Cellule CHO-P sono stati un gentile dono del Dr. Barbara Furie (Beth Israel Deaconess Medical Center, Harvard Medical School). Questo lavoro è stato sostenuto dal National Institute of Health di sovvenzione HL095722 a JMK Questo lavoro è stato supportato anche in parte da un Movember-Prostate Cancer Foundation Challenge Award per JMK

1. Cell Culture <ol><li> Leucemia promielocitica umana (HL-60) le cellule <ol><li> Culture cellule HL-60 nel 75 cm 2 palloni con 15 ml di Iscove Modified Medium (IMDM) di Dulbecco, supplementato con 20% (v / v) di siero fetale bovino (FBS), 1% (v / v) L-glutammina e 1 % (v / v) Penicillina-Streptomicina. </li><li> Cambiare supporto ogni 3 giorni aspirando metà del volume di sospensione cellulare e sostituendolo con mezzi IMDM completo. </li><li> Per carbossifluoresceina diacetato, estere succinimidyl (CFSE) colorazione, centrifuga HL-60 sospensione cellulare (400 xg, 5 min), risospendere in una soluzione 1 mM CFSE (preparato in PBS preriscaldata) e incubare per 15 min a 37 ° C. Poi centrifugare le cellule, aspirare il surnatante e risospendere le cellule in un mezzo fresco preriscaldato per 30 min. Lavare le cellule in PBS e poi utilizzare per esperimenti di laminazione (vedere Figura 1B per un&#39;immagine rappresentativa della CFSE colorati con cellule HL-60 su P-sel-superficie rivestita). </li></ol></li></ol><p class = &quot;jove_content&quot;&gt; Nota: CFSE colorazione è facoltativo, ed è presentato qui per dimostrare il fenomeno di laminazione nel canale microfluidica. Analisi dei parametri di laminazione presentati in questo manoscritto è stato eseguito su cellule non colorate utilizzando l&#39;imaging campo chiaro standard. <ol start="2"><li> Cellule endoteliali microvascolari polmonari (LMVECs) <ol><li> Coat 100 millimetri piastre di Petri con soluzione di gelatina 0,1% (v / v in PBS) e incubare a 37 ° C per almeno 30 min. </li><li> LMVECs Cultura in gelatina rivestite 100 millimetri piastre di Petri in mezzo completo di crescita endoteliale (endothelial basale medio-2 (EBM-2)), integrato con uno specifico kit di supplemento di crescita, vedi REAGENTI). Cambiare i media ogni altro giorno e cellule sub-cultura al raggiungimento di 80-90% di confluenza. </li><li> Per sub-coltura, lavare le cellule con PBS e poi staccare cellule con 4 ml di 1x Tripsina-EDTA per 3 min a 37 ° C e neutralizzare in un volume uguale di completi EBM-2 media. Trasferire la sospensione cellulare in una provetta da 15 ml e centrifuge (400 xg, 5 min). Dopo la centrifugazione, risospendere il pellet in 1 ml di mezzi completi endoteliali e contare le cellule con un emocitometro. Non oltre-passare le cellule, in quanto questo incide sulla loro morfologia e funzione utilizzare solo cellule sotto il passaggio 7 per tutti gli esperimenti. </li></ol></li></ol><ol start="3"><li> Ovaio di criceto cinese-P-selectina (CHO-P) Cells <ol><li> Cellule CHO-P, che sono cellule CHO trasfettate stabilmente per esprimere umana P-sel, sono stati forniti dai collaboratori (Beth Israel Deaconess Medical Center, Harvard Medical School) 11,12. </li><li> Cellule Cultura CHO-P nel T175 cm 2 boccette in 25 ml di F-12 media. </li><li> Per passaging, lavare le cellule con 10 ml di PBS per 4-5 secondi e poi trypsinize in 10 ml di 1x Tripsina-EDTA per 3 min a 37 ° C, seguito da neutralizzazione in mezzi completi. </li><li> Centrifugare la sospensione cellulare (400 xg, 5 min), aspirare accuratamente il surnatante, risospendere il pellet di cellule in 1 ml di mezzi completi e contare le cellule conun emocitometro. </li></ol></li></ol> 2. Il funzionamento del Integrated Multi-pozzetti sistema Microfluidic <ol><li> Assicurarsi che tutta l&#39;attrezzatura sia collegato correttamente e accendere i diversi moduli: il calcolatore, controllore, microscopio invertito, e fotocamera CCD. </li><li> Aprire il software di