Name: generating a fractal microstructure of laminin-111 to signal to cells
Uploaded: 2020-09-28T13:00:00.000+00:00
Duration: 6 min 56 s
Description: Watch this Scientific Journal Video about Generating a Fractal Microstructure of Laminin-111 to Signal to Cells at JoVE.com

Questo lavoro è stato finanziato dal Lawrence Livermore National Lab LDRD 18-ERD-062 (a C.R.). Grazie a John Muschler per il suo gentile dono delle cellule DgKO e DgKI. Grazie al personale del Laboratorio di Microscopio Elettronico dell'Università della California di Berkeley per i consigli e l'assistenza nella preparazione dei campioni di microscopia elettronica e nella raccolta dei dati.

1. Scongelamento e aliquota laminina-111
NOTA: La laminina-111 purificata è disponibile in commercio (tipicamente purificata dalla matrice EHS venduta come Matrigel), ma non tutte le fonti forniscono proteine intatte e non degradate.
<ol><li>Verificare che Ln1 non sia degradato mediante l'approvvigionamento di Ln1 e la cromatografia ad esclusione dimensionale26 o l'elettroforesi su gel di poliacrilammide26. Ln1 denaturato dovrebbe avere bande a 337 kDa, 197 kDa e 177 kDa, e Ln1 nativo dovrebbe avere una singola banda a 711 kDa.</li>
	<li>Scongelare Ln1 su ghiaccio in frigorifero per una notte e aliquotarlo in provette per microcentrifuga a basso legame proteico (tipicamente aliquota a 100 μg/provetta per le sovrapposizioni di coltura o 10 μg per i rivestimenti) utilizzando puntali a basso legame proteico e tecnica sterile27. Congelare le aliquote e conservare a -80 °C.</li>
</ol>2. Polimerizzazione della laminina mediante tamponi a basso pH

