Apprezziamo profondamente il dottor Takeshi Ikeya e personale tecnico Toyo Gosei, Tokyo, Giappone per la fornitura di piastre di coltura micropatterned termosensibili e pareri scientifici. Ringraziamo anche la signora Satomi Ogura, la signora Sae Suzuki, la signora Asuka Miyoshi e la signora Katsue Morii per l&#39;assistenza tecnica con la sperimentazione animale. Questo lavoro è stato sostenuto finanziariamente in parte dal JSPS KAKENHI Grant-in-Aid per la ricerca scientifica, il Centro per l&#39;Innovazione (COI) Programma e il programma di innovazione S- dal Giappone Scienza e della Tecnologia Agency (JST), e il JSPS Core- a-Core Programma, A. Ricerca reti avanzate.

Tutti gli studi su animali sono stati condotti con l&#39;approvazione della cura e l&#39;uso Comitato Animale dell&#39;Università di Tokyo, Tokyo, Giappone. 1. Preparazione delle cellule <ol><li> Per epatociti primari, seguire il protocollo per l&#39;isolamento di epatociti di ratto da una a due stadi modificati processo di digestione collagenasi 13,14. <ol><li> Anestetizzare Sprague Dawley (SD) ratti (maschi, 5 settimane di vita) sotto inalazione anestesia con isoflurano. Inserire un ratto in una camera collegata a una macchina per anestesia a fornire isoflurano per la camera. Estrarre il topo dopo essersi addormentati, e metterlo sul tavolo operatorio con ventilazione utilizzando una maschera. Flow Control isoflurano approssimativamente 0,4-0,7 L / min controllando le condizioni di ratto. Profumato i fegati di Sprague Dawley (SD) ratti (maschi, 5 settimane) dalla vena porta con una soluzione speciale composta da 8 g / l di cloruro di sodio (NaCl), 400 mg / l di cloruro di potassio (KCl), 78 mg / Lsodio diidrogeno fosfato diidrato (NaH 2 PO 4 · 2H 2 O), 151 mg / L sodio fosfato dibasico dodecaidrato (Na 2 HPO 4 · 12H 2 O), 2,38 g / L 2- [4- (2-idrossietil) -1 -piperazinile] acido etansolfonico (HEPES), 190 mg / L glicole etilenico tetraacetico (EGTA), 350 mg / L di carbonato acido di sodio (NaHCO3), e 900 mg / L di glucosio. </li><li> Far circolare soluzione di collagenasi attraverso il fegato. NOTA: La soluzione è composta da 500 mg / L collagenasi, 9,8 g / salina tampone di L Hank, 2,38 g / ml HEPES, 556 mg / ml idrato di cloruro di calcio (CaCl 2 · H 2 O), 350 mg / L NaHCO 3, e inibitore della tripsina / L 50 mg, con il pH regolato a 7,2. </li><li> Rimuovere il fegato con attenzione, e tritare delicatamente su un piatto con un bisturi, aggiungere Dulbecco Modified mezzo di Eagle (DMEM) supplementato con 10% di siero fetale bovino (FBS) e filtrare la sospensione cellulare attraverso un 100 micronrete di nylon. Per rimuovere i detriti aggiuntivo, centrifugare la sospensione cellulare a 20 g per 1 min. A questo punto, gli epatociti sono nel supernatante. </li><li> Ripetere la fase di centrifugazione due volte (per un totale di tre centrifugazioni). Infine, centrifugare epatociti a 50 g per 3 minuti per recuperarli in forma di pellet. </li><li> Risospendere epatociti ad una concentrazione di 4 × 10 5 cellule / ml in un mezzo di coltura speciale composto DMEM supplementato con 10% FBS, 1% Pen-Strep-Glut (PSQ), 1% dimetilsolfossido (DMSO), 0.1 mmol / L desametasone, 0,5 mg / ml di insulina, 10 mmol / L nicotinamide, 0,2 mmol / L ascorbato fosforilata (Asc-2P), e 10 ng / ml il fattore di crescita epidermico umano (hEGF) 15. Questo mezzo speciale è obbligatoria per preservare la funzione epatica in condizioni in vitro. </li></ol></li><li> Per ottenere MSC di ratto, eutanasia Sprague Dawley (SD) ratti (maschi, 5 settimane di vita) da eccessiva somministrazione di isoflurane. Resecare femore e tibia, e raccogliere le midollo osseo inserendo un ago G 22 nel pozzo dell&#39;osso per