Ringraziamo T. Shatova per l&#39;utile discussione sul design sperimentale e analisi dei dati. L&#39;assistenza e la competenza di G. Paradis, il personale della citometria a flusso principale presso l&#39;Istituto Koch, e il personale della Microsystems Technology Laboratory presso il Massachusetts Institute of Technology sono riconosciuti con gratitudine. Questo lavoro è stato sostenuto dal National Institutes of Health Grants RC1 EB011187-02, DE013023, DE016516, EB000351, e in parte dal National Cancer Institute Cancer Center Support (Core) concede P30-CA14051 e MPP-09Call-Langer-60.

1. Conservazione <ol><li> Conservare i serbatoi, i titolari, O-ring e dispositivi microfluidici in etanolo al 70%. Utilizzare un contenitore (ad esempio vaso o bicchiere) che ha un coperchio per evitare l&#39;evaporazione e la contaminazione da particelle di polvere o all&#39;esterno. Posizionare i dispositivi in ​​un contenitore (1), serbatoi e O-ring in un secondo contenitore (2), e titolari nel terzo (3). </li></ol> Nota: L&#39;uso di etanolo al 70% per il rimessaggio è di mantenere la sterilità. Se gli unici componenti della soluzione sono etanolo e acqua (cioè senza denaturanti), tutti i componenti di sistema dovrebbe essere pienamente compatibile e non si degrada nel tempo. <ol start="2"><li> Cambiare soluzione etanolica in contenitori (2) e (3) prima di ogni uso per evitare la contaminazione incrociata di tutti gli esperimenti e minimizzare la presenza di particelle indesiderate che possono causare l&#39;intasamento. </li></ol> 2. Esperimento Preparazione <ol><li> Contenitori Consiglio (2) e (3) in un bagno ad ultrasuoni for 5-10 minuti prima di ogni utilizzo. Questo aiuta a rimuovere eventuali particelle contaminanti da esperimenti precedenti. </li><li> Pulire lavoro in armadio biosicurezza con una soluzione di etanolo al 70%. </li><li> Spruzzare tutti i materiali (3 contenitori e pinzette) con una soluzione di etanolo al 70% prima di collocarli all&#39;interno dell&#39;armadio biosicurezza. </li></ol> 3. Montaggio <ol><li> Deporre 2-3 salviette low-lint nell&#39;area di lavoro. </li><li> Rimuovere i serbatoi di plastica dal loro contenitore con le pinzette e metterli sulle salviettine per favorire l&#39;evaporazione della soluzione di etanolo da superfici interne. Battere delicatamente i serbatoi in superficie o soffiare aria attraverso di loro per facilitare la rimozione della soluzione di etanolo. </li><li> Inserire gli O-ring nel loro alloggiamento del serbatoi. </li><li> Rimuovere il supporto e patatine da rispettivi contenitori e lasciare evaporare l&#39;etanolo (~ 1-2 min). </li><li> Utilizzare le pinzette per posizionare il chip faccia-up desiderato (ad esempio fori di accesso su) nel supporto. Sollevare il titolarecon il chip di visione orizzontale per assicurarsi che il chip sia posizionato orizzontalmente nel supporto e regolare, se necessario, con le pinzette. IMPORTANTE: Se il dispositivo non si inserisce correttamente nel suo supporto, c&#39;è il rischio che si romperà durante le fasi successive. </li><li> Quindi, posizionare delicatamente i serbatoi sul supporto e allinearle con le clip. Fare attenzione che O-ring non cadono fuori dai loro alloggiamenti durante questo processo. </li><li> Premere delicatamente verso il basso i serbatoi finché non scattano in posizione. Assicurarsi che entrambi i lati dei serbatoi siano sicure e che il chip sembra essere nella posizione corretta. </li></ol> 4. Preparazione cellulare <ol><li> Per cellule aderenti (linee primarie o creare): cellule piastra 1-2 giorni prima dell&#39;esperimento tale che essi non sono confluenti più del 80% il giorno dell&#39;esperimento. </li><li> Posizionare cellule in sospensione (in PBS o supporto rilevante) e puntare ad una concentrazione operativa di 1,0 x 10 6 cellule / ml a 1,0 x 10 7 cells / ml. NOTA: Non abbiamo osservato cambiamenti significativi nelle prestazioni di consegna a causa di concentrazione cellulare. </li><li> Mescolare cellule e la consegna materiale desiderato in un tubo separato per ottenere la concentrazione desiderata materiale per l&#39;esperimento. IMPORTANTE: Poiché