polydimethylsiloxane-polycarbonate-microfluidic-devices-for-cell

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

Academia Sinica

It has been shown previously that proline-directed protein kinase F(A) (PDPK F(A)) is overexpressed in various human malignancies compared with its expression in normal controls, and the suppression of overexpressed PDPK F(A) is capable of inhibiting the growth of various types of human carcinoma cells, suggesting a role for this PDPK in human malignancies. In this report, the authors combine immunohistologic, molecular, cellular, and clinicopathologic studies to demonstrate further an essential critical role for overexpressed PDPK F(A) in bladder carcinoma invasion, chemoresistance, and poor prognosis.

Association of overexpressed proline-directed protein kinase F(A) with chemoresistance, invasion, and recurrence in patients with bladder carcinoma.

Initial studies revealed that the multisubstrate proline-directed protein kinase F(A) (PDPK F(A)) is overexpressed in various types of human carcinomas relative to normal controls. Suppression of overexpressed PDPK F(A) inhibits the growth of cancer cells, suggesting a role of this PDPK in human malignancy. In this study, we combine immunohistologic, molecular, cellular, animal, and clinicopathologic studies to demonstrate an essential and critical role of PDPK F(A) in progression and poor prognosis of human colon carcinoma.

Association of proline-directed protein kinase FA with tumorigenesis, invasion, and poor prognosis of human colon carcinoma.

Berberine, a botanical alkaloid purified from Cortidis rhizoma, has effects in cardiovascular diseases, yet the mechanism is not fully understood. Foam cells play a critical role in the progression of atherosclerosis. This study aimed to investigate the effect of berberine on the formation of foam cells by macrophages and the underlying mechanism. Treatment with berberine markedly suppressed oxidized low-density lipoprotein (oxLDL)-mediated lipid accumulation, which was due to an increase in cholesterol efflux. Berberine enhanced the mRNA and protein expression of ATP-binding membrane cassette transport protein A1 (ABCA1) but did not alter the protein level of ABCG1 or other scavenger receptors. Additionally, functional inhibition of ABCA1 with a pharmacological inhibitor or neutralizing antibody abrogated the effects of berberine on cholesterol efflux and lipid accumulation. Moreover, berberine induced the nuclear translocation and activation of liver X receptor alpha (LXRalpha) but not its protein expression. Knockdown of LXRalpha mRNA expression by small interfering RNA abolished the berberine-mediated protective effects on ABCA1 protein expression and oxLDL-induced lipid accumulation in macrophages. These data suggest that berberine abrogates the formation of foam cells by macrophages by enhancing LXRalpha-ABCA1-dependent cholesterol efflux.

Anti-atherogenic effect of berberine on LXRalpha-ABCA1-dependent cholesterol efflux in macrophages.

This paper reports a microfluidic device capable of generating oxygen gradients for cell culture using spatially confined chemical reactions with minimal chemical consumption. The microfluidic cell culture device is constructed by single-layer polydimethylsiloxane (PDMS) microfluidic channels, in which the cells can be easily observed by microscopes. The device can control the oxygen gradients without the utilization of bulky pressurized gas cylinders, direct addition of oxygen scavenging agents, or tedious gas interconnections and sophisticated flow control. In addition, due to the efficient transportation of oxygen within the device using the spatially confined chemical reactions, the microfluidic cell culture device can be directly used in conventional cell incubators without altering their gaseous compositions. The oxygen gradients generated in the device are numerically simulated and experimentally characterized using an oxygen-sensitive fluorescence dye. In this paper, carcinomic human alveolar basal epithelial (A549) cells have been cultured in the microfluidic device with a growth medium and an anti-cancer drug (Tirapazamine, TPZ) under various oxygen gradients. The cell experiment results successfully demonstrate the hyperoxia-induced cell death and hypoxia-induced cytotoxicity of TPZ. In addition, the results confirm the great cell compatibility and stable oxygen gradient generation of the developed device. Consequently, the microfluidic cell culture device developed in this paper is promising to be exploited in biological labs with minimal instrumentation to study cellular responses under various oxygen gradients.

Generation of oxygen gradients in microfluidic devices for cell culture using spatially confined chemical reactions.

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Flow cytometry is a technique capable of optically characterizing biological particles in a high-throughput manner. In flow cytometry, three dimensional (3D) hydrodynamic focusing is critical for accurate and consistent measurements. Due to the advantages of microfluidic techniques, a number of microfluidic flow cytometers with 3D hydrodynamic focusing have been developed in recent decades. However, the existing devices consist of multiple layers of microfluidic channels and tedious fluidic interconnections. As a result, these devices often require complicated fabrication and professional operation. Consequently, the development of a robust and reliable microfluidic flow cytometer for practical biological applications is desired. This paper develops a microfluidic device with a single channel layer and single sheath-flow inlet capable of achieving 3D hydrodynamic focusing for flow cytometry. The sheath-flow stream is introduced perpendicular to the microfluidic channel to encircle the sample flow. In this paper, the flow fields are simulated using a computational fluidic dynamic (CFD) software, and the results show that the 3D hydrodynamic focusing can be successfully formed in the designed microfluidic device under proper flow conditions. The developed device is further characterized experimentally. First, confocal microscopy is exploited to investigate the flow fields. The resultant Z-stack confocal images show the cross-sectional view of 3D hydrodynamic with flow conditions that agree with the simulated ones. Furthermore, the flow cytometric detections of fluorescence beads are performed using the developed device with various flow rate combinations. The measurement results demonstrate that the device can achieve great detection performances, which are comparable to the conventional flow cytometer. In addition, the enumeration of fluorescence-labelled cells is also performed to show its practicality for biological applications. Consequently, the microfluidic flow cytometer developed in this paper provides a practical platform that can be used for routine analysis in biological laboratories. Additionally, the 3D hydrodynamic focusing channel design can also be applied to various applications that can advance the lab on a chip research.

Single channel layer, single sheath-flow inlet microfluidic flow cytometer with three-dimensional hydrodynamic focusing.

