivati dal gel dicollagene 17.
Il processo di allineamento e i successivi eventi di rimodellamento nei gel di fibrina procedono in modo complicato e poco compreso. Per caratterizzare meglio queste interazioni e il loro effetto sul comportamento cellulare e sul rimodellamento di ECM, abbiamo sviluppato una procedura basata sul metodo di espianto. In questo metodo, le espianto di fibroblasti sono posizionate su un gel di fibrina in diversi motivi geometrici. I gel sono mantenuti in un bioreattore18controllato dall'ambiente e montato al microscopio e il processo di compattazione e riallineamento delle fibre viene monitorato con microscopia DIC (Differential Interference Contrast) time-lapse. I campi di spostamento vengono quantificati con algoritmi personalizzati. I dati ottenuti da questi esperimenti hanno implicazioni ad ampio raggio per una serie di processi, tra cui l'ottimizzazione delle strategie di ingegneria tissutale, il miglioramento della guarigione delle ferite e il trattamento del rimodellamento patologico dei tessuti.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sigma-Aldrich</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Invitrogen</td>
			<td>3000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lonza</td>
			<td>DE14-701F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Molecular Probes</td>
			<td>F8858</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GIBCO</td>
			<td>A10483-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GIBCO</td>
			<td>11430-030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisher-Scientific</td>
			<td>SS264-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>A3428-25MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Biotense Bioreactor</td>
			<td style="width:85px;">ADMET</td>
			<td style="width:125px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ti-Eclipe Microscope</td>
			<td>Nikon</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"># 0 35 mm Glass Bottom Petri Dish</td>
			<td>MatTek</td>
			<td>P35G-0-20-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"># 0 35 mm Glass Top Petri Dish</td>
			<td>MatTek</td>
			<td>P35GTOP-0-20-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic Luer fittings, PVC tubing with Luer ends</td>
			<td>Cole-Parmer</td>
			<td>30600-65</td>
		</tr>
	</tbody>
</table>

observing and quantifying fibroblast-mediated fibrin gel compaction

Le cellule incorporate nei gel di collagene e fibrina attaccano ed esercitano forze di trazione sulle fibre del gel. Queste forze possono portare alla riorganizzazione locale e globale e al riallineamento della microstruttura del gel. Questo processo procede in modo complesso che dipende in parte dall'interazione tra la posizione delle celle, la geometria del gel e i vincoli meccanici sul gel. Per comprendere meglio come queste variabili producono modelli globali di allineamento delle fibre, utilizziamo la microscopia DIC (Time-Lapse Differential Interference Contrast) abbinata a un bioreattore controllato dall'ambiente per osservare il processo di compattazione tra espianto geometricamente distanziato (ammassi di fibroblasti). Le immagini vengono quindi analizzate con un algoritmo di elaborazione delle immagini personalizzato per ottenere mappe del ceppo. Le informazioni ottenute da questa tecnica possono essere utilizzate per sondare la meccanobiologia di varie interazioni cellula-matrice, che ha importanti implicazioni per la comprensione dei processi nella guarigione delle ferite, nello sviluppo di malattie e nelle applicazioni di ingegneria tissutale.

Il rimodellamento dei tessuti è un processo complesso che è guidato in parte da interazioni fisiche reciproche tra le cellule e la matrice circostante. Le cellule riorganizzano le fibre circostanti e generano tensione nella rete in fibra. L'allineamento delle fibre e dell'ambiente meccanico a sua volta controlla il comportamento delle cellule, in modo che sia le cellule che la matrice si riorganizzno globalmente per produrre tessuti rimodellati. In questo esperimento, le cellule delle espianto, inizialmente rotonde nel...

Watch this Scientific Journal Video about Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction at JoVE.com

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

La microscopia time-lapse e le tecniche di elaborazione delle immagini sono state utilizzate per osservare e analizzare la compattazione del gel mediata da fibroblasti e il riallineamento della fibra di fibrina in un bioreattore controllato dall'ambiente per un periodo di 48 ore.

Osservazione e quantificazione della compattazione del gel di fibrina mediata da fibroblasti

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

observing-and-quantifying-fibroblast-mediated-fibrin-gel-compaction

Research

JoVE Journal

Bioengineering

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

Preparazione di matrici di reclamo per Quantificare Contrazione Cellular

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

Ricerca

Bioingegneria

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

Fabbricazione di micro-tessuti utilizzando i moduli di Collagene Gel cellule contenenti

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Incapsulamento di cardiomiociti in un idrogel di fibrina per l&#39;ingegneria tissutale cardiaca

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Preparazione del 3D fibrina ponteggi per le applicazioni delle cellule staminali Cultura

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

Ingegneria fibrina a base di tessuto costrutti da miofibroblasti e applicazione di vincoli e la tensione per indurre Organizzazione cellulare e collagene (Re)

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

Caratterizzazione di leucociti-piastrine Rich fibrina, un biomateriale Novel

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (&gt; 4 &#181;m) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (&gt;4 &#181;m) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (&gt;100 &#181;m).

We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

Formare giganti di dimensioni Polymersomes Uso reidratazione Gel-assistita

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier ...

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

Una tecnica innovativa per la generazione e Observing Chemiluminescenza in un ambiente biologico

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Un Microfluidic flusso Camera Modello per la trasfusione di piastrine e Emostasi Misure Deposizione delle piastrine e la formazione di fibrina in tempo reale

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

Gel-seq: Un metodo per la preparazione di libreria simultanea di sequenziamento del DNA e RNA utilizzando matrici di idrogel

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...