<ol><li>Preparare il tampone di polimerizzazione polyLM: pesare e miscelare 1 mM CaCl2 e 20 mM di acetato di sodio in acqua ultrapura. Quindi regolare il pH a 4,0, utilizzando HCl o acido acetico. Filtrare sterile attraverso un filtro da 0,2 μm e conservare a 4 °C.</li>
	<li>Preparare il tampone di polimerizzazione di controllo: mescolare 1 mM di CalCl2 e 20 mM di Tris e regolare il pH a 7,0, utilizzando 10 N di HCl o 12 N di NaOH secondo necessità. Filtrare sterile attraverso un filtro da 0,2 μm e conservare a 4 °C. NOTA: Il protocollo può essere messo in pausa qui.</li>
	<li>Scongelare il numero necessario di aliquote di Ln-1 in frigorifero per una notte.</li>
	<li>Utilizzando la tecnica di coltura cellulare sterile, aggiungere il tampone appropriato (tampone di polimerizzazione polyLM dalla fase 2.1 o tampone di polimerizzazione di controllo dalla fase 2.2) alle aliquote di laminina a una concentrazione finale di 100 μg/mL (cioè aggiungere 0,5 mL ad aliquote da 50 μg) e miscelare delicatamente mediante pipettaggio.</li>
	<li>Se si utilizza laminina marcata con fluorescenza per la visualizzazione, ricostituire e aggiungere in rapporto 1:50 wt/wt (ad es. 2 μg/100 μg) e miscelare delicatamente mediante pipettaggio. NOTA: Se si utilizzano laminine come substrato per l'attacco cellulare, seguire i passaggi 2.6-2.8:</li>
	<li>Immediatamente dopo la diluizione in un tampone appropriato, trasferire le laminine su plastica o vetreria per colture e incubare per una notte in un incubatore per colture cellulari a 37 °C. Si noti che quando si ricoprono i vetrini coprioggetto, aggiungere un volume sufficientemente grande da produrre una goccia equilibrata (in genere, utilizzare 50 μL per un vetrino coprioggetti rotondo da 13 mm) e conservare in una camera umidificata per una notte a 37 °C.</li>
	<li>Utilizzando la tecnica sterile27, lavare con 1x soluzione salina tamponata con fosfato (PBS).</li>
	<li>Rimuovere il liquido con cura. Non lasciare che la superficie sia completamente asciutta. NOTA: Se si utilizzano laminine per coprire le cellule per l'induzione delle proteine del latte, seguire i passaggi 2.9-2.12:</li>
	<li>Coltura di cellule epiteliali mammarie. Colture di cellule knock-in di distroglicani MepG knock in (DgKI) o di cellule knockout di distroglicani MepG (DgKO) in cellule DMEM-F12 integrate con FBS al 2%, insulina 0,01 mg/mL, EGF 0,005 μg/mL, gentamicina 0,05 mg/mL e normocina 0,05 g/mL.</li>
	<li>Seme di cellule DgKO e DgKI per la stimolazione della laminina a 10.500 cellule/cm2 in piastre a pozzetti o piastre con fondo coprioggetto</li>
	<li>Incubare le laminine diluite in un incubatore per colture cellulari a 37 °C per 30 minuti.</li>
	<li>Centrifugare le aliquote di polyLM a 2.000 x g per 20 minuti, ma centrifugare LM a pH neutro a 20.000 x g per 20 minuti a causa delle dimensioni ridotte delle entità proteiche e della necessità di velocità più elevate per raccogliere in modo efficace.</li>
	<li>Utilizzando la tecnica sterile27, rimuovere delicatamente il surnatante e risospendere in un volume appropriato di terreno di induzione DMEM-F12 integrato con 3 μg/mL di prolattina, 2,8 μM di idrocortisone e 5 μg/mL di insulina</li>
	<li>Trasferire immediatamente la laminina nel terreno di coltura cellulare per stimolare le cellule. Abbiamo stimolato con successo le cellule con 50-800 μg/mL Ln1</li>
</ol>3. Polimerizzazione della laminina mediante glicoproteine isolate
<ol><li>Scongelare delicatamente il nidogeno-1 ricombinante e Ln1 sul ghiaccio in frigorifero per una notte.</li>
	<li>Miscelare Ln1 con nidogeno-1 in un rapporto 1:1 wt/wt nel terreno di coltura cellulare utilizzando puntali e provette a basso legame proteico e utilizzando una tecnica sterile. Utilizzare Ln1 puro nel terreno di coltura cellulare come controllo.</li>
	<li>Se si utilizza laminina marcata con fluorescenza per la visualizzazione, ricostituire e aggiungere in rapporto 1:50 wt/wt (ad esempio, 2 μg/100 μg). Miscelare delicatamente mediante pipettaggio.</li>
	<li>Incubare Ln1-nidogeno o Ln1 di controllo in un incubatore per colture cellulari a 37 °C per 30 min.</li>
	<li>Centrifugare i copolimeri Ln1-nidogeni a 2.000 x g per 30 min e il controllo Ln1 a 20.000 x g per 30 min.</li>
	<li>Rimuovere delicatamente il surnatante e risospendere nel terreno di coltura cellulare fino a una concentrazione finale di 100-800 μg di proteine totali/mL.</li>
	<li>Utilizzare immediatamente per stimolare le cellule, oppure trasferire su vetrini coprioggetti e lasciare aderire per una notte a 37 °C in incubatori per colture cellulari umidificati.</li>
