fabrication-multiplexed-artificial-cellular-microenvironment

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Kyoto University

Cell-based arrays offer powerful tools for genomics/proteomics and drug discovery, and are widely applicable for most cell lines. However, it is challenging to apply cell-based arrays for in vitro diagnosis due to limited amount of patient samples. Here, we utilized and demonstrated microfluidic image cytometry (MIC), capable of quantitative, single-cell profiling of multiple signaling molecules using only 300-3,000 cells from clinical brain tumor specimens for in vitro molecular diagnosis. First, we characterized the PI3K/AKT/mTOR pathway, which is often over-activated in the brain tumors, in U87 brain tumor cell lines by measuring EGFR, PTEN, pAKT, and pS6 with a MIC platform, and applied this measurement to clinical brain tumor specimens. In conjunction with statistical analysis, we were able to characterize extensive inter- and intra-tumoral molecular heterogeneity.

Microfluidic image cytometry.

Delivery of intact transcription factor by using self-assembled supramolecular nanoparticles.

&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE; (&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;) &#x662F;&#x7528;&#x4E8E;&#x836F;&#x7269;&#x7684;&#x53D1;&#x73B0;&#x3001; &#x53D1;&#x80B2;&#x751F;&#x7269;&#x5B66;&#x548C;&#x75BE;&#x75C5;&#x7814;&#x7A76;&#x7684;&#x6709;&#x7528;&#x8D44;&#x6E90;&#x3002;&#x7EC6;&#x80DE;&#x5FAE;&#x73AF;&#x5883;&#x63D0;&#x793A;&#x53D1;&#x6325;&#x5173;&#x952E;&#x7684;&#x4F5C;&#x7528;&#xFF0C;&#x5728;&#x8C03;&#x8282; esc &#x952E;&#x7684;&#x529F;&#x80FD;&#xFF0C;&#x4F46;&#x5B83;&#x4E00;&#x9879;&#x6311;&#x6218;&#x5B83;&#x4EEC;&#x63A7;&#x5236;&#x4E0E;&#x5408;&#x6210;&#x7EC4;&#x4EF6;&#x3002;&#x7EB3;&#x7C73;&#x7EA4;&#x7EF4;&#x4E3E;&#x884C;&#x521B;&#x5EFA;&#x4EBA;&#x5DE5;&#x7EC6;&#x80DE;&#x63D0;&#x793A;&#x63A7;&#x5236;&#x7EC6;&#x80DE;&#x7C98;&#x9644;&#x53CA;&#x7EC6;&#x80DE;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x7684;&#x6F5C;&#x529B;&#x3002;&#x9F20;&#x6807; ESC (&#x5353;) &#x4E0A;&#x7535;&#x7EBA;&#x7EB3;&#x7C73;&#x7EA4;&#x7EF4;&#x7531; polymethylglutarimide (PMGI)&#xFF0C;&#x8FD9;&#x662F;&#x4E00;&#x79CD;&#x5408;&#x6210;&#x7684;&#x70ED;&#x5851;&#x6027;&#x805A;&#x5408;&#x7269;&#x7A33;&#x5B9A;&#x57F9;&#x517B;&#x6761;&#x4EF6;&#x4E0B;&#x57F9;&#x517B;&#x3002;&#x62D3;&#x6251;&#x7ED3;&#x6784;&#x548C; PMGI &#x7EB3;&#x7C73;&#x7EA4;&#x7EF4;&#x5BC6;&#x5EA6;&#x662F;&#x5173;&#x952E;&#x56E0;&#x7D20;&#x3002;mESCs &#x5BF9;&#x7EB3;&#x7C73;&#x7EA4;&#x7EF4;&#x589E;&#x957F;&#x7387;&#x662F;&#x5AB2;&#x7F8E;&#x4F20;&#x7EDF;&#x57F9;&#x517B;&#x548C;&#x4FDD;&#x7559;&#x4ED6;&#x4EEC;&#x7684;&#x6F5C;&#x80FD;&#x3002;&#x6B64;&#x5916;&#xFF0C;self-renewed &#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x5206;&#x5316;&#x4E3A;&#x6240;&#x6709;&#x4E09;&#x4E2A;&#x80DA;&#x5C42;&#x4ECE;&#x800C;&#x63D0;&#x4F9B;&#x53EF;&#x9760;&#x7684;&#x65B9;&#x5F0F;&#x6269;&#x5927; mESCs &#x65E0;&#x8FDB;&#x7EB8;&#x5668;&#x7684;&#x5355;&#x5143;&#x683C;&#x3002;