Cytom&eacute;trie en flux est une technique capable de caract&eacute;riser optiquement les particules biologiques de mani&egrave;re &agrave; haut d&eacute;bit. En cytom&eacute;trie en flux, trois dimensions (3D) hydrodynamiques focalisation est critique pour des mesures pr&eacute;cises et coh&eacute;rentes. En raison des avantages techniques microfluidique, un certain nombre de microfluidique cytom&egrave;tres avec focalisation hydrodynamique 3D ont &eacute;t&eacute; d&eacute;velopp&eacute; dans les derni&egrave;res d&eacute;cennies. Cependant, les dispositifs existants sont constitu&eacute;s de plusieurs couches de canaux microfluidiques et interconnexions fluidiques fastidieuses. En cons&eacute;quence, ces appareils n&eacute;cessitent souvent complexe de fabrication et le fonctionnement professionnel. Par cons&eacute;quent, le d&eacute;veloppement d&#39;un cytom&egrave;tre de flux microfluidique robuste et fiable pour des applications pratiques biologiques est souhait&eacute;. Cet article d&eacute;veloppe un dispositif microfluidique avec une couche de canal unique et entr&eacute;e de gaine-flux unique capable d&#39;atteindre une focalisation hydrodynamique 3D pour la cytom&eacute;trie en flux. La gaine-flux est introduite perpendiculairement au canal microfluidique &agrave; encercler le d&eacute;bit de l&#39;&eacute;chantillon. Dans le pr&eacute;sent document, les champs d&#39;&eacute;coulement sont simul&eacute;es en utilisant un calcul fluidique dynamique (CFD) logiciel, et les r&eacute;sultats montrent que la focalisation hydrodynamique 3D peut &ecirc;tre correctement form&eacute;e dans le dispositif microfluidique con&ccedil;us dans des conditions d&#39;&eacute;coulement. Le dispositif d&eacute;velopp&eacute; se caract&eacute;rise encore exp&eacute;rimentalement. Tout d&#39;abord, confocal microscopy est exploit&eacute;e pour enqu&ecirc;ter sur les champs d&#39;&eacute;coulement. Les images confocales de Z-pile r&eacute;sultantes montrent la vue en coupe de la 3D hydrodynamique avec des conditions d&#39;&eacute;coulement qui rejoins ceux simul&eacute;s. En outre, les d&eacute;tections de cytom&eacute;trie en flux de perles de fluorescence sont effectu&eacute;es &agrave; l&#39;aide de l&#39;appareil d&eacute;velopp&eacute; avec diverses combinaisons de taux d&#39;&eacute;coulement. Les r&eacute;sultats des mesures d&eacute;montrent que le dispositif peut atteindre des performances de d&eacute;tection grande, qui sont comparables au cytom&egrave;tre classiques. En outre, l&#39;&eacute;num&eacute;ration des cellules marqu&eacute;es fluorescence est &eacute;galement r&eacute;alis&eacute;e pour montrer sa praticit&eacute; pour applications biologiques. Par cons&eacute;quent, le cytom&egrave;tre de microfluidique d&eacute;velopp&eacute; dans cet article fournit une plate-forme pratique qui peut &ecirc;tre utilis&eacute;e pour l&#39;analyse de routine dans les laboratoires biologiques. En outre, la conception de canal focalisation hydrodynamique 3D aussi peut &ecirc;tre appliqu&eacute;e qu&#39;aux diff&eacute;rentes applications qui peuvent avancer au laboratoire sur une recherche de la puce.

Couche de canal unique, gaine simple-flux d&#39;admission microfluidique cytom&egrave;tre avec focalisation hydrodynamique tridimensionnel.

Durchflusszytometrie ist eine Technik, die optisch Charakterisierung biologischer Partikel in gewissem Sinne Hochdurchsatz-f&auml;hig. Drei dimensionale (3D) hydrodynamische Fokussierung ist Durchflusszytometrie f&uuml;r genaue und konsistente Messungen von entscheidender Bedeutung. Aufgrund der Vorteile der Microfluidic Techniken wurden eine Reihe von mikrofluidische flie&szlig;en Cytometers mit 3D hydrodynamische Fokussierung in den letzten Jahrzehnten entwickelt. Jedoch bestehen die vorhandenen Ger&auml;te mehrerer Ebenen mikrofluidische Kan&auml;le und m&uuml;hsame fluidische Verbindungsleitungen. Infolgedessen m&uuml;ssen diese Ger&auml;te oft komplizierte Fertigung und professionelle Bedienung. Daher ist die Entwicklung der eine robuste und zuverl&auml;ssige mikrofluidische Flow Cytometer f&uuml;r praktische biologische Anwendungen erw&uuml;nscht. Dieses Papier entwickelt eine mikrofluidische Ger&auml;t mit single-Channel-Schicht und einzigen Mantel-Flow Meeresarm 3D hydrodynamische Fokussierung f&uuml;r Durchflusszytometrie erreicht werden k&ouml;nnen. Der Mantel-Flow-Stream ist eingef&uuml;hrten senkrecht zu der Microfluidic-Kanal, der Durchsatz der Probe einzukreisen. In diesem Papier die Fluss-Felder werden mit einem computational fluidische dynamische (CFD) Software simuliert und die Ergebnisse zeigen, dass die 3D hydrodynamische Fokussierung erfolgreich im gestaltete mikrofluidische Ger&auml;t unter richtigen Str&ouml;mungsverh&auml;ltnisse gebildet werden kann. Das entwickelte Ger&auml;t ist experimentell weiter charakterisiert. Erste, konfokale Mikroskopie wird ausgenutzt, um die Str&ouml;mung-Felder zu untersuchen. Die resultierende konfokale Z-Stapel-Bilder zeigen die Schnittansicht von 3D hydrodynamische mit Str&ouml;mungsverh&auml;ltnisse, die mit den simulierten zustimmen. Dar&uuml;ber hinaus werden die flie&szlig;en Cytometric Erkennungen von Fluoreszenz-Perlen mit das entwickelte Ger&auml;t mit verschiedenen Kombinationen von Fluss-Tarif durchgef&uuml;hrt. Die Messergebnisse zeigen, dass das Ger&auml;t gro&szlig;e Erkennung Auff&uuml;hrungen, erreichen kann, die vergleichbar mit den herk&ouml;mmlichen Fluss Cytometer sind. Dar&uuml;ber hinaus ist auch die Aufz&auml;hlung der Fluoreszenz-gekennzeichnet Zellen durchgef&uuml;hrt, um seine Praktikabilit&auml;t f&uuml;r biologische Anwendungen zeigen. Daher enth&auml;lt der Microfluidic Flow Cytometer entwickelt in diesem Papier eine praktische Plattform, die f&uuml;r Routineanalysen in biologischen Laboratorien verwendet werden kann. Dar&uuml;ber hinaus kann das 3D hydrodynamische Fokussierung Kanaldesign auch zu verschiedenen Anwendungen angewendet werden, die das Labor auf einem Chip-Forschung voranbringen k&ouml;nnen.