	<li>Rimuovere il terreno di coltura dai vetrini coprioggetti, lavare i vetrini coprioggetti con 1x PBS, quindi fissare con il 10% di formalina (cioè il 4% di formaldeide nel PBS) per 15 minuti a temperatura ambiente.</li>
</ol>4. Polimerizzazione della laminina utilizzando cellule che esprimono distroglicani
<ol><li>Coltura di cellule epiteliali mammarie. Colture di cellule knock-in di distroglicani MepG knock in (DgKI) o di cellule knockout di distroglicani MepG (DgKO) in cellule DMEM-F12 integrate con FBS al 2%, insulina 0,01 mg/mL, EGF 0,005 μg/mL, gentamicina 0,05 mg/mL e normocina 0,05 g/mL.</li>
	<li>Semina le cellule DgKI a 5.000 cellule/cm2 e le cellule DgKO a 8.000 cellule/cm2 a causa delle differenze nei tassi di crescita. Coltura a 37 °C in incubatori umidificati con cambi di terreno ogni 2-3 giorni e passaggi ogni 4-5 giorni utilizzando una rigorosa tecnica sterile. Contare attentamente le cellule con un emocitometro o un contatore automaticodi cellule 27 per garantire tassi di crescita adeguati.</li>
	<li>Cellule seme per la stimolazione della laminina a 10.500 cellule/cm2 in piastre a pozzetti o piastre con fondo coprioggetto e coltura per 2 giorni, utilizzando cellule DgKI per generare cellule frattali Ln1 e DgKO come controlli negativi. Scongelare delicatamente Ln-1 per una notte su ghiaccio in frigorifero, quindi mescolare con terreno di induzione di DMEM-F12 integrato con 3 μg/mL di prolattina, 2,8 μM di idrocortisone e 5 μg/mL di insulina. Per una capsula da 35 mm, utilizzare 1 mL di terreno a induzione con 50-800 μg di Ln1.</li>
	<li>Se si utilizza Ln1 fluorescente, miscelare con un rapporto peso/peso di 50:1.</li>
	<li>Rimuovere i terreni dalle cellule e coprire con laminina e terreni di induzione di DMEM-F12 integrati con 3 μg/mL di prolattina, 2,8 μM di idrocortisone e 5 μg/mL di insulina</li>
	<li>Coltivare le cellule per 2 giorni, quindi fissare con formalina al 10%.</li>
</ol>5. Caratterizzazione della microstruttura della laminina mediante microscopia ottica
<ol><li>Raccogliere campioni fissi e lavare 3 volte con 1x PBS a temperatura ambiente per 5 minuti.</li>
	<li>Bloccare e permeabilizzare i campioni in 1x PBS con Triton X-100 allo 0,5%, albumina sierica bovina al 3%, siero di capra al 10% e frammento di anticorpo bloccante antitopo da 40 μg/mL per 1 ora a temperatura ambiente in una camera umidificata. NOTA: Triton X-100 è pericoloso. Maneggiare con guanti e protezioni per gli occhi e smaltire in modo appropriato.</li>
	<li>Miscelare l'anticorpo anti-laminina a una diluizione di 1:100 con 1x PBS con Triton X-100 allo 0,5% e BSA al 3% e aggiungere ai campioni per 2 ore a temperatura ambiente o per una notte a 4 °C in una camera umidificata (in genere utilizzare scatole per puntali con una salvietta da laboratorio umidificata sul fondo).</li>
	<li>Rimuovere l'anticorpo primario e lavare 3 volte con 1x PBS con Triton X-100 allo 0,5% e BSA al 3%.</li>
	<li>Aggiungere un anticorpo secondario appropriato a una diluizione di 1:500 ai campioni in PBS 1x con Triton X-100 allo 0,5% e BSA al 3% e incubare a temperatura ambiente al buio in una camera umidificata.</li>
	<li>Rimuovere l'anticorpo secondario e lavare 3 volte con 1x PBS con 0,5% Triton X-100 e 3% BSA.</li>
	<li>Per i campioni contenenti cellule, aggiungere 6 μM di falloidina per 45 minuti, seguiti da 3 lavaggi con PBS con Triton X-100 allo 0,5% e BSA al 3% e seguiti da 3 lavaggi con PBS. Quindi colorare con DAPI 0,1 μg/mL in PBS per 5 minuti.</li>
	<li>Montare i campioni, se necessario.</li>
	<li>Immagine della struttura ln-1 mediante microscopia confocale ad alta risoluzione. Utilizzare un microscopio a scansione laser dotato di obiettivi a immersione (Plan Apochromat 40X/1.1NA ad immersione in acqua o Plan Apochromat 63X/1.4NA ad immersione in olio).</li>
</ol>6. Caratterizzazione di costrutti lamininici tramite dimensione di conteggio a scatola
<ol><li>Analizza le proprietà frattali utilizzando gli algoritmi di dimensione di conteggio delle caselle disponibili nei toolbox Matlab o in ImageJ. Questi metodi tracciano le immagini di soglia della laminina e tracciano su un log log il numero di scatole contenenti Ln1 rispetto alla dimensione delle scatole. La pendenza della relazione tra questi parametri è la dimensione del conteggio delle caselle: le geometrie non frattali avranno una dimensione di 0, 1 o 2, mentre le geometrie frattali hanno una dimensione non intera28. NOTA: Ln1 trattato con acido, trattato con glicoproteine e attaccato alle cellule DgKI ha tutti una dimensione frattale di ~1,7, mentre Ln1 di controllo ha una dimensione frattale di 2,0.</li>
</ol>7. Caratterizzazione di costrutti lamininici mediante microscopia elettronica
<ol><li>Risospendere le laminine nei terreni di coltura cellulare e lasciare adsorbire i wafer di silice per 90 minuti in un ambiente umido a 37 °C.</li>