lavarlo con 10 ml di DMEM supplementato con 10% FBS. Raccogliere le cellule mediante filtrazione attraverso una rete di nylon 100 micron. </li><li> Seme le cellule su 10 piatti cm di coltura con DMEM contenente 10% FBS e 1% di penicillina / streptomicina. Per gli esperimenti sferoidali, utilizzare MSC a 5 passaggi. </li></ol> 2. Preparazione di Spheroids Cell 3D <ol><li> Commercialmente ottenere piastre di coltura micropatterned, in cui le aree adesive cellulari sono regolarmente schierati a 100 micron di diametro in modo bidimensionale, circondata da aree non adesivi rivestiti dalla matrice PEG. NOTA: per il trapianto di cellule, un ulteriore rivestimento con la PIPAAm polimero termosensibile è necessario per consentire il distacco cellulare raffreddando le piastre (vedi passo 5.1). Fluttuazioni di temperatura possono indurre cambiamenti nella chimica di questo polimero8-10. A 37 ° C, PIPAAm è leggermente idrofoba, permettendo alle cellule di essere coltivate in condizioni normali. Una diminuzione della temperatura sotto 32 ° C risultati in rapida idratazione del polimero, che porta al distacco spontaneo delle cellule. </li><li> Seme epatociti o MSC in piastre da 12 pozzetti micropatterned ad una densità di 4 x 10 5 cellule / pozzetto loro andincubate a 37 ° C in atmosfera umidificata contenente il 5% di CO 2. Le cellule si accumulano sulle aree di adesione micropatterned e gradualmente formare sferoidi rotonde in 2 giorni. NOTA: Per la preparazione di cellule di controllo in una coltura monostrato, utilizzare piastre da 12 pozzetti normali e sementi le cellule ad una densità identico, seguendo una procedura simile a quella descritta sopra. </li></ol> 3. Preparazione del Polyplex Nanomicelles <ol><li> Sintetizzare un copolimero a blocchi, PEG-PASP (DET) (poli [N &#39;- [N - (2-amminoetil) -2-amminoetil] aspartamide]) (PEG Mw = 12,000, grado di polimerizzazione (DP) di PASP (DET) segmento = 59), e un omopolimero che è composto solo segmento cationico [PASP (DET)] (DP = 55), seguendo le procedure precedentemente descritte dagli autori 11, 16, 17. </li><li> Preparare una soluzione mista di PEG-PASP (DET) copolimero a blocchi e il PASP (DET) omopolimero con un rapporto molare uguale di gruppi amminici residui in tampone HEPES 10 mM (pH 7,3), regolando la concentrazione di polimero a 33,3 ug / ml e 19.1 mg / ml, rispettivamente. NOTA: Le concentrazioni di polimero sopra descritte sono valori rappresentativi per preparare nanomicelles con un rapporto molare residua del totale dei gruppi amminici in due polimeri ai gruppi fosfato nella (N / rapporto P) pDNA 10. Il rapporto N / P può variare a seconda del tipo di cellula e lo scopo (per dettagli vedi Discussione). L&#39;utilizzo combinato dei due polimeri può ottenere sia efficace schermatura PEG e il funzionamento del PASP (DET) per migliorare endosomalfuggire (per dettagli vedi Discussione) 18. </li><li> Preparare la codifica pDNA luciferasi Gaussia, GL4 luciferasi, o eritropoietina clonando il segmento che esprime i rispettivi geni nel plasmide pCAG-GS (http://www.cdb.riken.jp/pcs/protocol/vector/map/m36.html ) per ottenere espressione sotto il CAG promotore / enhancer utilizzando un kit commerciale secondo il protocollo del produttore. Amplificare il pDNA in un ceppo di Escherichia coli competente e purificarlo utilizzando un plasmide sistema di purificazione del DNA privo di endotossine. Determinare la concentrazione pDNA in assorbanza a 260 nm per ottenere una soluzione / ml 150 mg in 10 mM tampone HEPES (pH 7,3). </li><li> Per preparare i nanomicelles Polyplex, mescolare la soluzione pDNA (150 ug / ml in tampone HEPES 10 mM) e la soluzione premiscelata dei due polimeri ad un rapporto di 2: 1 (in volume). </li></ol> 4. Gene Trasfezione in Spheroids <ol><li> Incubare le cellule (epatocitio MSC) per 72 ore dopo la semina sulle piastre micropatterned per consentire la formazione di sferoidi maturi. Per gene trasfezione, aggiungere 100 ml di soluzione di Polyplex nanomicelle (contenenti 10 mg di pDNA) per ogni bene dopo la sostituzione del mezzo di coltura con 1 ml di terreno fresco. Continuare l&#39;incubazione con la soluzione nanomicelle per 24 ore. </li><li> Per un controllo utilizzando un trasfezione reattivo a base lipidica, mescolare soluzioni pDNA con il reagente con un rapporto peso / reagente pDNA del 3. Regolare la dose finale di pDNA essere uguale sia per il reattivo a base di lipidi e metodi nanomicelle. </li></ol> 5. Recupero e trapianto di cellule Spheroids <ol><li> Sostituire il terreno di coltura con 200 ml di PBS freddo e posizionare le piastre sul ghiaccio. NOTA: In generale, i sferoidi possono staccarsi in circa 15 minuti e può essere recuperata sotto forma di una sospensione per il trapianto. </li><li> Aspirare delicatamente le cellule in 200 microlitrisospensione con una siringa con un 23 G 27 o G ago per iniezioni in vivo. </li></ol> 6. Valutazione di Transgene Expression <ol><li> Per la valutazione in vitro di espressione di luciferasi Gaussia secreto nel mezzo di coltura, raccogliere 50 - 100 ml del mezzo proprio 24 ore dopo aver sostituito con terreno fresco. Stimare l&#39;espressione luciferasi mediante un sistema di dosaggio renilla luciferasi commerciale e un luminometro secondo il protocollo del produttore. NOTA: La luciferasi Gaussia rimane stabile nel terreno di coltura per più di una settimana. Così, per valutare l&#39;efficienza in tempo reale della espressione del transgene, sostituire il supporto con quello fresco prima di raccogliere il fluido da misurare. I tempi del cambiamento mezzo può essere flessibile. </li><li> Per la valutazione in vivo dell&#39;espressione del transgene in animali ospiti dopo il trapianto di cellule, anestetizzare BALB / c topi nudi (femminile; 7 settimanevecchio) sotto inalazione anestesia con isoflurano. <ol><li> Inserire un topo in una camera collegata a una macchina per anestesia a fornire isoflurano per la camera. Estrarre il mouse dopo essersi addormentati, e metterlo sul tavolo operatorio con ventilazione utilizzando una maschera. Flow Control isoflurano approssimativamente 0,2-0,5 L / min controllando le condizioni del mouse. </li><li> Iniettare 200 microlitri della sospensione cellulare contenente sferoidi trasfettate con i pDNA luciferasi che esprimono GL4 (come descritto in 4.1) nel tessuto sottocutaneo della regione addominale. </li><li> Subito dopo l&#39;iniezione di D-luciferina (150 mg / kg; via endovenosa), misurare l&#39;espressione della luciferasi utilizzando sistema di imaging IVIS secondo il protocollo del produttore. </li></ol></li><li> Per valutare gli effetti terapeutici del trapianto di cellule, iniettare 200 ml di sospensione cellulare contenente sferoidi trasfettate con i pDNA-eritropoietina codificazione neltessuto sottocutaneo della regione addominale. <ol><li> Raccogliere i campioni di sangue da emorragia submandibolare di ottenere circa 200 ml di sangue 19. Misurare l&#39;emoglobina ed ematocrito utilizzando un analizzatore di campione di sangue. NOTA: Il numero di cellule per il trapianto è regolato dal numero seminate sui piatti. Purtroppo, è difficile determinare il numero di cellule esatto perché il numero all&#39;interno sferoidi non può essere misurata. </li></ol></li><li> Dopo gli esperimenti, inserire i topi su una piastra elettrica collegato con un regolatore di temperatura fino a quando il risveglio dall&#39;anestesia. </li></ol>

