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>

<ol>
	<li>Carey, S. P., Kraning-Rush, C. M., Williams, R. M., Reinhart-King, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+control+of+invasive+tumor+cell+behavior+by+extracellular+matrix+microarchitecture.">Biophysical control of invasive tumor cell behavior by extracellular matrix microarchitecture.</a> Biomaterials. 33 (16), 4157-4165 (2012).</li><li>Wang, K., 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=Stiffening+and+unfolding+of+early+deposited-fibronectin+increase+proangiogenic+factor+secretion+by+breast+cancer-associated+stromal+cells.">Stiffening and unfolding of early deposited-fibronectin increase proangiogenic factor secretion by breast cancer-associated stromal cells.</a> Biomaterials. 54, 63-71 (2015).</li><li>Bruekers, S. M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin-fiber+architecture+influences+cell+spreading+and+differentiation.">Fibrin-fiber architecture influences cell spreading and differentiation.</a> Cell Adhesion &amp; Migration. 10 (5), 495-504 (2016).</li><li>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=Integrating+Activities+of+Laminins+that+Drive+Basement+Membrane+Assembly+and+Function.">Integrating Activities of Laminins that Drive Basement Membrane Assembly and Function.</a> Curr Top Membr. 76, 1-30 (2015).</li><li>Colognato, H., Winkelmann, D. A., 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=Laminin+Polymerization+Induces+a+Receptor-Cytoskeleton+Network.">Laminin Polymerization Induces a Receptor-Cytoskeleton Network.</a> The Journal of Cell Biology. 145 (3), 619-631 (1999).</li><li>Powell, S. K., Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+laminins+and+their+cellular+receptors.">Neuronal laminins and their cellular receptors.</a> International Journal of Biochemistry &amp; Cell Biology. 29 (3), 401-414 (1997).</li><li>Li, M. L., 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=Influence+of+a+reconstituted+basement+membrane+and+its+components+on+casein+gene+expression+and+secretion+in+mouse+mammary+epithelial+cells.">Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 84 (1), 136-140 (1987).</li><li>Fiore, A., 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=Laminin-111+and+the+Level+of+Nuclear+Actin+Regulate+Epithelial+Quiescence+via+Exportin-6.">Laminin-111 and the Level of Nuclear Actin Regulate Epithelial Quiescence via Exportin-6.</a> Cell Rep. 19 (10), 2102-2115 (2017).</li><li>Inman, J. L., Robertson, C., Mott, J. D., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+gland+development:+cell+fate+specification,+stem+cells+and+the+microenvironment.">Mammary gland development: cell fate specification, stem cells and the microenvironment.</a> Development. 142 (6), 1028-1042 (2015).</li><li>Weaver, V. M., 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=Reversion+of+the+malignant+phenotype+of+human+breast+cells+in+three-dimensional+culture+and+in+vivo+by+integrin+blocking+antibodies.">Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.</a> Journal of Cell Biology. 137 (1), 231-245 (1997).</li><li>Furuta, S., Ren, G., Mao, J. -. H., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+signals+initiate+the+reciprocal+loop+that+informs+breast-specific+gene+expression+and+homeostasis+by+activating+NO,+p53+and+microRNAs.">Laminin signals initiate the reciprocal loop that informs breast-specific gene expression and homeostasis by activating NO, p53 and microRNAs.</a> eLife. 7, 26148 (2018).</li><li>Brown, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophic+phenotype+induced+in+vitro+by+antibody+blockade+of+muscle+alpha-dystroglycan-laminin+interaction.">Dystrophic phenotype induced in vitro by antibody blockade of muscle alpha-dystroglycan-laminin interaction.</a> Journal of Cell Science. 112 (2), 209 (1999).</li><li>Calof, A. L., Campanero, M. R., O'Rear, J. J., Yurchenco, P. D., Landert, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain-specific+activation+of+neuronal+migration+and+neurite+outgrowth-promoting+activities+of+laminin.">Domain-specific activation of neuronal migration and neurite outgrowth-promoting activities of laminin.</a> Neuron. 13 (1), 117-130 (1994).</li><li>Li, S., 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=Laminin-sulfatide+binding+initiates+basement+membrane+assembly+and+enables+receptor+signaling+in+Schwann+cells+and+fibroblasts.">Laminin-sulfatide binding initiates basement membrane assembly and enables receptor signaling in Schwann cells and fibroblasts.</a> The Journal of Cell Biology. 169 (1), 179-189 (2005).</li><li>Sonnenberg, A., 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=Integrin+recognition+of+different+cell-binding+fragments+of+laminin+(P1,+E3,+E8)+and+evidence+that+alpha+6+beta+1+but+not+alpha+6+beta+4+functions+as+a+major+receptor+for+fragment+E8.">Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8.</a> The Journal of Cell Biology. 110 (6), 2145-2155 (1990).</li><li>Sorokin, L., Sonnenberg, A., Aumailley, M., Timpl, R., Ekblom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+the+laminin+E8+cell-binding+site+by+an+integrin+possessing+the+alpha+6+subunit+is+essential+for+epithelial+polarization+in+developing+kidney+tubules.">Recognition of the laminin E8 cell-binding site by an integrin possessing the alpha 6 subunit is essential for epithelial polarization in developing kidney tubules.</a> The Journal of Cell Biology. 111 (3), 1265-1273 (1990).</li><li>Horejs, C. -. M., 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=Biologically-active+laminin-111+fragment+that+modulates+the+epithelial-to-mesenchymal+transition+in+embryonic+stem+cells.">Biologically-active laminin-111 fragment that modulates the epithelial-to-mesenchymal transition in embryonic stem cells.