	<li>Fissare le matrici in glutaraldeide al 2,5% in tampone cacodilato di sodio 0,1 M a pH 7,2. NOTA: La glutaraldeide è pericolosa e deve essere maneggiata in una cappa aspirante con guanti e protezioni per gli occhi e smaltita come rifiuto pericoloso.</li>
	<li>Matrici Postfix in tetrossido di osmio all'1% in tampone cacodilato di sodio 0,1 M a pH 7,2. NOTA: Il tetrossido di osmio è pericoloso e deve essere maneggiato in una cappa aspirante con guanti e protezioni per gli occhi e smaltito in modo appropriato. Il cacodilato è pericoloso e deve essere maneggiato in una cappa aspirante con guanti e protezione per gli occhi e smaltito in modo appropriato.</li>
	<li>Lavare 3 volte in tampone di cacodilato di sodio a pH 7,2.</li>
	<li>Disidratare con concentrazioni crescenti di etanolo.</li>
	<li>Matrici di rivestimento sputter con un sottile strato d'oro.</li>
	<li>Immagine mediante microscopia elettronica a scansione.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+a+complex+protein+mixture+required+for+optimal+growth+of+cell+culture.">Matrigel: a complex protein mixture required for optimal growth of cell culture.</a> Proteomics. 10 (9), 1886-1890 (2010).</li><li>Palmero, C. 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=The+follicular+thyroid+cell+line+PCCL3+responds+differently+to+laminin+and+to+polylaminin,+a+polymer+of+laminin+assembled+in+acidic+pH.">The follicular thyroid cell line PCCL3 responds differently to laminin and to polylaminin, a polymer of laminin assembled in acidic pH.</a> Molecular and Cellular Endocrinology. 376 (1), 12-22 (2013).</li><li>Hochman-Mendez, C., Lacerda de Menezes, J. R., Sholl-Franco, A., Coelho-Sampaio, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polylaminin+recognition+by+retinal+cells.">Polylaminin recognition by retinal cells.</a> Journal of Neuroscience Research. 92 (1), 24-34 (2014).</li><li>Leal-Lopes, C., Grazioli, G., Mares-Guia, T. R., Coelho-Sampaio, T., Sogayar, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerized+laminin+incorporation+into+alginate-based+microcapsules+reduces+pericapsular+overgrowth+and+inflammation.">Polymerized laminin incorporation into alginate-based microcapsules reduces pericapsular overgrowth and inflammation.</a> Journal of Tissue Engineering and Regenerative Medicine. 13 (10), 1912-1922 (2019).</li><li>Paccola Mesquita, F. C., Hochman-Mendez, C., Morrissey, J., Sampaio, L. C., Taylor, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+as+a+Potent+Substrate+for+Large-Scale+Expansion+of+Human+Induced+Pluripotent+Stem+Cells+in+a+Closed+Cell+Expansion+System.">Laminin as a Potent Substrate for Large-Scale Expansion of Human Induced Pluripotent Stem Cells in a Closed Cell Expansion System.</a> Stem Cells International. 2019, 9 (2019).</li><li>Walker, J. M., Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Protein Protocols Handbook. , 61-67 (2002).</li><li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells: A Manual of Basic Techniques. 7th edition. , (2016).</li><li>Mandelbrot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Fractal Geometry of Nature. , (1982).</li><li>Schittny, J. C., Yurchenco, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terminal+short+arm+domains+of+basement+membrane+laminin+are+critical+for+its+self-assembly.">Terminal short arm domains of basement membrane laminin are critical for its self-assembly.</a> Journal of Cell Biology. 110 (3), 825-832 (1990).</li><li>Schuger, L., Yurchenco, P., Relan, N. K., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+fragment+E4+inhibition+studies:+basement+membrane+assembly+and+embryonic+lung+epithelial+cell+polarization+requires+laminin+polymerization.">Laminin fragment E4 inhibition studies: basement membrane assembly and embryonic lung epithelial cell polarization requires laminin polymerization.</a> International Journal of Developmental Biology. 42 (2), 217-220 (1998).</li><li>Colognato, H., Yurchenco, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Form+and+function:+the+laminin+family+of+heterotrimers.">Form and function: the laminin family of heterotrimers.</a> Developmental Dynamics. 218 (2), 213-234 (2000).</li><li>Kabaeva, Z., Meekhof, K. E., Michele, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcolemma+instability+during+mechanical+activity+in+Large+myd+cardiac+myocytes+with+loss+of+dystroglycan+extracellular+matrix+receptor+function.">Sarcolemma instability during mechanical activity in Large myd cardiac myocytes with loss of dystroglycan extracellular matrix receptor function.</a> Human Molecular Genetics. 20 (17), 3346-3355 (2011).</li></ol>