imaging, assicurarsi che il modulo piastra multi-bene e il modulo di imaging siano correttamente presentate sullo schermo. </li><li> Collegare i tubi alla trappola del vapore (collegato al controller), e anche li collegano all&#39;interfaccia pressione. </li><li> Posizionare la piastra multi-pozzetto nella piastra di riscaldamento / adattatore. Aggiungere nei pozzetti (descritti sotto) e attaccare un interfaccia sulla parte superiore della piastra. Posizionare la piastra per l&#39;imaging sul palco automatizzato. </li><li> L&#39;interfaccia attribuisce alla parte superiore della piastra e applica una pressione pneumatica dal controller all&#39;inizio dei pozzi, guidando il fluido attraverso i canali microfluidica alla portata definita, facilmente controllato utilizzando la piastra multi-pozzettoschermo di modulo in modalità manuale. </li><li> Reagenti nel flusso canale attraverso un&#39;area di osservazione, situato tra i pozzetti. Dimensioni del canale microfluidica sono 350 micron di larghezza x 70 micron di altezza. La lunghezza del canale lineare è 1 mm e il fondo dei canali comprende un coprioggetti da 180 micron, che è compatibile con campo chiaro, fase, fluorescenza e microscopia confocale. </li><li> Acquisire video utilizzando una camera CCD (acquisizione flusso, 11 fotogrammi / sec) e analizzare tramite software compatibile. </li></ol> 3. Rivestimento di canali microfluidica con un substrato di proteine ​​o di un monostrato di cellule <ol><li> Rivestimento canale microfluidica con fibronectina o P-/E-selectin <ol><li> Preparare 1 ml di 20 mg / ml soluzione fibronectina in PBS. Modificare il volume in base al numero di canali da verniciare (utilizzare 25-50 ml di fibronectina per canale). </li><li> Aggiungere 25-50 ml di soluzione di fibronectina ad ogni ingresso bene. Applicare forza di taglio di 2 dine / cm 2per 5 min a perfusione canale. Si prega di notare il tallone di liquido che compare nella presa bene. Incubare per 30-45 minuti a temperatura ambiente </li><li> Aspirare la soluzione da pozzi (non aspirare direttamente dal cerchio centrale che alimenta il canale) 1,13. Aggiungere 200-500 ml di PBS in presa e lavare canale con PBS applicando flusso di taglio di 2 dyn / cm 2 per 5 min. Il canale è ora correttamente rivestito con fibronectina e pronto per essere utilizzato. </li><li> Per ricoprire con P-o E-SEL, preparare una soluzione 5 mg / ml di proteina ricombinante umana desiderata in PBS, e rivestire i canali come descritto sopra, con 1 ora di incubazione a 37 ° C per un rivestimento superficiale. </li></ol></li><li> Creazione di CHO-P o LMVEC monostrato all&#39;interno del canale microfluidica <ol><li> Trypsinize delicatamente le cellule dai piatti di coltura per 3 minuti, spegnere utilizzando un volume di 2 volte di supporti pieni e centrifugare (5 min a 400 xg). Risospendere le cellule con 10 ml di supporti pieni e centrifugare (5 min a 400 xg) again. </li><li> Contare le cellule per determinare la concentrazione delle cellule in sospensione. Per garantire la formazione di un monostrato confluente LMVEC all&#39;interno del canale, portare la concentrazione cellulare a 15-20 milioni di cellule / ml. Per un confluente CHO-P monostrato cellulare, utilizzare 50-60 milioni di cellule / ml. Utilizzare 25-50 ml di sospensione cellulare per ogni canale - determinare il numero di cellule iniziale usato per l&#39;esperimento di conseguenza. </li><li> Aggiungere 25-50 ml di sospensione cellulare in concentrazione adeguata al bocchettone. Posizionare la piastra sul palco microscopio e introdurre cellule nel canale (2 dyn / cm 2) finché le cellule si osservano sullo schermo riempiendo tutto canali, e quindi arrestare il flusso. </li><li> Riempire sia uscita e ingresso con 200 ml di LMVEC sia pieno o CHO media. Lasciate che le cellule si depositano ed aderiscono per 3 ore in incubatore (37 ° C, 5% CO 