Einkanal-Schicht, einzelne Mantel-Flow Meeresarm mikrofluidische Fluss Cytometer mit dreidimensionalen hydrodynamische Fokussierung.

Citometria a flusso &egrave; una tecnica in grado di caratterizzare otticamente particelle biologiche in un modo ad alta velocit&agrave;. In citometria a flusso, tre dimensioni (3D) idrodinamico concentrandosi &egrave; critico per misure accurate e coerenti. Dovuto i vantaggi delle tecniche di microfluidica, un certo numero di citometri flusso microfluidica con focalizzazione idrodinamica 3D &egrave; stato sviluppato negli ultimi decenni. Tuttavia, i dispositivi esistenti costituiti da pi&ugrave; strati di canali microfluidici e interconnessioni fluidici noiose. Come risultato, questi dispositivi richiedono spesso complicata fabbricazione e funzionamento professionale. Di conseguenza, lo sviluppo di un citometro a flusso microfluidica robusto e affidabile per applicazioni pratiche biologiche &egrave; desiderato. Questa carta si sviluppa un dispositivo microfluidico con un canale singolo strato e singola guaina-portata ingresso in grado di raggiungere la focalizzazione idrodinamica 3D per citometria a flusso. Il flusso di flusso guaina &egrave; introdotto perpendicolarmente al canale microfluidico per accerchiare il flusso del campione. In questo documento, i campi di flusso sono simulati usando un calcolo fluido dinamico (CFD) software, e i risultati mostrano che la focalizzazione idrodinamica 3D pu&ograve; essere correttamente formata nel dispositivo microfluidico progettato in condizioni di flusso adeguato. Il dispositivo sviluppato pi&ugrave; ulteriormente &egrave; caratterizzato sperimentalmente. Microscopia confocale, prima &egrave; sfruttata per indagare i campi di flusso. Le risultante immagini confocale Z-stack Visualizza la vista di sezione trasversale del 3D idrodinamico con condizioni di flusso che concordano con quelli simulati. Inoltre, i rilevamenti cytometric di flusso di perline di fluorescenza vengono eseguiti utilizzando il dispositivo sviluppato con varie combinazioni di tasso di flusso. I risultati della misurazione dimostrano che il dispositivo pu&ograve; raggiungere prestazioni di grande rivelazione, che sono paragonabili per il citometro a flusso convenzionale. Inoltre, l&#39;enumerazione delle cellule marcate con fluorescenza viene eseguita anche per mostrare la sua praticit&agrave; per applicazioni biologiche. Di conseguenza, il citometro a flusso microfluidica sviluppato in questo libro fornisce una piattaforma pratica che pu&ograve; essere utilizzata per analisi di routine nei laboratori biologici. Inoltre, la struttura canale focalizzazione idrodinamica 3D pu&ograve; essere applicata anche alle varie applicazioni che possono avanzare il laboratorio su una ricerca di chip.

Strato singolo canale, guaina-flusso singolo ingresso microfluidica citometro a flusso con focalizzazione idrodinamica tridimensionale.