Questo lavoro è stato eseguito sotto gli auspici del Dipartimento dell'Energia degli Stati Uniti dal Lawrence Livermore National Laboratory nell'ambito del contratto DE-AC52-07NA27344. Versione IM LLNL-JRNL-799443.

Le laminine rappresentano un elemento chiave dell'ECM nei sistemi epiteliali, muscolari e neurali e hanno dimostrato in vitro di svolgere ruoli essenziali nella regolazione della differenziazione funzionale delle cellule. Evidenze crescenti indicano che la struttura della laminina mostrata alle cellule regola la sua segnalazione e il fenotipo cellularerisultante 15,16, quindi i metodi per controllare la microstruttura di Ln1 dovrebbero essere di interesse per i b...

A differenza dei fattori di crescita o delle citochine, le proteine della matrice extracellulare (ECM) possono assemblarsi in reti strutturali. Poiché la disfunzione dell'ECM è un elemento chiave di molte condizioni patologiche, i meccanismi attraverso i quali le cellule percepiscono e trasducono i segnali dalla composizione, dalla microstruttura e dalla biomeccanica dell'ECM sono bersagli interessanti per lo sviluppo terapeutico. Prove crescenti suggeriscono che la microstruttura di queste reti svolge un ruolo importante nel modo in cui queste proteine segnalano alle cellule. Ad oggi, questo legame tra la microstruttura dell'ECM e la funzione cellulare è stato dimostrato per il collagene I1, la fibronectina2 e la fibrina3.
La laminina-111 (Ln1) è una proteina trimerica a forma di croce che è un componente strutturale chiave della matrice extracellulare che entra in contatto con le cellule epiteliali4, le cellule muscolari5 e le cellule neurali6. Nella ghiandola mammaria, Ln1 è necessario per la differenziazione funzionale delle cellule epiteliali della ghiandola mammaria in cellule produttrici di latte 7,8,9 e Ln1 è cruciale per l'induzione della reversione tumorale10,11. Ln1 e altre laminine sono necessarie per lo sviluppo delle reti corticali di actina e dell'organizzazione del sarcolemma nel muscolo12,13 e per la migrazione delle cellule neurali e la crescita dei neuriti13 . Pertanto, la comprensione dei meccanismi attraverso i quali la laminina invia segnali alle cellule è un'area di ricerca attiva.
Ln1 contiene più domini adesivi per un'ampia gamma di recettori, tra cui integrine, sindecani, distroglicani, LBP-110 e LamR (Figura 1A)4,14. Il frammento E8, che contiene i domini globulari c-terminali della laminina α1, è necessario per legare il distroglicano e le integrine α6β1 e α3β115 ed è necessario per il differenziamento funzionale delle cellule epiteliali 15,16. Al contrario, i bracci corti mostrano una diversa attività biologica17, il che significa che la disponibilità per il riconoscimento di questi diversi domini Ln1 dopo la formazione della rete potrebbe alterare il comportamento cellulare.
Recentemente abbiamo dimostrato che Ln1 disposto in una rete polimerica mostra proprietà frattali (cioè, una struttura che è auto-similare su più scale di lunghezza)18. Le reti frattali segnalano in modo diverso alle cellule epiteliali rispetto a Ln1, che manca di questa disposizione19. Questa rete frattale indica una particolare struttura di assemblaggio, in cui il braccio lungo è preferenzialmente esposto alle celle18. Come risultato di questa diversa esposizione del braccio lungo, le cellule epiteliali subiscono una differenziazione funzionale in cellule produttrici di latte con giunzioni strette ben organizzate e soppressione delle fibre di stress dell'actina19.
Descriviamo 3 metodi per generare reti Ln1 da interazioni di legame braccio corto-braccio corto, che mostrano un'elevata visualizzazione del braccio lungo Ln1: 1) mediante assemblaggio senza cellule con tamponi acidi, 2) mediante co-incubazione con glicoproteine, o 3) mediante interazione con distroglicani di superficie cellulare. Questi tre metodi generano microstrutture di laminina simili attraverso tre meccanismi molto diversi, consentendo flessibilità nella progettazione sperimentale. Lavori precedenti suggeriscono che questi tre metodi potrebbero rappresentare la robustezza biologica: negli epiteli della ghiandola mammaria, la superficie cellulare distroglicana, le glicoproteine o la laminina strutturata artificialmente possono indurre la differenziazione funzionale19. Pertanto, i ricercatori possono scegliere tra questi metodi in base all'argomento dello studio: le cellule che esprimono il distroglicano non mostrano alcuna sensibilità alla microstruttura di Ln-1, poiché il distroglicano nella membrana cellulare induce una corretta polimerizzazione in una rete frattale19,20. Matrigel, una miscela di laminine e glicoproteine21, rappresenta la fonte più economica di Ln-1 e offre la microstruttura necessaria per indurre le cellule mammarie knockout dei distroglicani a differenziarsi20, ma contiene una complessa miscela di proteine con variabilità da lotto a lotto. PolyLM rappresenta il sistema più pulito e riduzionista che fornisce questa funzione, ma a un costo maggiore e a una minore efficacia nell'indurre il differenziamento delle cellule mammarie rispetto a Matrigel19.
Questi tre metodi hanno una struttura a rete frattale caratteristica di aggregazione limitata alla diffusione 18,19 e mostrano un'attività biologica alterata rispetto alla laminina aggregata in più sistemi sperimentali 22,23,24,25. Studi futuri che utilizzano questi metodi potrebbero identificare i recettori e la segnalazione necessari per la differenziazione epiteliale e determinare il ripristino delle normali interazioni cellula-matrice nelle cellule in microambienti anormali20, come i tumori.