il metodo di consegna descritto si basa sulla diffusione per facilitare la consegna, una concentrazione di materiale superiore produrrà consegna superiore. Se possibile, si raccomanda di utilizzare una soluzione 1 mM del materiale desiderato per prove iniziali. Questa concentrazione può essere titolato al esperimenti futuri come necessario. La concentrazione più bassa segnalato utilizzato con questo dispositivo è 10 nM 21. </li></ol> 5. Funzionamento <ol><li> Pipettare miscela di cellule e la consegna materiale in un serbatoio (disegno corrente ha un max 150 microlitri). NOTA: La maggior parte dei disegni di periferica sono completamente reversibili, pertanto la direzione del flusso attraverso i canali non importa. I campioni possono essere caricati in uno dei due serbatoi. </li&gt; <li> Collegare il tubo di pressione al serbatoio riempito e serrare il dado per garantire la corretta tenuta (dito stretto è spesso sufficiente). </li><li> Regolare la pressione al livello desiderato sul regolatore. Questo controlla la velocità con cui le cellule attraverso il dispositivo. Si noti che il flusso delle cellule non inizia fino a quando si preme il pulsante. </li></ol> NOTA: Nel lavoro precedente, azoto o aria compressa hanno lavorato altrettanto bene come gas di trasporto. <ol start="4"><li> Sollevare dispositivo all&#39;altezza degli occhi e orientarlo in modo che il liquido nel serbatoio è facilmente visibile. NOTA: Traccia colonna di liquido per spegnere il sistema prima che il serbatoio si svuota. </li><li> Premere il pulsante per pressurizzare il serbatoio e iniziare il flusso delle cellule. </li><li> Quando il livello del liquido è circa 2 mm dal fondo del serbatoio, girare rapidamente il regolatore a 0 psi per arrestare il flusso. IMPORTANTE: Se uno non riesce a fermare il flusso prima che il serbatoio si svuota, si rischia l&#39;espulsione del campione dal collection serbatoio. Si noti inoltre che se la colonna di fluido non si muove ad un ritmo apprezzabile, o ha rallentato notevolmente rispetto alla sua portata iniziale (ad esempio 3x lento), il dispositivo montato è probabilmente sporco e deve essere sostituito. La gestione di un dispositivo intasato può portare alla morte delle cellule superiore. </li><li> Raccogliere le cellule trattate dal serbatoio appropriato e metterli nel tubo di raccolta / piastra desideri. IMPORTANTE: Non diluire le cellule raccolte in tutti i buffer in questa fase, le cellule porati continueranno ad assorbimento di materiale per un massimo di 10 min 20. Dopo questa finestra è passato, diluire le cellule nel desiderato media / buffer. </li><li> Per raccogliere le cellule trattate più o provare condizioni sperimentali alternativi, ripetere i passaggi 5,1-5,7 come necessario. Ricordiamo che i chip sono reversibili, quindi i campioni possono essere montati in uno dei serbatoi. Assicurati di scambiare i chip come necessario se si intasano. Eliminare i dispositivi ostruiti. </li></ol> 6. Smontaggio <ol><li&gt; Staccare delicatamente i serbatoi del titolare principale, mettendo da parte le armi della clip. </li><li> Collocare ogni parte nel contenitore di archiviazione appropriato (descritto nel paragrafo 1). </li><li> Posizionare chip utilizzati in un contenitore separato per lo smaltimento. </li></ol>

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Mater. 16, 961-966 (2004).</li><li>Michalet, X., 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=Quantum+Dots+for+Live+Cells+in+Vivo+Imaging,+and+Diagnostics.">Quantum Dots for Live Cells in Vivo Imaging, and Diagnostics.</a> Science. 307, 538-544 (2005).</li><li>Dahan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+Dynamics+of+Glycine+Receptors+Revealed+by+Single-Quantum+Dot+Tracking.">Diffusion Dynamics of Glycine Receptors Revealed by Single-Quantum Dot Tracking.</a> Science. 302, 442-445 (2003).</li><li>Slowing, I. I., Trewyn, B. G., Lin, V. S. 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Gli autori Armon Azionei, Robert Langer, e Klavs F. Jensen sono azionisti di SQZ Biotecnologie Società che produce i dispositivi microfluidici utilizzati in questo articolo.