&#x30D5;&#x30ED;&#x30FC;&#x30B5;&#x30A4;&#x30C8;&#x30E1;&#x30C8;&#x30EA;&#x30FC;&#x5149;&#x5B66;&#x7684;&#x751F;&#x7269;&#x5B66;&#x7684;&#x7C92;&#x5B50;&#x3092;&#x9AD8;&#x30B9;&#x30EB;&#x30FC;&#x30D7;&#x30C3;&#x30C8;&#x306B;&#x7279;&#x5FB4;&#x306E;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x6280;&#x8853;&#x3067;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x30D5;&#x30ED;&#x30FC;&#x30B5;&#x30A4;&#x30C8;&#x30E1;&#x30C8;&#x30EA;&#x30FC;&#x3067; 3 &#x6B21;&#x5143; (3 D) &#x6D41;&#x4F53;&#x4E2D;&#x5FC3;&#x306E;&#x6B63;&#x78BA;&#x3067;&#x4E00;&#x8CAB;&#x6027;&#x306E;&#x3042;&#x308B;&#x6E2C;&#x5B9A;&#x304C;&#x91CD;&#x8981;&#x3067;&#x3059;&#x3002;&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x6D41;&#x4F53;&#x6280;&#x8853;&#x306E;&#x5229;&#x70B9;&#x306E;&#x305F;&#x3081;&#x3001;&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x6D41;&#x4F53; cytometers &#x304C; 3 D &#x306E;&#x6D41;&#x4F53;&#x529B;&#x5B66;&#x7684;&#x7126;&#x70B9;&#x306E;&#x6570;&#x306F;&#x3001;&#x6700;&#x8FD1;&#x6570;&#x5341;&#x5E74;&#x3067;&#x958B;&#x767A;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;&#x65E2;&#x5B58;&#x306E;&#x30C7;&#x30D0;&#x30A4;&#x30B9;&#x306F;&#x3001;&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x6D41;&#x8DEF;&#x3068;&#x9000;&#x5C48;&#x306A;&#x6D41;&#x4F53;&#x76F8;&#x4E92;&#x63A5;&#x7D9A;&#x306E;&#x8907;&#x6570;&#x306E;&#x5C64;&#x3067;&#x69CB;&#x6210;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x7D50;&#x679C;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x30C7;&#x30D0;&#x30A4;&#x30B9;&#x306F;&#x3001;&#x3057;&#x3070;&#x3057;&#x3070;&#x8907;&#x96D1;&#x306A;&#x4F5C;&#x88FD;&#x3068;&#x30D7;&#x30ED;&#x306E;&#x64CD;&#x4F5C;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x305D;&#x306E;&#x7D50;&#x679C;&#x3001;&#x5805;&#x7262;&#x3067;&#x4FE1;&#x983C;&#x6027;&#x306E;&#x9AD8;&#x3044;&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x6D41;&#x4F53;&#x6D41;&#x308C;&#x306E; cytometer &#x5B9F;&#x7528;&#x7684;&#x306A;&#x751F;&#x7269;&#x5B66;&#x7684;&#x5FDC;&#x7528;&#x306E;&#x305F;&#x3081;&#x306E;&#x958B;&#x767A;&#x304C;&#x671B;&#x307E;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x672C;&#x7A3F;&#x3067;&#x306F;&#x3001;&#x5358;&#x4E00;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x5C64;&#x3068; 1 &#x3064;&#x306E;&#x30B7;&#x30FC;&#x30B9; &#x30D5;&#x30ED;&#x30FC;&#x5165;&#x53E3;&#x30D5;&#x30ED;&#x30FC;&#x30B5;&#x30A4;&#x30C8;&#x30E1;&#x30C8;&#x30EA;&#x30FC;&#x7528; 3 D &#x6D41;&#x4F53;&#x7126;&#x70B9;&#x3092;&#x9054;&#x6210;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x6D41;&#x4F53;&#x30C7;&#x30D0;&#x30A4;&#x30B9;&#x3092;&#x958B;&#x767A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x30B7;&#x30FC;&#x30B9; &#x30D5;&#x30ED;&#x30FC; &#x30B9;&#x30C8;&#x30EA;&#x30FC;&#x30E0; &#x30B5;&#x30F3;&#x30D7;&#x30EB; &#x30D5;&#x30ED;&#x30FC;&#x3092;&#x53D6;&#x308A;&#x56F2;&#x3080;&#x3088;&#x3046;&#x306B;&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x6D41;&#x8DEF;&#x306B;&#x5C0E;&#x5165;&#x3055;&#x308C;&#x305F;&#x5782;&#x76F4;&#x3067;&#x3059;&#x3002;&#x672C;&#x7A3F;&#x306F;, &#x6D41;&#x308C;&#x5834;&#x8A08;&#x7B97;&#x6D41;&#x4F53;&#x306E;&#x52D5;&#x7684; (CFD) &#x30BD;&#x30D5;&#x30C8;&#x30A6;&#x30A7;&#x30A2;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x30B7;&#x30DF;&#x30E5;&#x30EC;&#x30FC;&#x30C8;&#x3057;&#x3066;&#x3044;&#x308B;&#x3057;&#x3001;3 D &#x306E;&#x6D41;&#x4F53;&#x529B;&#x5B66;&#x7684;&#x7126;&#x70B9;&#x304C;&#x6B63;&#x5E38;&#x306B;&#x9069;&#x5207;&#x306A;&#x6D41;&#x308C;&#x306E;&#x6761;&#x4EF6;&#x306E;&#x4E0B;&#x3067;&#x8A2D;&#x8A08;&#x3055;&#x308C;&#x305F;&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x6D41;&#x4F53;&#x30C7;&#x30D0;&#x30A4;&#x30B9;&#x3067;&#x5F62;&#x6210;&#x3055;&#x308C;&#x308B;&#x3053;&#x3068;&#x306E;&#x7D50;&#x679C;&#x3057;&#x307E;&#x3059;&#x3002;&#x958B;&#x767A;&#x3057;&#x305F;&#x30C7;&#x30D0;&#x30A4;&#x30B9;&#x306F;&#x3001;&#x3055;&#x3089;&#x306B;&#x5B9F;&#x9A13;&#x7684;&#x7279;&#x5FB4;&#x3067;&#x3059;&#x3002;&#x6700;&#x521D;&#x306B;&#x3001;&#x5171;&#x7126;&#x70B9;&#x9855;&#x5FAE;&#x93E1;&#x6D41;&#x308C;&#x5834;&#x3092;&#x8ABF;&#x67FB;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x60AA;&#x7528;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;Z &#x30B9;&#x30BF;&#x30C3;&#x30AF;&#x5171;&#x7126;&#x70B9;&#x753B;&#x50CF; 3 D &#x306E;&#x65AD;&#x9762;&#x306F;&#x3001;&#x6A21;&#x64EC;&#x6D41;&#x308C;&#x306E;&#x6761;&#x4EF6;&#x3068;&#x6D41;&#x4F53;&#x529B;&#x5B66;&#x7684;&#x8868;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x86CD;&#x5149;&#x30D3;&#x30FC;&#x30BA;&#x306E;&#x6D41;&#x308C;&#x30D5;&#x30ED;&#x30FC;&#x30B5;&#x30A4;&#x30C8;&#x30E1;&#x30C8;&#x30EA;&#x30FC;&#x306B;&#x3088;&#x308B;&#x691C;&#x51FA;&#x306F;&#x3001;&#x3055;&#x307E;&#x3056;&#x307E;&#x306A;&#x30D5;&#x30ED;&#x30FC;&#x901F;&#x5EA6;&#x3092;&#x7D44;&#x307F;&#x5408;&#x308F;&#x305B;&#x3066;&#x958B;&#x767A;&#x3057;&#x305F;&#x30C7;&#x30D0;&#x30A4;&#x30B9;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x5B9F;&#x884C;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x6E2C;&#x5B9A;&#x7D50;&#x679C;&#x306F;&#x3001;&#x30C7;&#x30D0;&#x30A4;&#x30B9;&#x304C;&#x5F93;&#x6765;&#x306E;&#x30D5;&#x30ED;&#x30FC;&#x30B5;&#x30A4;&#x30C8;&#x306B;&#x5339;&#x6575;&#x3059;&#x308B;&#x5049;&#x5927;&#x306A;&#x691C;&#x51FA;&#x6027;&#x80FD;&#x3092;&#x9054;&#x6210;&#x3067;&#x304D;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x86CD;&#x5149;&#x6A19;&#x8B58;&#x7D30;&#x80DE;&#x306E;&#x5217;&#x6319;&#x4F53;&#x306F;&#x3001;&#x5B9F;&#x7528;&#x6027;&#x306E;&#x751F;&#x7269;&#x5B66;&#x7684;&#x30A2;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3092;&#x8868;&#x793A;&#x3059;&#x308B;&#x3082;&#x5B9F;&#x884C;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x305D;&#x306E;&#x7D50;&#x679C;&#x3001;&#x672C;&#x7A3F;&#x3067;&#x306F;&#x958B;&#x767A;&#x3057;&#x305F;&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x6D41;&#x4F53;&#x6D41;&#x308C;&#x306E; cytometer &#x30EB;&#x30FC;&#x30C1;&#x30F3;&#x306B;&#x304A;&#x3051;&#x308B;&#x751F;&#x7269;&#x5B66;&#x7684;&#x7814;&#x7A76;&#x306E;&#x305F;&#x3081;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x5B9F;&#x7528;&#x7684;&#x306A;&#x30D7;&#x30E9;&#x30C3;&#x30C8;&#x30D5;&#x30A9;&#x30FC;&#x30E0;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;3 D &#x306E;&#x6D41;&#x4F53;&#x529B;&#x5B66;&#x7684;&#x7126;&#x70B9;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x8A2D;&#x8A08;&#x6F14;&#x7FD2;&#x30C1;&#x30C3;&#x30D7;&#x306E;&#x7814;&#x7A76;&#x3092;&#x9032;&#x3081;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x69D8;&#x3005; &#x306A;&#x30A2;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306B;&#x3082;&#x9069;&#x7528;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;