<table><tbody><tr><td>10% formalin</td><td>Sigma Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Anti-Laminin primary antibody</td><td>Sigma Aldrich</td><td>L9393</td><td></td></tr><tr><td>Anti-Rabbit Alexafluor 488 secondary antibody</td><td>Thermo</td><td>A32731</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A9418</td><td></td></tr><tr><td>CaCl2</td><td>Sigma Aldrich</td><td>C4901 or similar</td><td></td></tr><tr><td>Cell Culture Incubator</td><td>Thermo</td><td>Heracell 150 or similar</td><td></td></tr><tr><td>Centrifuge for microcentrifuge tubes</td><td>Eppendorf</td><td>5418 or similar</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM 710 or equivalent</td><td></td></tr><tr><td>Coverslips</td><td>Thermo</td><td>25CIR-1 or similar</td><td></td></tr><tr><td>DMEM-F12, Liquid, High Glucose, +HEPES, L-glutamine, +Phenol red</td><td>Thermo</td><td>11330107</td><td></td></tr><tr><td>EGF (Epidermal Growth Factor)</td><td>Sigma Aldrich</td><td>11376454001</td><td></td></tr><tr><td>Fluorescently Labeled Laminin</td><td>Cytoskeleton Inc</td><td>LMN02</td><td></td></tr><tr><td>Gentamicin</td><td>VWR</td><td>VWRV0304-10G</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma Aldrich</td><td>G5882</td><td></td></tr><tr><td>HCl</td><td>Sigma Aldrich</td><td>320331 or similar</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H0888-5G</td><td></td></tr><tr><td>Insulin</td><td>Sigma Aldrich</td><td>16634-50mg</td><td></td></tr><tr><td>MepG Dystroglycan Knockout Cells</td><td>LBNL</td><td>N/A</td><td></td></tr><tr><td>NaOH</td><td>Sigma Aldrich</td><td>S8045 or similar</td><td></td></tr><tr><td>Normocin</td><td>Invivogen</td><td>ant-nr-1</td><td></td></tr><tr><td>Osmium Tetroxide</td><td>Sigma Aldrich</td><td>75633</td><td></td></tr><tr><td>Ovine Prolactin</td><td>Los Angeles Biomedical Research Institute</td><td>Ovine Pituitary Prolactin</td><td></td></tr><tr><td>Phalloidin</td><td>Thermo</td><td>A22287</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Thermo</td><td>10010023 or similar</td><td></td></tr><tr><td>Purified Laminin-111</td><td>Sigma Aldrich</td><td>L2020-1MG</td><td></td></tr><tr><td>Recombinant human Nidogen-1, carrier free</td><td>R&amp;amp;D systems</td><td>2570-ND-050</td><td></td></tr><tr><td>Sodium Acetate</td><td>Sigma Aldrich</td><td>S2889 or similar</td><td></td></tr><tr><td>Sodium Cacodylate</td><td>Sigma Aldrich</td><td>C0250</td><td></td></tr><tr><td>Sterile filters</td><td>Millipore</td><td>SLGP033RS or similar</td><td></td></tr><tr><td>Tris</td><td>Sigma Aldrich</td><td>252859 or similar</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Ultra-low protein binding tubes</td><td>VWR</td><td>76322-516, 76322-522 or similar</td><td></td></tr><tr><td>Ultrapure water</td><td>Thermo</td><td>15230253 or similar</td><td></td></tr></tbody></table>