2). </li><li> Seguendo la incubazione 3 hr, lavare il canale con piena supporti (2 dyn / cm 2, 10-15 min) per rimuovere distaccaticellule. Le cellule dovrebbero ora appaiono completamente confluenti e il canale è ora pronto per l&#39;uso. A seconda iniziale densità di semina cellulare, ulteriori 2-3 ore di tempo di assestamento può essere necessaria per assicurare una copertura completa della superficie con le cellule. </li></ol></li></ol> 4. LMVEC attivazione pro-infiammatoria e anticorpo Blocco di P-/E-selectin <ol><li> Preparare una soluzione di TNF-α (10 ng / ml) in LMVEC mezzi basale. </li><li> Per indurre l&#39;attivazione infiammatoria LMVEC nei canali, aggiungere 100 microlitri della soluzione di TNF-α al bocchettone e introdurre la soluzione nel canale applicando flusso di taglio di 2 dyn / cm 2 per 5 min. Per i canali di controllo (non attivato EC), aggiungere 100 ml di LMVEC supporti basale a bocchettone e introdurre nel canale (2 dyn / cm 2 per 5 min). Canale è ora pronto per un saggio di rotolamento. </li><li> Per bloccare P-sel e E-sel su LMVECs e cellule CHO-P, introdurre neutralizzando P-sel (clone AK4, 5 mg / ml in basal media) o E-sel (P2H3 clone, 5 ug / ml in mezzi basale) anticorpi nel canale e incubare per 1 ora a 37 ° C. Successivamente, lavare canali con i media basale (2 dyn / cm 2 per 5 min). I canali sono ora pronti per un saggio di rotolamento. </li></ol> 5. HL-60 di Rolling saggio sul substrato / Cellulari monostrato-Coated canali microfluidica <ol><li> Esaminare attentamente i canali al microscopio per confermare che i canali siano rivestite (nel caso del rivestimento con cellule, un monostrato di cellule confluenti completamente deve osservare). </li><li> Per preparare HL-60 sospensione cellulare per gli esperimenti di rotolamento, centrifuga HL-60 sospensione cellulare (5 min a 400 xg) e lavare una volta con terreni di base. Contare le cellule e risospendere in IMDM (supporti basali, contenenti Ca 2 + e Mg 2 +) per creare una sospensione di cellule HL-60 con 5 milioni di cellule / ml. Utilizzare 25-50 ml di sospensione cellulare per ogni canale per eseguire il test di rotolamento. </li><li> Aggiungere 25-50 ml di Sosp cellularension alla presa bene, piastra posto all&#39;interno del portatarga a temperatura controllata (37 ° C) e posto sul palco microscopio. Quindi, introdurre cellule nel canale applicando forza di taglio di 2 dine / cm 2 (celle devono essere osservate entro 10-15 secondi che scorre dalla presa di ingresso). </li><li> Per esaminare la risposta rotolamento in funzione della sollecitazione di taglio, ridurre taglio a 0,25 dyn / cm 2 e acquisire video 20-30 sec (tramite la funzione &quot;acquisizione stream&quot;) in ogni taglio desiderata (shear aumentare gradualmente da 0,25 fino a 5 dyn / cm 2. E &#39;anche possibile utilizzare cesoie superiori). </li><li> Acquisire video utilizzando una camera CCD (acquisizione flusso, 11 fotogrammi / sec) e analizzare i percorsi di laminazione e di laminazione velocità via software compatibile. </li></ol> 6. Citometria a flusso per rilevare l&#39;espressione di molecole di superficie <ol><li> Dopo tripsinizzazione, preparare una sospensione cellulare (con 1-2 x 10 5 cellule / campione) di tipo cellulare desiderato (HL-60, CHO-P o LMVECs) in PBS (- / -), supplementato con 2% FBS. Lavare le cellule due volte e portare il volume del campione a 50 microlitri (usando lo stesso tampone). </li><li> Incubare ogni campione con il fluoroforo coniugato desiderato Ab (vedi tabella allegata per informazioni dettagliate) a 4 ° C per 20 min (coprire con un foglio di alluminio). </li><li> Lavare le cellule due volte (stesso tampone) e portare il volume finale della sospensione cellulare tinto a 200 microlitri. Analizzare campioni usando un citometro a flusso per rilevare l&#39;espressione di molecole di superficie. </li></ol>