Taluni aspetti della procedura sperimentale descritta (cioè fattori diversi progettazione di chip e velocità operativa) possono avere bisogno di essere ottimizzato a seconda del tipo di cellula e materiale consegna il sistema viene applicato. La discussione che segue indirizzi alcuni dei fattori più comuni da considerare quando si progetta esperimenti. Per migliorare il segnale di consegna per i composti fluorescente, si ha la necessità di affrontare le fonti di fluorescenza di ...

Consegna delle macromolecole nel citoplasma delle cellule è un passo fondamentale in applicazioni terapeutiche e di ricerca. Nanoparticelle mediata consegna, per esempio, ha dimostrato una potenziale nella terapia genica 1,2, mentre la consegna proteina è un mezzo promettente di influenzare la funzione cellulare sia clinico 3 e 4 impostazioni di laboratorio. Altri materiali, come ad esempio farmaci a piccole molecole, i punti quantici, o nanoparticelle d&#39;oro, sono di interesse in applicazioni che vanno dal cancro terapeutica 5,6 all&#39;etichettatura intracellulare 7,8 e singola molecola di monitoraggio 9. La membrana cellulare è sostanzialmente impermeabile alle macromolecole. Molte tecniche esistenti utilizzano nanoparticelle polimeriche 10,11, liposomi 12 o modificazioni chimiche 13 per facilitare rottura della membrana o la consegna intracellulare. In questi metodi, la vitalità efficienza consegna e cellulari sono spesso dipendente dalla struttura del secoloe molecola target e il tipo di cella. Questi metodi possono essere efficaci nella fornitura di materiali di struttura uniforme, come acidi nucleici, ma spesso sono poco adatti per la consegna dei materiali più strutturalmente diverse, come le proteine ​​14,15 e nanomateriali 7. Inoltre, il meccanismo di interruzione endosome che la maggior parte di questi metodi si basano su è spesso inefficiente, lasciando quindi molto materiale intrappolato in strutture vescicole 16. Infine, i metodi che sono spesso sviluppati per essere utilizzati con linee cellulari stabilizzate non si traducono bene alle cellule primarie. Metodi di membrana all&#39;incorporazione, come elettroporazione 17,18 e sonoporazione 19, sono una valida alternativa in alcune applicazioni, tuttavia, sono noti per causare la vitalità cellulare bassi e possono essere limitate dal responsabile del materiale consegna bersaglio. Rapida deformazione meccanica delle cellule, un approccio microfluidica alla consegna, ha recentemente demonstrated suoi vantaggi rispetto alle tecniche attuali nel contesto di riprogrammazione cellulare 20 e consegna nanomaterial 21. Questo metodo si basa sulla distruzione meccanica o la membrana cellulare per facilitare la consegna citosolico dei materiali presenti nel buffer circostante. Il sistema ha dimostrato che consenta potenziale in precedenza sfidare tipi di cellule (ad esempio cellule immunitarie primarie e cellule staminali) e materiali (ad esempio anticorpi e nanotubi di carbonio). Qui, la procedura generale di utilizzare questi dispositivi per la consegna intracellulare di macromolecole bersaglio è descritto. La procedura è generalizzabile a maggior parte dei tipi di cellule e materiali di consegna, tuttavia, è consigliabile una condurre una breve ottimizzazione delle condizioni, come descritto nelle nostre linee guida progettuali precedentemente segnalati 20, per tutte le applicazioni precedentemente non. Ad oggi, il sistema è stato usato con successo per la consegna di RNA, DNA, nanoparticelle di oro, punti quantici, i nanotubi di carbonio, proteines, e polimeri destrano 20,21.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:375px;">Device Holder &#38; Plastic reservoir</td>
			<td style="width:136px;">SQZ Biotechnologies</td>
			<td style="width:149px;">Holder</td>
			<td style="width:117px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LSRFortessa Analyzer</td>
			<td>Becton Dickinson</td>
			<td>N/A</td>
			<td>Flow cytometry machine used at the Koch Institute Core Facilities</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microfluidic device</td>
			<td>SQZ Biotechnologies</td>
			<td>Cell Squeeze</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">O-Rings</td>
			<td>McMaster</td>
			<td>9452K311</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pressure system to operate device</td>
			<td>SQZ Biotechnologies</td>
			<td>Pressure System</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrasound bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
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cell squeezing as a robust, microfluidic intracellular delivery platform