1 &#x3064;&#x306E;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x5C64;&#x306E;&#x4E09;&#x6B21;&#x5143;&#x6D41;&#x4F53;&#x529B;&#x5B66;&#x3092;&#x4E2D;&#x5FC3;&#x3068; 1 &#x3064;&#x306E;&#x30B7;&#x30FC;&#x30B9; &#x30D5;&#x30ED;&#x30FC;&#x5165;&#x53E3;&#x6D41;&#x4F53;&#x30D5;&#x30ED;&#x30FC;&#x30B5;&#x30A4;&#x30C8;&#x3002;

Cytometry &#xAD11;&#xD559; &#xB192;&#xC740; &#xCC98;&#xB9AC;&#xB7C9; &#xBC29;&#xBC95;&#xC73C;&#xB85C; &#xC0DD;&#xBB3C; &#xC785;&#xC790;&#xB97C; &#xD2B9;&#xC131;&#xD654; &#xD558;&#xB294; &#xAE30;&#xC220; &#xC774;&#xB2E4;. Cytometry, &#xCD08;&#xC810; 3 &#xCC28;&#xC6D0; (3D) &#xC720;&#xCCB4;&#xC5ED;&#xD559;&#xC744; &#xC815;&#xD655; &#xD558; &#xACE0; &#xC77C;&#xAD00; &#xB41C; &#xCE21;&#xC815;&#xC5D0; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD55C; &#xBB38;&#xC81C;&#xC785;&#xB2C8;&#xB2E4;. &#xBBF8;&#xC138; &#xAE30;&#xC220;&#xC758; &#xC7A5;&#xC810;&#xC73C;&#xB85C; &#xC778;&#xD574; &#xB2E4;&#xC591; &#xD55C; 3 &#xCC28;&#xC6D0; &#xC720;&#xCCB4; &#xCD08;&#xC810; &#xBBF8;&#xC138; &#xD750;&#xB984; cytometers &#xCD5C;&#xADFC; &#xC218;&#xC2ED; &#xB144; &#xB3D9;&#xC548;&#xC5D0;&#xC11C; &#xAC1C;&#xBC1C; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xAE30;&#xC874; &#xC7A5;&#xCE58; &#xACB0;&#xD569; &#xCC44;&#xB110;&#xACFC; &#xC9C0;&#xB8E8;&#xD55C; &#xC720;&#xCCB4; &#xC0C1;&#xD638; &#xC5F0;&#xACB0;&#xC758; &#xC5EC;&#xB7EC; &#xB808;&#xC774;&#xC5B4; &#xAD6C;&#xC131; &#xB429;&#xB2C8;&#xB2E4;. &#xADF8; &#xACB0;&#xACFC;, &#xC774;&#xB7EC;&#xD55C; &#xC7A5;&#xCE58;&#xB294; &#xC885;&#xC885; &#xBCF5;&#xC7A1; &#xD55C; &#xC81C;&#xC870; &#xBC0F; &#xC804;&#xBB38; &#xC791;&#xC5C5; &#xD544;&#xC694;&#xD569;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, &#xC0DD;&#xBB3C;&#xD559;&#xC801; &#xC751;&#xC6A9;&#xC5D0; &#xB300; &#xD55C; &#xAC15;&#xB825; &#xD558; &#xACE0; &#xC548;&#xC815;&#xC801;&#xC778; &#xBBF8;&#xC138; &#xD750;&#xB984; cytometer &#xAC1C;&#xBC1C; &#xD544;&#xC694;. &#xC774; &#xC885;&#xC774; &#xB2E8;&#xC77C; &#xCC44;&#xB110; &#xACC4;&#xCE35; &#xBC0F; &#xB2E8;&#xC77C; &#xCE7C; &#xC9D1; &#xD750;&#xB984; &#xC785;&#xAD6C; cytometry&#xC5D0; &#xB300; &#xD55C; 3 &#xCC28;&#xC6D0; &#xC720;&#xCCB4; &#xCD08;&#xC810;&#xC744; &#xB2EC;&#xC131; &#xAC00;&#xB2A5;&#xD55C; &#xBBF8;&#xC138; &#xC7A5;&#xCE58; &#xAC1C;&#xBC1C;. &#xCE7C; &#xC9D1; &#xD750;&#xB984; &#xC2A4;&#xD2B8;&#xB9BC; &#xC0D8;&#xD50C; &#xD750;&#xB984;&#xC744; &#xB458;&#xB7EC;&#xC2F8; &#xC790; &#xBBF8;&#xC138; &#xCC44;&#xB110;&#xC5D0; &#xC18C;&#xAC1C; &#xB41C; &#xC218;&#xC9C1;&#xC785;&#xB2C8;&#xB2E4;. &#xC774; &#xC2E0;&#xBB38;&#xC5D0; &#xD750;&#xB984; &#xD544;&#xB4DC;&#xB97C; &#xC804;&#xC0B0; &#xC720;&#xCCB4; &#xB3D9;&#xC801; (CFD) &#xC18C;&#xD504;&#xD2B8;&#xC6E8;&#xC5B4;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC2DC;&#xBBAC;&#xB808;&#xC774;&#xC158; &#xB418; &#xACE0; &#xACB0;&#xACFC; 3 &#xCC28;&#xC6D0; &#xC720;&#xCCB4; &#xCD08;&#xC810;&#xC744; &#xD615;&#xC131; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xC131;&#xACF5;&#xC801;&#xC73C;&#xB85C; &#xC801;&#xC808; &#xD55C; &#xD750;&#xB984; &#xC870;&#xAC74; &#xD558;&#xC5D0;&#xC11C; &#xC124;&#xACC4; &#xB41C; &#xBBF8;&#xC138; &#xC7A5;&#xCE58;. &#xAC1C;&#xBC1C;&#xB41C; &#xB41C; &#xC7A5;&#xCE58;&#xB294; &#xCD94;&#xAC00; &#xC2E4;&#xD5D8;&#xC801;&#xC73C;&#xB85C; &#xD2B9;&#xC9D5;&#xC785;&#xB2C8;&#xB2E4;. &#xCCAB;&#xC9F8;, confocal &#xD604;&#xBBF8;&#xACBD; &#xAC80;&#xC0AC; &#xBC95;&#xC740; &#xD750;&#xB984; &#xD544;&#xB4DC; &#xC870;&#xC0AC;&#xB97C; &#xC545;&#xC6A9;. &#xACB0;&#xACFC; Z &#xC2A4;&#xD0DD; confocal &#xC774;&#xBBF8;&#xC9C0; hydrodynamic &#xC2DC;&#xBBAC;&#xB808;&#xC774;&#xC158; &#xB41C; &#xAC83; &#xB4E4;&#xC5D0; &#xB3D9;&#xC758; &#xD750;&#xB984; &#xC870;&#xAC74; 3d &#xD6A1;&#xB2E8;&#xBA74; &#xBDF0;&#xB97C; &#xD45C;&#xC2DC; &#xD569;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, &#xD615;&#xAD11; &#xAD6C;&#xC2AC;&#xC758; &#xD750;&#xB984; cytometric &#xD0D0;&#xC9C0; &#xB2E4;&#xC591; &#xD55C; &#xD750;&#xB984; &#xC18D;&#xB3C4; &#xC870;&#xD569;&#xC73C;&#xB85C; &#xAC1C;&#xBC1C;&#xB41C; &#xB41C; &#xC7A5;&#xCE58;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC218;&#xD589; &#xB429;&#xB2C8;&#xB2E4;. &#xCE21;&#xC815; &#xACB0;&#xACFC; &#xC785;&#xC99D; &#xC7A5;&#xCE58; &#xC88B;&#xC740; &#xD0D0;&#xC9C0; &#xACF5;&#xC5F0;, &#xAE30;&#xC874;&#xC758; &#xD750;&#xB984; cytometer &#xBE44;&#xAD50;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xC5BB;&#xC744; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, &#xD615;&#xAD11; &#xD45C;&#xC2DC; &#xC140;&#xC758; &#xC5F4;&#xAC70; &#xC0DD;&#xBB3C; &#xC751;&#xC6A9; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC5D0; &#xB300; &#xD55C; &#xADF8;&#xAC83;&#xC758; &#xC2E4;&#xC6A9;&#xC131;&#xC744; &#xBCF4;&#xC5EC;&#xB3C4; &#xC218;&#xD589; &#xB429;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, &#xBCF8;&#xC774; &#xB17C;&#xBB38;&#xC5D0;&#xC11C; &#xAC1C;&#xBC1C;&#xD55C; &#xBBF8;&#xC138; &#xD750;&#xB984; cytometer &#xC0DD;&#xBB3C;&#xD559; &#xC2E4;&#xD5D8;&#xC2E4;&#xC5D0; &#xC788;&#xB294; &#xC77C;&#xC0C1;&#xC801;&#xC778; &#xBD84;&#xC11D;&#xC744; &#xC704;&#xD574; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xB294; &#xC2E4;&#xC6A9;&#xC801;&#xC778; &#xD50C;&#xB7AB;&#xD3FC;&#xC744; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, 3 &#xCC28;&#xC6D0; &#xC720;&#xCCB4; &#xCD08;&#xC810; &#xCC44;&#xB110; &#xC124;&#xACC4; &#xCE69; &#xC5F0;&#xAD6C; &#xC2E4;&#xD5D8;&#xC2E4;&#xC744; &#xBBF8;&#xB9AC; &#xC218; &#xC788;&#xB294; &#xB2E4;&#xC591; &#xD55C; &#xC751;&#xC6A9; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC5D0;&#xB3C4; &#xC801;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xB2E8;&#xC77C; &#xCC44;&#xB110; &#xACC4;&#xCE35;, &#xB2E8;&#xC77C; &#xCE7C; &#xC9D1; &#xD750;&#xB984; &#xC785;&#xAD6C; &#xBBF8;&#xC138; &#xD750;&#xB984; cytometer 3 &#xCC28;&#xC6D0; &#xC720;&#xCCB4; &#xCD08;&#xC810;.