<ol>
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A model for an antibody-based microfluidic screening system.</a> Microelectronic Eng. 98, 668-671 (2012).</li><li>Perozziello, G., 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=Microfluidic+devices+modulate+tumor+cell+line+susceptibility+to+NK+cell+recognition.">Microfluidic devices modulate tumor cell line susceptibility to NK cell recognition.</a> Small. 8, 2886-2894 (2012).</li><li>Perozziello, G., 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=Microfluidic+biofunctionalisation+protocols+to+form+multivalent+interactions+for+cell+rolling+and+phenotype+modification+investigations.">Microfluidic biofunctionalisation protocols to form multivalent interactions for cell rolling and phenotype modification investigations.</a> Electrophoresis. , (2013).</li><li>Simone, G., 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=A+facile+in+situ+microfluidic+method+for+creating+multivalent+surfaces:+toward+functional+glycomics.">A facile in situ microfluidic method for creating multivalent surfaces: toward functional glycomics.</a> Lab Chip. 12, 1500-1507 (2012).</li><li>Sarkar, D., 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=Engineered+cell+homing.">Engineered cell homing.</a> Blood. 118, e184-e191 (2011).</li><li>Cheng, Z., 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=Targeted+Migration+of+Mesenchymal+Stem+Cells+Modified+With+CXCR4+Gene+to+Infarcted+Myocardium+Improves+Cardiac+Performance.">Targeted Migration of Mesenchymal Stem Cells Modified With CXCR4 Gene to Infarcted Myocardium Improves Cardiac Performance.</a> Mol. Ther. 16, 571-579 (2008).</li><li>Enoki, 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=Enhanced+mesenchymal+cell+engraftment+by+IGF-1+improves+left+ventricular+function+in+rats+undergoing+myocardial+infarction.">Enhanced mesenchymal cell engraftment by IGF-1 improves left ventricular function in rats undergoing myocardial infarction.</a> Int. J. Cardiol. 138, 9-18 (2010).</li></ol>