Rapida deformazione meccanica delle cellule è emerso come un metodo promettente, libero da vettori per la consegna intracellulare di macromolecole e dei nanomateriali. Questa tecnologia ha dimostrato il potenziale per affrontare applicazioni precedentemente impegnative, tra cui, la consegna alle cellule immunitarie primarie, riprogrammazione cellulare, nanotubi di carbonio, e la consegna quantum dot. Questa piattaforma microfluidica libero da vettori deduce rottura meccanica della membrana cellulare per facilitare la consegna citosolico del materiale bersaglio. Qui, si descrive il metodo dettagliato di uso per questi dispositivi microfluidici compresi, montaggio del dispositivo, preparazione cellulare, e il funzionamento del sistema. Questo approccio consegna richiede una breve ottimizzazione di tipo di dispositivo e le condizioni operative per le applicazioni precedentemente rimanere sconosciuti. Le istruzioni fornite sono generalizzabili alla maggior parte dei tipi di cellule e materiali di consegna in quanto questo sistema non richiede buffer specializzati o passi modificazione chimica / coniugazione. Questo lavoro fornisce anches raccomandazioni su come migliorare le prestazioni del dispositivo e problemi-shoot potenziali problematiche relative a intasamento, l&#39;efficienza di consegna bassi, e la vitalità cellulare.

Figura 1 contiene uno schema descrittivo del sistema di erogazione microfluidica. Figure 2a-b illustrano i risultati tipici della trattando le cellule HeLa con disegni diversi dispositivi in presenza di coniugato fluorescente 3 kDa destrano 20. Se la procedura viene seguita correttamente, le prestazioni del sistema sarà sensibile al tipo di dispositivo e velocità operativa. Pertanto, si dovrebbe ottimizzare queste condizioni per una data applicazione prima di procedere a es...

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Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapida deformazione meccanica delle cellule è emerso come un metodo promettente, libero da vettori per la consegna intracellulare di macromolecole e dei nanomateriali. Questo protocollo fornisce la procedura dettagliata su come utilizzare il sistema per una vasta gamma di applicazioni.

Spremere cellulare come un robusto, Microfluidic intracellulare Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

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12.7K Views.  Massachusetts Institute of Technology.  Rapida deformazione meccanica delle cellule è emerso come un metodo promettente, libero da vettori per la consegna intracellulare di macromolecole e dei nanomateriali. Questo protocollo fornisce la procedura dettagliata su come utilizzare il sistema per una vasta gamma di applicazioni. 

Video: Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Microcapsule di alginato come piattaforma 3D per la propagazione e la differenziazione delle cellule staminali embrionali umane (hESC) per diverse linee

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

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High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

High-throughput generatore di espressione delle proteine ​​Utilizzando una piattaforma microfluidica

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

Il Chip Multi-organo - Una piattaforma Microfluidic per lungo termine co-coltura Multi-tessuto

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Una piattaforma Microfluidic per l&#39;isolamento Single-cell high-throughput e cultura

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Piattaforma microfluidica con multiplex di rilevamento elettronico per spaziale monitoraggio di particelle

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

La morfologia dei capelli di radice di semenzali di Arabidopsis in una piattaforma di Microfluidic di due-strato di imaging

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

Una piattaforma microfluidica per Imaging longitudinale in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Una piattaforma di Microfluidic ad altissima velocità di trasmissione per il sequenziamento del genoma di cella singola

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

Una piattaforma microfluidica multistrato per la conduzione di un'espressione genica prolungata senza cellule

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Una piattaforma microfluidica per stimolare i condrociti con la compressione dinamica

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...