Citometr&iacute;a de flujo es una t&eacute;cnica capaz de caracterizar &oacute;pticamente las part&iacute;culas biol&oacute;gicas de una manera de alto rendimiento. En citometr&iacute;a de flujo, tres dimensiones (3D) hidrodin&aacute;mico enfoque es cr&iacute;tico para la medici&oacute;n precisa y coherente. Debido a las ventajas de las t&eacute;cnicas de microfluidos, un n&uacute;mero de microfluidos cit&oacute;metros de flujo con enfoque hidrodin&aacute;mico 3D se han desarrollado en las &uacute;ltimas d&eacute;cadas. Sin embargo, los dispositivos existentes consisten en varias capas de microfluidos canales y tediosas interconexiones flu&iacute;dicas. Como resultado, estos dispositivos requieren a menudo complicada fabricaci&oacute;n y operaci&oacute;n profesional. En consecuencia, se desea el desarrollo de un cit&oacute;metro de flujo de microfluidos robusta y fiable para aplicaciones pr&aacute;cticas biol&oacute;gicas. Este art&iacute;culo desarrolla un dispositivo microflu&iacute;dico con una capa de monocanal y entrada de vaina-flujo &uacute;nico capaz de lograr el enfoque hidrodin&aacute;mico 3D para citometr&iacute;a de flujo. La corriente de flujo vaina es introducido perpendicular al canal microflu&iacute;dicos para rodear el caudal de muestreo. En este trabajo, los campos de flujo son simulados utilizando un software din&aacute;mico de flu&iacute;dico computacional (CFD), y los resultados muestran que el enfoque hidrodin&aacute;mico 3D puede ser correctamente formado en el dispositivo microflu&iacute;dico dise&ntilde;ado bajo condiciones de flujo apropiado. El dispositivo desarrollado se caracteriza experimentalmente. Microscop&iacute;a confocal, primera es explotado para investigar los campos de flujo. Las im&aacute;genes resultantes del Z-pila confocales Mostrar la vista transversal de 3D hidrodin&aacute;mico con condiciones de flujo que de acuerdo con los simulados. Adem&aacute;s, las detecciones de citometr&iacute;a de flujo de granos de la fluorescencia se realizan utilizando el dispositivo desarrollado con varias combinaciones de tasa de flujo. Los resultados de medici&oacute;n demuestran que el dispositivo puede lograr actuaciones de detecci&oacute;n grandes, que son comparables con el cit&oacute;metro de flujo convencional. Adem&aacute;s, la enumeraci&oacute;n de c&eacute;lulas marcado con fluorescencia tambi&eacute;n se realiza para mostrar su practicidad para aplicaciones biol&oacute;gicas. En consecuencia, el cit&oacute;metro de flujo de microfluidos desarrollado en este trabajo proporciona una plataforma pr&aacute;ctica que puede ser utilizada para el an&aacute;lisis de rutina en laboratorios biol&oacute;gicos. Adem&aacute;s, el 3D dise&ntilde;o hidrodin&aacute;mico de canal enfoque puede aplicarse tambi&eacute;n a varias aplicaciones que pueden avanzar el laboratorio en una investigaci&oacute;n de la viruta.