Gli autori dichiarano assenza di conflitto di interessi.

Una delle sfide principali nella traduzione di successo della terapia a base di cellule esogena è l&#39;incapacità di fornire in modo efficiente le celle a siti di lesioni e infiammazioni con alta efficienza attecchimento 3. Laminazione cella rappresenta un passo fondamentale nel processo di homing delle cellule, facilitando la decelerazione di cellule sulle pareti dei vasi sanguigni, portando infine alla loro ferma adesione e trasmigrazione attraverso l&#39;endotelio nel tessuto 5. Una migliore ...

Una delle sfide principali nella traduzione clinica di successo della terapia a base di cellule è la consegna inefficiente o il targeting di cellule sistemica infuse ai siti desiderati 3,4. Di conseguenza, vi è una costante ricerca di metodi per migliorare homing delle cellule, e specificamente cellula rotolamento, come strategia per migliorare la terapia cellulare. Cella a rotazione sui vasi sanguigni è un passo fondamentale nella cascata di homing delle cellule, definito classicamente per i leucociti che vengono reclutati per siti di malattia 5. Questa fase è regolata da interazioni specifiche tra selectine endoteliali, cioè-P ed E-selectina (-P e E-sel), e loro ligandi contatore sulla superficie dei leucociti 5,6. Una migliore comprensione e una migliore efficienza di homing delle cellule, e in particolare la fase di laminazione, sono di grande importanza nella ricerca di nuove piattaforme per migliorare la terapia cellulare. Fino ad oggi questo è stato ottenuto utilizzando camere di flusso piastra parallele (PPFCs), comprendente due plat piattoes con una guarnizione tra loro, con una apertura di afflusso e deflusso trova alla piastra superiore, attraverso la quale una sospensione cellulare è perfuso utilizzando una siringa pompa 7,8, 9. La superficie della piastra inferiore può essere rivestito con un monostrato di cellule competenti / substrati e l&#39;interazione tra cellule perfusi e la superficie sotto flusso di taglio è poi esplorato 7. Tuttavia, PPFC è un volume basso, reattivo-consumo, e metodo abbastanza noioso, con formazione di bolle, perdite, e il flusso scarsamente controllato presenta gravi inconvenienti. Una tecnica alternativa alla PPFC tradizionale è un sistema di microfluidica piastra multi-pozzetto, permettendo prestazioni più elevate velocità di analisi cellulari (fino a 10 volte superiore rispetto PPFCs) sotto flusso preciso e taglio controllato da computer, con basso consumo di reagente 1,10. Esperimenti di laminazione delle cellule vengono eseguiti all&#39;interno dei canali microfluidica, che può essere rivestito con monostrati cellulari o substrati ingegnerizzati e USI ripresong un microscopio, con proprietà di rotolamento prontamente analizzati utilizzando un software adatto. In questo studio, abbiamo dimostrato le capacità di questo multi-well sistema di microfluidica piastra di studiare le proprietà di rotolamento di leucemia promielocitica umana (HL-60) le cellule su superfici diverse. HL-60 rotola su supporti come-P ed E-sel, nonché su monostrati di cellule che esprimono diversi recettori di rotolamento, è stato analizzato. Inoltre, l&#39;anticorpo (Ab) di blocco è stato utilizzato per dimostrare coinvolgimento diretto di selectine specifici nel mediare il movimento di rotolamento di HL-60 su tali superfici. Esperimenti di rotolamento sono stati effettuati con maggiore velocità, sotto flusso di taglio stabile, con il minimo consumo di reagente / cellulare, che permette l&#39;analisi efficiente dei parametri di laminazione chiave come velocità di rotazione, il numero di cellule di rotolamento, e rotolamento proprietà del percorso.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cells</td>
		</tr>
		<tr>
			<td>Human Lung Microvascular Endothelial Cells</td>
			<td>Lonza</td>
			<td>CC-2527</td>
			<td></td>
		</tr>
		<tr>
			<td>P-selectin-expressing Chinese Hamster Ovary Cells (CHO-P)</td>
			<td>Kind gift by Dr. Barbara Furie11,12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HL-60 Cells</td>
			<td>ATCC</td>
			<td>CCL-240</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cell Culture Reagents</td>
		</tr>
		<tr>
			<td>Endothelial Basal Medium</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr>
			<td>EBM-2 Media</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Basal Medium Supplements</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 MV SingleQuots</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM - Iscove&#39;s Modified Dulbecco&#39;s Medium 1x</td>
			<td>Gibco</td>
			<td>12440</td>
			<td></td>
		</tr>
		<tr>
			<td>F-12 (1x) Nutrient Mixture (Ham)</td>
			<td>Gibco</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin (P/S)</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (L/G) 200 mM</td>
			<td>Gibco</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>Sa550</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>BD Falcon</td>
			<td>BD-353003</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Cell Culture Dish, Tissue-Culture Treated Polystyrene</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (15 ml polypropylene conical tubes)</td>
			<td>MedSupply Partners</td>
			<td>TC1500</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 Flasks</td>
			<td>BD Falcon</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin Solution (2%)</td>
			<td>Sigma</td>
			<td>G1393</td>
			<td></td>
		</tr>
		<tr>
			<td>dPBS (without calcium chloride and magnesium chloride)</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA Solution (10x)</td>
			<td>Sigma</td>
			<td>T4174</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Antibodies</td>
		</tr>
		<tr>
			<td>Anti-hE-Selectin/CD62E</td>
			<td>R&#38;D Systems</td>
			<td>BBA21</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Conjugated Mouse IgG1</td>
			<td>R&#38;D Systems</td>
			<td>BBA21</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-hP-Selectin</td>
			<td>R&#38;D Systems</td>
			<td>BBA34</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Conjugated Mouse IgG1</td>
			<td>R&#38;D Systems</td>
			<td>BBA34</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse IgG&#173;1 &#954; Isotype Control</td>
			<td>BD Bioscience</td>
			<td>555748</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-SLeX /CD15s Ab, Clone: 5F18</td>
			<td>Santa Cruz</td>
			<td>SC70545</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Conjugated</td>
			<td>Santa Cruz</td>
			<td>SC70545</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Mouse IgM-FITC Isotype Control</td>
			<td>Santa Cruz</td>
			<td>SC2859</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse Anti-Human CD162, Clone: KPL-1</td>
			<td>BD Pharmingen</td>
			<td>556055</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse IgG1 k Isotype Control</td>
			<td>BD Pharmingen</td>
			<td>550617</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-P-Selectin Ab (AK4)</td>
			<td>Santa Cruz</td>
			<td>SC19996</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-E-Selectin Ab, Clone P2H3</td>
			<td>Millipore</td>
			<td>MAB2150</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG1 Isotype Control</td>
			<td>Santa Cruz</td>
			<td>SC3877</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Other Reagents</td>
		</tr>
		<tr>
			<td>Recombinant Human TNF-alpha</td>
			<td>PeproTech</td>
			<td>300-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Trace CFSE Cell Proliferation Kit - For Flow Cytometry</td>
			<td>Invitrogen</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr>
			<td>Human P-selectin-FC recombinant protein</td>
			<td>R&#38;D Systems</td>
			<td>137-PS-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Human E-selectin-FC recombinant protein</td>
			<td>R&#38;D Systems</td>
			<td>724-ES-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin Human, Plasma</td>
			<td>Invitrogen</td>
			<td>33016-015</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Bioflux 1000</td>
			<td>Fluxion Biosciences</td>
			<td>Bioflux Montage was the software used to run the experiments and analyze the data</td>
			<td></td>
		</tr>
		<tr>
			<td>BioFlux 48-well plates</td>
			<td>Fluxion Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Accuri C6 Flow Cytometer</td>
			<td>BD Bioscience</td>
			<td>CFlow Plus was the software used to run the experiments and analyze the data</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti-S</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CoolSnap HQ2 CCD camera</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