generating a fractal microstructure of laminin-111 to signal to cells

La laminina-111 (Ln1) è una parte essenziale della matrice extracellulare negli epiteli, nei muscoli e nei sistemi neurali. Abbiamo precedentemente dimostrato che la microstruttura di Ln1 altera il modo in cui segnala alle cellule, probabilmente perché l'assemblaggio di Ln1 in reti espone diversi domini adesivi. In questo protocollo, descriviamo tre metodi per generare Ln1 polimerizzato.

La laminina-111 è una proteina trimerica con tre domini autoassemblanti all'estremità dei suoi bracci corti (Figura 1A). Se trattati adeguatamente, i bracci corti possono polimerizzare in un reticolo esagonale (Figura 1B,1C), che mostra proprietà frattali aggregate limitate alla diffusione su scale spaziali più grandi (Figura 2). La laminina incubata in tamponi a pH neutro contenenti calcio forma piccoli aggrega...

Watch this Scientific Journal Video about Generating a Fractal Microstructure of Laminin-111 to Signal to Cells at JoVE.com

Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

Descriviamo tre metodi per generare polimeri Ln1 con proprietà frattali che segnalano alle cellule in modo diverso rispetto a Ln1 non polimerizzato.

Generazione di una microstruttura frattale di laminina-111 per segnalare alle cellule

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the ...

generating-a-fractal-microstructure-of-laminin-111-to-signal-to-cells

Research

JoVE Journal

Bioengineering

924 Views.  Texas Heart Institute. Descriviamo tre metodi per generare polimeri Ln1 con proprietà frattali che segnalano alle cellule in modo diverso rispetto a Ln1 non polimerizzato.

Video: Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

La creazione di substrati Patterned bidimensionali per confinamento di proteine ​​e cellule

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Ricerca

Bioingegneria

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

Studi strutturali di macromolecole in soluzione con piccolo angolo x-ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

Biochimica

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

Nanofibre ECM proteine ​​e Nanostrutture Engineered Uso Assembly-Surface avviato

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microambienti sandwich di sfruttare Cellulari / Materiale Interazioni

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparazione del 3D collagene Gel e microcanali per lo studio delle interazioni 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Tessuti epiteliali Ingegneria tridimensionali integrati all&#39;interno di matrice extracellulare

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

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Array Ligand Nano-cluster in un doppio strato lipidico supportati

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Generazione di modelli per microscopia a trazione micropattern

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the cells themselves. Here, we present the method of Light-Induced Molecular Adsorption of Proteins (LIMAP) using the PRIMO system as a patterning technique to produce micro-patterned extracellular matrix (ECM) substrates using a single or combination of proteins. The method enables printing of ECM patterns in micron resolution with excellent reproducibility. We provide a step-by-step protocol and demonstrate how this can be applied to study the processes of neuronal pathfinding. LIMAP has significant advantages over existing micro-printing methods in terms of the ease of patterning more than one component and the ability to generate a pattern with any geometry or gradient. The protocol can easily be adapted to study the contribution of almost any chemical component towards cell fate and cell behavior. Finally, we discuss common issues that can arise and how these can be avoided.