</a> Proceedings of the National Academy of Sciences. 111 (16), 5908-5913 (2014).</li><li>Hochman-Mendez, C., Cantini, M., Moratal, D., Salmeron-Sanchez, M., 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=A+Fractal+Nature+for+Polymerized+Laminin.">A Fractal Nature for Polymerized Laminin.</a> PLOS ONE. 9 (10), 109388 (2014).</li><li>Kent, A. 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=The+microstructure+of+laminin-111+compensates+for+dystroglycan+loss+in+mammary+epithelial+cells+in+downstream+expression+of+milk+proteins.">The microstructure of laminin-111 compensates for dystroglycan loss in mammary epithelial cells in downstream expression of milk proteins.</a> Biomaterials. 218, 119337 (2019).</li><li>Weir, M. L., 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=Dystroglycan+loss+disrupts+polarity+and+beta-casein+induction+in+mammary+epithelial+cells+by+perturbing+laminin+anchoring.">Dystroglycan loss disrupts polarity and beta-casein induction in mammary epithelial cells by perturbing laminin anchoring.</a> Journal of Cell Science. 119, 4047-4058 (2006).</li><li>Hughes, C. S., Postovit, L. M., Lajoie, G. 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 border="0" cellpadding="0" cellspacing="0" style="width:665px;" width="665">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10% formalin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">HT501128</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Laminin primary antibody</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">L9393</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-Rabbit Alexafluor 488 secondary antibody</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">A32731</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">C4901 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell Culture Incubator</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">Heracell 150 or similar</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge for microcentrifuge tubes</td>
			<td style="width:121px;">Eppendorf</td>
			<td style="width:161px;">5418 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Confocal Microscope</td>
			<td style="width:121px;">Zeiss</td>
			<td style="width:161px;">LSM 710 or equivalent</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coverslips</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">25CIR-1 or similar</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DMEM-F12, Liquid, High Glucose, +HEPES, L-glutamine, +Phenol red</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">11330107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EGF (Epidermal Growth Factor)</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">11376454001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescently Labeled Laminin</td>
			<td style="width:121px;">Cytoskeleton Inc</td>
			<td style="width:161px;">LMN02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gentamicin</td>
			<td style="width:121px;">VWR</td>
			<td style="width:161px;">VWRV0304-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutaraldehyde</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">G5882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HCl</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">320331 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrocortisone</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td style="width:161px;">H0888-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">16634-50mg</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MepG Dystroglycan Knockout Cells</td>
			<td style="width:121px;">LBNL</td>
			<td style="width:161px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaOH</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">S8045 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Normocin</td>
			<td style="width:121px;">Invivogen</td>
			<td style="width:161px;">ant-nr-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Osmium Tetroxide</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">75633</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Ovine Prolactin</td>
			<td style="width:121px;">Los Angeles Biomedical Research Institute</td>
			<td style="width:161px;">Ovine Pituitary Prolactin</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phalloidin</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">A22287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">10010023 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Purified Laminin-111</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">L2020-1MG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Recombinant human Nidogen-1, carrier free</td>
			<td style="width:121px;">R&#38;D systems</td>
			<td style="width:161px;">2570-ND-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Acetate</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">S2889 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Cacodylate</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">C0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile filters</td>
			<td style="width:121px;">Millipore</td>
			<td style="width:161px;">SLGP033RS or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">252859 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td style="width:161px;">X100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultra-low protein binding tubes</td>
			<td style="width:121px;">VWR</td>
			<td style="width:161px;">76322-516, 76322-522 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultrapure water</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">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