systematic analysis of in vitro cell rolling using a multi-well plate microfluidic system

Una sfida importante per terapia cellulare è l&#39;incapacità di indirizzare sistemicamente una grande quantità di cellule vitali con alta efficienza per tessuti di interesse dopo infusione endovenosa o intraarteriosa. Di conseguenza, l&#39;aumento homing delle cellule è attualmente studiata come una strategia per migliorare la terapia cellulare. Cella rotolamento sull&#39;endotelio vascolare è un passo importante nel processo di homing delle cellule e può essere sondato in vitro utilizzando una camera di flusso a piatti paralleli (PPFC). Tuttavia, questo è estremamente noioso, basso dosaggio produttività, con condizioni di flusso scarsamente controllato. Invece, abbiamo utilizzato un sistema di microfluidica piastra multi-pozzetto che consente lo studio delle proprietà di rotolamento cellulare in un throughput più elevato sotto controllato con precisione, fisiologicamente rilevante flusso di taglio 1,2. In questo articolo, vi mostriamo come le proprietà di rotolamento di HL-60 (leucemia promielocitica umana), le cellule su superfici E-selectina-rivestite-P e così come su superfici monostrato rivestite cellulari possono essere readily esaminato. Per meglio simulare condizioni infiammatorie, la superficie canale microfluidico è stata rivestita con le cellule endoteliali (EC), che sono stati poi attivati ​​con Tumor Necrosis Factor-α (TNF-α), aumentando notevolmente le interazioni con cellule HL-60 in condizioni dinamiche. La velocità aumentata e piattaforma integrata di analisi software multi-parametro, che permette una rapida analisi di parametri quali la velocità di rotolamento e il percorso di rotolamento, sono vantaggi importanti per la valutazione delle cellule laminazione di proprietà in-vitro. Consentire l&#39;analisi rapida e accurata di approcci di ingegneria progettato per avere un impatto rotolamento delle cellule e homing, questa piattaforma può aiutare terapia anticipo cell-based esogeno.

Cellule HL-60 rotolano sulle superfici-P ed E-selectina, ma non su fibronectina Cellule HL-60 sono considerati &quot;rulli&quot; gold standard quanto esprimono una varietà di ligandi homing, compresi i leganti rotolamento P-sel-1 glicoproteina ligando (PSGL-1) e Sialyl-Lewis X (sLex) 5,14 (Figura 1A ). La proteina di superficie PSGL-1 agisce come impalcatura per il SLeX tetra-saccaridi, che mediano l&#39;interazione specifica con P ed E-sel, che sono up-regolati sul...