Our overall aim is to understand how cells sense extracellular cues that lead to directed axonal growth. Here, we describe the methodology of Light-Induced Molecular Adsorption of Proteins, used to produce defined micro-patterns of extracellular matrix components in order to study specific events that govern axon outgrowth and pathfinding.

Adsordimento molecolare indotto dalla luce delle proteine utilizzando il sistema PRIMO per micro-patterning per studiare le risposte cellulari alle proteine a matrice extracellulare

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the ...

Neuroscienze

Fabricating Engineered Nanofibrillar Collagen with Directed Patterning

Production of Nanofibrillar Patterned Collagen for Tissue Engineering

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are designed to emulate the biophysical properties of native tissue, providing cellular phenotypic and morphological guidance that contributes to the restoration of the regenerative tissue niche. Collagen, a prevalent extracellular matrix protein, is a common component of these regenerative biomaterials due to its biocompatibility and other favorable properties. The current study describes a novel and straightforward method for the fabrication of engineered nanofibrillar collagen with directed fibril patterning. Through the manipulation of shear stress, temperature, and pH, collagen fibrillogenesis and alignment are precisely controlled without requiring specialized equipment. This approach allows for the creation of nanofibrillar collagen biomaterials that mimic the native structure of tissues exhibiting either anisotropic or isotropic characteristics. The flexibility in collagen nanofibril patterning not only facilitates the study of nanoscale patterning on cell behavior but also offers diverse possibilities for patterned tissue engineering applications.

The following protocol presents a shear-based extrusion method for the fabrication of collagen hydrogels with nanoscale patterned fibrils, which can be applied to a broad range of tissue engineering applications.

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are ...

Generazione di una microstruttura frattale di laminina-111 per segnalare alle cellule

Riepilogo

Esplora Altri Video

Capitoli in questo video

La creazione di substrati Patterned bidimensionali per confinamento di proteine e cellule

Studi strutturali di macromolecole in soluzione con piccolo angolo x-ray Scattering

Nanofibre ECM proteine e Nanostrutture Engineered Uso Assembly-Surface avviato

Microambienti sandwich di sfruttare Cellulari / Materiale Interazioni

Preparazione del 3D collagene Gel e microcanali per lo studio delle interazioni 3D

Tessuti epiteliali Ingegneria tridimensionali integrati all'interno di matrice extracellulare

Array Ligand Nano-cluster in un doppio strato lipidico supportati

Generazione di modelli per microscopia a trazione micropattern

Adsordimento molecolare indotto dalla luce delle proteine utilizzando il sistema PRIMO per micro-patterning per studiare le risposte cellulari alle proteine a matrice extracellulare

Production of Nanofibrillar Patterned Collagen for Tissue Engineering

Generazione di una microstruttura frattale di laminina-111 per segnalare alle cellule

Riepilogo

Esplora Altri Video

Capitoli in questo video

La creazione di substrati Patterned bidimensionali per confinamento di proteine ​​e cellule

Studi strutturali di macromolecole in soluzione con piccolo angolo x-ray Scattering

Nanofibre ECM proteine ​​e Nanostrutture Engineered Uso Assembly-Surface avviato

Microambienti sandwich di sfruttare Cellulari / Materiale Interazioni

Preparazione del 3D collagene Gel e microcanali per lo studio delle interazioni 3D

Tessuti epiteliali Ingegneria tridimensionali integrati all'interno di matrice extracellulare

Array Ligand Nano-cluster in un doppio strato lipidico supportati

Generazione di modelli per microscopia a trazione micropattern

Adsordimento molecolare indotto dalla luce delle proteine utilizzando il sistema PRIMO per micro-patterning per studiare le risposte cellulari alle proteine a matrice extracellulare

Production of Nanofibrillar Patterned Collagen for Tissue Engineering

La creazione di substrati Patterned bidimensionali per confinamento di proteine e cellule

Nanofibre ECM proteine e Nanostrutture Engineered Uso Assembly-Surface avviato