Capa monocanal, vaina-flujo &uacute;nico entrada microflu&iacute;dicos cit&oacute;metro de flujo con enfoque hidrodin&aacute;mico tridimensional.

Oxygen tension plays an important role in regulating various cellular functions in both normal physiology and disease states. Therefore, drug testing using conventional in vitro cell models under normoxia often possesses limited prediction capability. A traditional method of setting an oxygen tension in a liquid medium is by saturating it with a gas mixture at the desired level of oxygen, which requires bulky gas cylinders, sophisticated control, and tedious interconnections. Moreover, only a single oxygen tension can be tested at the same time. In this paper, we develop a microfluidic cell culture array platform capable of performing cell culture and drug testing under various oxygen tensions simultaneously. The device is fabricated using an elastomeric material, polydimethylsiloxane (PDMS) and the well-developed multi-layer soft lithography (MSL) technique. The prototype device has 4 × 4 wells, arranged in the same dimensions as a conventional 96-well plate, for cell culture. The oxygen tensions are controlled by spatially confined oxygen scavenging chemical reactions underneath the wells using microfluidics. The platform takes advantage of microfluidic phenomena while exhibiting the combinatorial diversities achieved by microarrays. Importantly, the platform is compatible with existing cell incubators and high-throughput instruments (liquid handling systems and plate readers) for cost-effective setup and straightforward operation. Utilizing the developed platform, we successfully perform drug testing using an anti-cancer drug, triapazamine (TPZ), on adenocarcinomic human alveolar basal epithelial cell line (A549) under three oxygen tensions ranging from 1.4% to normoxia. The developed platform is promising to provide a more meaningful in vitro cell model for various biomedical applications while maintaining desired high throughput capabilities.

A microfluidic cell culture array with various oxygen tensions.

Culture of cells as three-dimensional (3D) aggregates, named spheroids, possesses great potential to improve in vitro cell models for basic biomedical research. However, such cell spheroid models are often complicated, cumbersome, and expensive compared to conventional Petri-dish cell cultures. In this work, we developed a simple microfluidic device for cell spheroid formation, culture, and harvesting. Using this device, cells could form uniformly sized spheroids due to strong cell-cell interactions and the spatial confinement of microfluidic culture chambers. We demonstrated cell spheroid formation and culture in the designed devices using embryonic stem cells, carcinoma cells, and fibroblasts. We further scaled up the device capable of simultaneously forming and culturing 5000 spheroids in a single chip. Finally, we demonstrated harvesting of the cultured spheroids from the device with a simple setup. The harvested spheroids possess great integrity, and the cells can be exploited for further flow cytometry assays due to the ample cell numbers.

A microfluidic device for uniform-sized cell spheroids formation, culture, harvesting and flow cytometry analysis.

In this paper, we develop a microfluidic device capable of generating nitric oxide (NO) gradients for cell culture using spatially controlled chemical reactions. NO plays an essential role in various biological activities, including nervous, immune, and cardiovascular systems. The device developed in this paper can control NO gradients without utilizing expensive and hazardous high purity NO gas sources or direct addition of NO donors. Consequently, the device provides an efficient, cost-effective, robust, and stable platform to generate NO gradients for cell culture studies. In the experiments, NO gradients are first characterized using a NO-sensitive fluorescence dye, and cell experiments using aortic smooth muscle cells are conducted. The results demonstrate that the device can alter the intracellular NO concentrations and further affect the Ca(2+) concentration oscillation for the cells. The device developed in this paper provides a powerful platform for researchers better study the biological roles of NO and its spatial distribution using in vitro cell models with minimal instrumentation.

Generation of nitric oxide gradients in microfluidic devices for cell culture using spatially controlled chemical reactions.

This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) hybrid microfluidic device capable of performing cell culture under combinations of chemical and oxygen gradients. The microfluidic device is constructed of two PDMS layers with microfluidic channel patterns separated by a thin PDMS membrane. The top layer contains an embedded PC film and a serpentine channel for a spatially confined oxygen scavenging chemical reaction to generate an oxygen gradient in the bottom layer for cell culture. Using the chemical reaction method, the device can be operated with a small amount of chemicals, without bulky gas cylinders and sophisticated flow control schemes. Furthermore, it can be directly used in conventional incubators with syringe pumps to simplify the system setup. The bottom layer contains arrangements of serpentine channels for chemical gradient generation and a cell culture chamber in the downstream. The generated chemical and oxygen gradients are experimentally characterized using a fluorescein solution and an oxygen-sensitive fluorescent dye, respectively. For demonstration, a 48 hour cell-based drug test and a cell migration assay using human lung adenocarcinoma epithelial cells (A549) are conducted under various combinations of the chemical and oxygen gradients in the experiments. The drug testing results show an increase in A549 cell apoptosis due to the hypoxia-activated cytotoxicity of tirapazamine (TPZ) and also suggest great cell compatibility and gradient controllability of the device. In addition, the A549 cell migration assay results demonstrate an aerotactic behavior of the A549 cells and suggest that the oxygen gradient plays an essential role in guiding cell migration. The migration results, under combinations of chemokine and oxygen gradients, cannot be simply superposed with single gradient results. The device is promising to advance the control of in vitro microenvironments, to better study cellular responses under various physiological conditions for biomedical applications.

A polydimethylsiloxane-polycarbonate hybrid microfluidic device capable of generating perpendicular chemical and oxygen gradients for cell culture studies.