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The authors declare that they have no competing financial interests.

In questo protocollo, è fondamentale per mantenere la struttura 3D della sferoidi durante le fasi di introduzione del gene e recupero sferoidale. È essenziale mantenere un microambienti favorevoli per le celle per evitare la morte cellulare o perdita di attività cellulare. Ad esempio, la secrezione di albumina, una funzione rappresentativa innata di epatociti, è ben conservata in sferoidi epatociti, mentre gli epatociti in coltura monostrato convenzionali perdono rapidamente la loro capacità secretoria pochi giorni...

La terapia di trapianto cellulare ha attirato l&#39;attenzione diffusa per il trattamento di varie malattie incurabili. L&#39;attività e l&#39;emivita dei fattori bioattivi che sono secreti dalle cellule trapiantate sono essenziali per migliorare l&#39;efficacia terapeutica di un sistema di trapianto di cellule. La modificazione genetica delle cellule prima del trapianto è una tecnica utile per regolare e manipolare funzioni cellulari, compresa la secrezione dei fattori bioattivi. E &#39;anche importante mantenere un microambiente favorevole per le cellule per evitare la morte cellulare o perdita di attività cellulare. Tridimensionale coltura (3D) cellule sferoidale, in quale cella-cellula interazioni sono ben conservate, è promettente per questo scopo, ad esempio, per migliorare la secrezione di albumina da epatociti primari e promuovendo multi-lineage differenziazione da cellule staminali mesenchimali (MSC ) 1-7. In questo studio, un sistema di nuova combinazione di spheroid cultura e trasfezione genica viene utilizzata per servire da piattaforma per il trapianto di cellule geneticamente modificate. Per creare cellule sferoidali, viene utilizzato un sistema di coltura sferoide su piastre di coltura micropatterned. Su queste piastre, aree di adesione cellulare di 100 micron di diametro sono regolarmente disposte in maniera bidimensionali e sono circondati da zone non adesivi rivestiti da una matrice PEG 3. Seminando un adeguato numero di cellule, array di sferoidi 3D di 100 micron di diametro sono formati corrispondenti al letto coltura micropatterned. Gli sferoidi siano recuperati senza distruggerne la struttura 3D utilizzando piastre di coltura cellulare termosensibili, che sono stati rivestiti con un polimero termosensibile, poli (iso-propylacrylamide) (PIPAAm) 8-10. L&#39;architettura micropatterned è costruito sulle piastre termosensibili (su misura costruito). Semplicemente abbassando la temperatura delle piastre, gli sferoidi sono staccati dal letto coltura e disperdonod in tampone fosfato (PBS). Pertanto, un gran numero di sferoidi con dimensioni uniformi di 100 micron può essere ottenuto sotto forma di una sospensione iniettabile. <img alt="Figura 1" p="" src="/files/ftp_upload/52384/52384fig1.jpg" /> Figura 1. Rappresentazione schematica del sistema di coltura sferoide su un piatto micropatterned. La modificazione genetica è ottenuta mediante trasfezione genica utilizzando il vettore non virale gene originale, nanomicelle Polyplex. E &#39;composto da DNA plasmidico (pDNA) e polietilene glicole (PEG) blocchi -polycation copolimeri 11. Questi hanno una struttura core-shell caratteristica costituito da un guscio di PEG e un nucleo interno di pDNA condensata, permettendo sicuro ed efficace introduzione del gene nelle cellule a scopo terapeutico 11. <a href="https://www.jove.com/files/ftp_upload/52384/52384fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di Thi</a>s figura. <img alt="Figura 2" src="/files/ftp_upload/52384/52384fig2.jpg" /> Figura 2. Struttura del nanomicelle Polyplex formata dal complesso di acidi nucleici e copolimeri a blocchi PEG-block-policatione. In questo studio, il vantaggio principale di questa tecnica è che la struttura sferoidale non interrompere durante gene trasfezione dai nanomicelles. Dopo trasfezioni nanomicelle-mediata di ratto sferoidi epatociti primari, l&#39;espressione del transgene prolungata è ottenuto per più di un mese con la secrezione di albumina continuo dagli epatociti ad un livello paragonabile a quello di sferoidi untransfected 12. L&#39;espressione del transgene e la secrezione di albumina da sferoidi sono mantenuti dopo il recupero dalle piastre termosensibili. E &#39;evidente che nanomicelles possono facilitare tranquillamente introduzione gene senza compromettere le funzioni innate della HEPatocytes. Pertanto, la combinazione di cellule sferoidali coltivate su piastre micropatterned termosensibili con introduzione del gene utilizzando nanomicelles è una piattaforma promettente per il trapianto di cellule geneticamente modificate. <a href="https://www.jove.com/files/ftp_upload/52384/52384fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pen-Strep-Glut</td>
			<td>GIBCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Wako Pure Chemical Industries</td>
			<td>041-18861</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Wako Pure Chemical Industries</td>
			<td>141-01202</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s buffered salt and L-ascorbic acid 2-phosphate (Asc-2P)</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>Human epidermal growth factor (hEGF)</td>
			<td>Toyobo</td>
			<td>PT10015</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-able multi-well plates</td>
			<td>Toyo Gosei</td>
			<td>PP-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermosensitive cell culture plates (Upcell)</td>
			<td>CellSeed Inc</td>
			<td></td>
			<td>The micropatterned architecture is constructed on the thermosensitive plates (custom-built by Toyo Gosei)</td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent (FuGENE HD)</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Renilla Luciferase Assay System</td>
			<td>Promega</td>
			<td>E2810</td>
			<td></td>
		</tr>
		<tr>
			<td>pGL4 Luciferase Reporter Vector</td>
			<td>Promega</td>
			<td>E6651</td>
			<td></td>
		</tr>
		<tr>
			<td>pDNA expressing Gaussia luciferase</td>
			<td>New England BioLabs</td>
			<td>N8082S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse erhthropoietin-expressing vector</td>
			<td>Origene</td>
			<td>MC208445</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG-GS</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Laboratory for Pluripotent Cell Studies, Center for Developmental Biology, RIKEN</td>
		</tr>
		<tr>
			<td>Escherichia coli DH5&#945; competent cells</td>
			<td>Takara</td>
			<td>9057</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotoxin-free plasmid DNA purification system</td>
			<td>Nippon Genetics</td>
			<td>NucleoBond Xtra EF</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase</td>
			<td>Wako Pure Chemical Industries</td>
			<td>639-00951</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin inhibitor</td>
			<td>GIBCO</td>
			<td>R-007-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td>GloMax&#8482; 96 Microplate Luminometer</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Imaging System</td>
			<td>Xenogen Corp.</td>
			<td>Xenogen IVIS Spectrum in vivo imaging system</td>
			<td></td>
		</tr>
		<tr>
			<td>blood sample analyzer</td>
			<td>Sysmex</td>
			<td>pocH-100i Automated Hematology Analyzer</td>
			<td></td>
		</tr>
	</tbody>
</table>