Watch this Scientific Journal Video about Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System at JoVE.com

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

Questo studio ha utilizzato un sistema di microfluidica piastra multi-pozzetto, aumentando notevolmente la velocità di rotolamento studi cella in flusso di taglio fisiologicamente rilevanti. Data l&#39;importanza della laminazione di cellule nella cella multi-step homing a cascata e l&#39;importanza di homing delle cellule dopo la consegna sistematica delle popolazioni esogeni di cellule nei pazienti, questo sistema offre un potenziale come piattaforma di screening per migliorare la terapia cellulare.

Analisi sistematica delle<em&gt; In Vitro</em&gt; Cella rotolamento Utilizzando un piatto multi-ben Sistema Microfluidic

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

systematic-analysis-vitro-cell-rolling-using-multi-well-plate

Research

JoVE Journal

Bioengineering

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

11.9K Views.  Brigham and Women's Hospital.  Questo studio ha utilizzato un sistema di microfluidica piastra multi-pozzetto, aumentando notevolmente la velocità di rotolamento studi cella in flusso di taglio fisiologicamente rilevanti. Data l'importanza della laminazione di cellule nella cella multi-step homing a cascata e l'importanza di homing delle cellule dopo la consegna sistematica delle popolazioni esogeni di cellule nei pazienti, questo sistema offre un potenziale come piattaforma di screening per ...

Video: Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

Dispositivo Microfluidic per ricreare un microambiente tumorale<em&gt; In Vitro</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Ricerca

Bioingegneria

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

Studiare Traiettorie cellulare Rolling Patterns Receptor asimmetrica

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

L&#39;uso di un high-throughput<em&gt; In Vitro</em&gt; Microfluidic sistema per sviluppare orale biofilm Multi-specie

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Oculare Test (EIT) per identificazione del pericolo di sostanze chimiche irritanti oculari utilizzando Ricostruito umana Cornea come epiteliale (RHCE) Tessuto Modello

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

A Method for Determination and Simulation of Permeability and Diffusion in a 3D Tissue Model in a Membrane Insert System for Multi-well Plates

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as well as substance testing. These models are mostly cultivated in membrane-insert systems, their permeability toward different substances being an essential factor. Typically, applied methods for determination of these parameters usually require large sample sizes (e.g., Franz diffusion cell) or laborious equipment (e.g., fluorescence recovery after photobleaching (FRAP)). This study presents a method for determining permeability coefficients directly in membrane-insert systems with diameter sizes of 4.26 mm and 12.2 mm (cultivation area). The method was validated with agarose and collagen gels as well as a collagen cell model representing skin models. The permeation processes of substances with different molecular sizes and permeation through different cell models (consisting of collagen gel, fibroblast, and HaCaT) were accurately described.
Moreover, to support the above experimental method, a simulation was established. The simulation fits the experimental data well for substances with small molecular size, up to 14 x 10-10 m Stokes radius (4,000 MW), and is therefore a promising tool to describe the system. Furthermore, the simulation can considerably reduce experimental efforts and is robust enough to be extended or adapted to more complex setups.

A method for determination of permeability in a membrane insert system for multi-well plates and in silico parameter optimization for the calculation of diffusion coefficients using simulation are presented.

Un metodo per la determinazione e la simulazione di permeabilità e la diffusione in un modello 3D del tessuto in una membrana inserire sistema per piastre multi-pozzetto

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as ...

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

Here we describe a protocol for the automated segmentation of fluorescently labeled tissues on slides using a widefield high-content analysis system (WHCAS). This protocol has wide-ranging applications in any field which involves the quantitation of fluorescent markers in biological tissues including the biological sciences, medical engineering, and health sciences.

Scansione automatizzata Slide e segmentazione nei tessuti fluorescente-identificati utilizzando un sistema di analisi di Widefield ad alto contenuto

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

Analisi dell'espressione genica di cellule endoteliali esposte per Shear Stress utilizzando più flusso parallelo-piastra Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties.

Live Cell analisi della sollecitazione di taglio su Pseudomonas aeruginosa utilizzando un sistema di microfluidica superiore-Throughput automatizzato

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

Stabilire coculture basate su una singola cellula in modo deterministico con un chip microfluidico

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...