We developed a microfluidic device to culture cellular spheroids of controlled sizes and suitable for live cell imaging by selective plane illumination microscopy (SPIM). We cocultured human umbilical vein endothelial cells (HUVECs) within the spheroids formed by hepatocellular carcinoma cells, and studied the distributions of the HUVECs over time. We observed that the migration of HUVECs depended on the size of spheroids. In the spheroids of ∼200 μm diameters, HUVECs migrated outwards to the edges within 48 h; while in the spheroids of ∼250 μm diameters, there was no outward migration of the HUVECs up to 72 h. In addition, we studied the effects of pro-angiogenic factors, namely, vascular endothelial growth factor (VEGF) and fibroblast growth factor (β-FGF), on the migration of HUVECs in the carcinoma cell spheroid. The outward migration of HUVECs in 200 μm spheroids was hindered by the treatment with VEGF and β-FGF. Moreover, some of the HUVECs formed hollow lumen within 72 h under VEGF and β-FGF treatment. The combination of SPIM and microfluidic devices gives high resolution in both spatial and temporal domains. The observation of HUVECs in spheroids provides us insight on tumor vascularization, an ideal disease model for drug screening and fundamental studies.

Migration and vascular lumen formation of endothelial cells in cancer cell spheroids of various sizes.

This paper reports a polydimethylsiloxane (PDMS) SlipChip for in vitro cell culture applications, multiple-treatment assays, cell co-cultures, and cytokine detection assays. The PDMS SlipChip is composed of two PDMS layers with microfluidic channels on each surface that are separated by a thin silicone fluid (Si-fluid) layer. The integration of Si-fluid enables the two PDMS layers to be slid to different positions; therefore, the channel patterns can be re-arranged for various applications. The SlipChip design significantly reduces the complexity of sample handling, transportation, and treatment processes. To apply the developed SlipChip for cell culture applications, human lung adenocarcinoma epithelial cells (A549) and lung fibroblasts (MRC-5) were cultured to examine the biocompatibility of the developed PDMS SlipChip. Moreover, embryonic pluripotent stem cells (ES-D3) were also cultured in the device to evaluate the retention of their stemness in the device. The experimental results show that cell morphology, viability and proliferation are not affected when the cells are cultured in the SlipChip, indicating that the device is highly compatible with mammalian cell culture. In addition, the stemness of the ES-D3 cells was highly retained after they were cultured in the device, suggesting the feasibility of using the SlipChip for stem cell research. Various cell experiments, such as simultaneous triple staining of cells and co-culture of MRC-5 with A549 cells, were also performed to demonstrate the functionalities of the PDMS SlipChip. Furthermore, we used a cytokine detection assay to evaluate the effect of endotoxin (lipopolysaccharides, LPS) treatment on the cytokine secretion of A549 cells using the SlipChip. The developed PDMS SlipChip provides a straightforward and effective platform for various on-chip in vitro cell cultures and consequent analysis, which is promising for a number of cell biology studies and biomedical applications.

Polydimethylsiloxane SlipChip for mammalian cell culture applications.

This paper reports a two-layered polydimethylsiloxane microfluidic device-Flip channel, capable of forming uniform-sized embryoid bodies (EBs) and performing stem cell differentiation within the same device after flipping the microfluidic channel. The size of EBs can be well controlled by designing the device geometries, and EBs with multiple sizes can be formed within a single device to study EB size-dependent stem cell differentiation. During operation of the device, cells are positioned in the designed positions. As a result, observation and monitoring specific population of cells can be achieved for further analysis. In addition, after flipping the microfluidic channel, stem cell differentiation from the EBs can be performed on an unconfined flat surface that is desired for various differentiation processes. In the experiments, murine embryonic stem cells (ES-D3) are cultured and formed EBs inside the developed device. The size of EBs is well controlled inside the device, and the neural differentiation is performed on the formed EBs after flipping the channel. The EB size-dependent stem cell differentiation is studied using the device to demonstrate its functions. The device provides a useful tool to study stem cell differentiation without complicated device fabrication and tedious cell handling under better-controlled microenvironments.

Flip channel: A microfluidic device for uniform-sized embryoid body formation and differentiation.

Three-dimensional (3D) tumor spheroid possesses great potential as an in vitro model to improve predictive capacity for pre-clinical drug testing. In this paper, we combine advantages of flow cytometry and microfluidics to perform drug testing and analysis on a large number (5000) of uniform sized tumor spheroids. The spheroids are formed, cultured, and treated with drugs inside a microfluidic device. The spheroids can then be harvested from the device without tedious operation. Due to the ample cell numbers, the spheroids can be dissociated into single cells for flow cytometry analysis. Flow cytometry provides statistical information in single cell resolution that makes it feasible to better investigate drug functions on the cells in more in vivo-like 3D formation. In the experiments, human hepatocellular carcinoma cells (HepG2) are exploited to form tumor spheroids within the microfluidic device, and three anti-cancer drugs: Cisplatin, Resveratrol, and Tirapazamine (TPZ), and their combinations are tested on the tumor spheroids with two different sizes. The experimental results suggest the cell culture format (2D monolayer vs. 3D spheroid) and spheroid size play critical roles in drug responses, and also demonstrate the advantages of bridging the two techniques in pharmaceutical drug screening applications.

Drug testing and flow cytometry analysis on a large number of uniform sized tumor spheroids using a microfluidic device.

In various physiological activities, cells experience stresses along their in-plane direction when facing substrate deformation. Capability of continuous monitoring elasticity of live cell layers during a period is highly desired to investigate cell property variation during various transformations under normal or disease states. This paper reports time-lapsed measurement of live cell layer in-plane elasticity using a pressure sensor embedded microfluidic device. The sensor converts pressure-induced deformation of a flexible membrane to electrical signals. When cells are cultured on top of the membrane, flexural rigidity of the composite membrane increases and further changes the output electrical signals. In the experiments, human embryonic lung fibroblast (MRC-5) cells are cultured and analyzed to estimate the in-plane elasticity. In addition, the cells are treated with a growth factor to simulate lung fibrosis to study the effects of cell transformation on the elasticity variation. For comparison, elasticity measurement on the cells by atomic force microscopy (AFM) is also performed. The experimental results confirm highly anisotropic configuration and material properties of cells. Furthermore, the in-plane elasticity can be monitored during the cell transformation after the growth factor stimulation. Consequently, the developed microfluidic device provides a powerful tool to study physical properties of cells for fundamental biophysics and biomedical researches.

Chien-Chung Peng

Research Center for Applied Sciences

Academia Sinica

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

Chien-Chung Peng

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Research Center for Applied Sciences

Academia Sinica

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

Research Center for Applied Sciences