gene transfection toward spheroid cells on micropatterned culture plates for genetically-modified cell transplantation

Per migliorare l&#39;efficacia terapeutica del trapianto di cellule, è stato sviluppato un sistema di trapianto di geneticamente modificati, sferoidi iniettabili. Gli sferoidi cellule vengono preparati in un sistema di coltura su piastre micropatterned rivestiti con un polimero termosensibile. Un certo numero di sferoidi sono formate sulle piastre, corrispondenti alle aree di adesione cellulare di 100 micron di diametro che sono regolarmente schierati in modo bidimensionale, circondate da aree non-adesive che sono rivestiti da un polietilenglicole (PEG) di matrice. Gli sferoidi possono essere facilmente recuperati da una sospensione liquida abbassando la temperatura delle piastre, e la loro struttura è ben mantenuto facendoli passare attraverso gli aghi di iniezione con una sufficientemente grosso calibro (over 27 G). La modificazione genetica è ottenuta gene trasfezione utilizzando il vettore originale non virale gene, nanomicelle Polyplex, che è in grado di introdurre geni nelle cellule senza distruggere la struttura sferoidale. Per primsferoidi epatociti ary trasfettate con un gene luciferasi che esprimono, la luciferasi è sostenibile ottenuti in animali trapiantati, con funzione di epatociti conservata, come indicato dall&#39;espressione albumina. Questo sistema può essere applicato ad una varietà di tipi cellulari, comprese le cellule staminali mesenchimali.

Gene trasfezione del Gaussia luciferasi che esprimono pDNA è stata eseguita in sferoidi formate dagli epatociti e cellule staminali mesenchimali con nanomicelles Polyplex o trasfezione reattivo a base di lipidi di controllo 12. I nanomicelles indotti quasi nessun cambiamento nella struttura sferoidale rispetto sferoidi non transfettate sulle piastre micropatterned, mentre il reagente di controllo interrotto significativamente la struttura un giorno dopo la trasfezione (Figura 3). Dopo trasfe...

Watch this Scientific Journal Video about Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation at JoVE.com

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

Gene Transfection verso Celle sferoide su lastre micropatterned coltura per trapianto di cellule geneticamente modificati

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

gene-transfection-toward-spheroid-cells-on-micropatterned-culture

Research

JoVE Journal

Bioengineering

8.7K Views.  The University of Tokyo. This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

Video: Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Superfici Micropatterned per studiare le interazioni acido ialuronico con le cellule tumorali

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Ricerca

Bioingegneria

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Una migliore visualizzazione e analisi quantitativa degli effetti del farmaco utilizzando cellule Micropatterned

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Fabbricazione di idrogel Micropatterned per i sistemi Cultura neurali utilizzando Dynamic Fotolitografia proiezione Mask

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

Rotazione Cultura sistemi cellulari per colture cellulari umane: cellule del trofoblasto umano come modello

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

Massa Citometria: Protocollo per l&#39;ottimizzazione giornaliera e campioni di cellule in esecuzione su un citometro Messa CyTOF

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Trapianto di progenitori miogenici pluripotenti derivati da cellule staminali indotte simili a mesoangioblasti nei modelli di topo di rigenerazione muscolare

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Stretching celle micropatterned su una membrana PDMS

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

Microfluidic genipina Deposizione Tecnica per la cultura estesa di micropatterned vascolari muscolari Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Segmentazione della crescita delle cellule endoteliali in piastre a 6 pozzi su uno shaker orbitale per studi meccanobiologici

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

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, ...