914 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

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Progettazione di un bioreattore per la pressione ciclica Ex Vivo Studio delle valvole cardiache aortica

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Ricerca

Bioingegneria

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Una pelle piena Difetto modello per valutare Vascolarizzazione di Biomateriali<em&gt; In Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

Un sistema integrato di attivare remotamente la trasduzione del segnale intracellulare di fotoattivazione Upconversion Nanoparticle-mediata della chinasi

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

Generazione di paesaggi chimici dinamici e controllati per studiare il comportamento microbico

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, biomimetic surfaces mimicking the microstructure of leaf surface, were used to study the effects of leaf microstructure on leaf-environment interactions. However, no such tool exists for roots. We developed a tool allowing the synthetic mimicry of the root surface microstructure into an artificial surface. We relied on the soft lithography method, known for leaf surface microstructure replication, using a two-step process. The first step is the more challenging one as it involves the biological tissue. Here, we used a different polymer and curing strategy, relying on the strong, rigid, polyurethane, cured by UV for the root molding. This allowed us to achieve a reliable negative image of the root surface microstructure including the delicate, challenging features such as root hairs. We then used this negative image as a template to achieve the root surface microstructure replication using both the well-established polydimethyl siloxane (PDMS) as well as a cellulose derivative, ethyl cellulose, which represents a closer mimic of the root and which can also be degraded by cellulase enzymes secreted by microorganisms. This newly formed platform can be used to study the microstructural effects of the surface in root-microorganism interactions in a similar manner to what has previously been shown in leaves. Additionally, the system enables us to track the microorganism&#8217;s locations, relative to surface features, and in the future its activity, in the form of cellulase secretion.

Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.

Replica biomimetica della microstruttura superficiale della radice mediante alterazione della litografia morbida

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Misurazione del potenziale d'azione ottico a singola cellula in cardiomiociti derivati da cellule staminali pluripotenti indotte umane

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Un metodo di coltura per mantenere le cellule staminali ematopoietiche umane quiescenti

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.

Assemblaggio e funzionamento di un dispositivo acoustofluidico per una maggiore consegna di composti molecolari alle cellule

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Tessuto connettivo ingegnerizzato umano derivato da fibroblasti per applicazioni di screening

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of &#34;molecular surgery&#34; to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical &#946;-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual &#946;-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level (&#34;molecular surgery&#34;), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of &#946;-barrel fluorescent proteins.

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

Scambio specifico di prolina da parte di analoghi della prolina in proteine fluorescenti: come la "chirurgia molecolare" della spina dorsale influisce sul ripiegamento e sulla stabilità

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often ...