&#x5316;&#x5B66;&#x5B9A;&#x4E49;&#x68DA;&#x67B6;&#x7528;&#x9759;&#x7535;&#x7EBA;&#x4E1D;&#x5408;&#x6210;&#x7EB3;&#x7C73;&#x7EA4;&#x7EF4;&#x4EE5;&#x7EF4;&#x6301;&#x5C0F;&#x9F20;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x57F9;&#x517B;&#x65E0;&#x8FDB;&#x7EB8;&#x5668;&#x7684;&#x6761;&#x4EF6;&#x4E0B;&#x521B;&#x5EFA;&#x7684;&#x3002;

Embryonic stem cells (ESCs) are useful resources for drug discovery, developmental biology and disease studies. Cellular microenvironmental cues play critical roles in regulating ESC functions, but it is challenging to control them with synthetic components. Nanofibers hold a potential to create artificial cellular cues for controlling cell adhesion and cell-cell interactions. Mouse ESC (mESC) were cultured on electrospun nanofibers made from polymethylglutarimide (PMGI), which is a synthetic thermoplastic polymer stable under culture conditions. Both topology and the density of PMGI nanofibers were key factors. mESCs on nanofibers had a growth rate comparable to those cultured conventionally and retained their pluripotency. Furthermore, self-renewed ESCs differentiated into all three germ layers thereby providing a reliable way to expand mESCs without feeder cells.

Chemically-defined scaffolds created with electrospun synthetic nanofibers to maintain mouse embryonic stem cell culture under feeder-free conditions.

Cellules souches embryonnaires (CSE) sont des ressources utiles pour la d&eacute;couverte, biologie du d&eacute;veloppement et des &eacute;tudes de la maladie. Des signaux cellulaires micro-environnementales jouent un r&ocirc;le crucial dans la r&eacute;gulation des fonctions de l&#39;ESC, mais il est difficile des pour combattre avec des composants synth&eacute;tiques. Nanofibres d&eacute;tiennent un potentiel de cr&eacute;ation artificielles signaux cellulaires pour contr&ocirc;ler les interactions entre les cellules adh&eacute;rence et cellules. Souris ESC (mESC) ont &eacute;t&eacute; cultiv&eacute;s sur des nanofibres &eacute;lectrofil&eacute;es issus de polymethylglutarimide (PMGI), qui est un polym&egrave;re synth&eacute;tique thermoplastique stables dans des conditions de culture. Topologie et la densit&eacute; des nanofibres PMGI &eacute;taient tous deux des facteurs cl&eacute;s. mESCs sur nanofibres avait un taux de croissance comparable &agrave; ceux cultiv&eacute;s de mani&egrave;re conventionnelle et conserv&eacute; leur pluripotence. En outre, ces self-renewed se diff&eacute;rencient en toutes les trois couches de germe offrant ainsi un moyen fiable pour &eacute;largir les mESCs sans cellules nourrici&egrave;res.

&Eacute;chafaudages chimiquement d&eacute;fini cr&eacute;&eacute;s avec &eacute;lectrofil&eacute;es synth&eacute;tique nanofibres pour maintenir la culture de cellules souches embryonnaires de souris en conditions exemptes d&#39;engraissement.

Embryonale Stammzellen (WSR) sind n&uuml;tzliche Ressourcen f&uuml;r Drogeentdeckung, Entwicklungsbiologie und Krankheit-Studien. Zellul&auml;re microenvironmental Signale spielen wichtige Rollen bei der Regulierung der ESC-Funktionen, aber es ist schwierig, um sie mit synthetischen Komponenten zu kontrollieren. Nanofasern halten eine Potenzial zur Schaffung von k&uuml;nstlichen zellul&auml;re Signale zur Steuerung der Zelle Adh&auml;sion und Zell-Zell-Interaktionen. Maus-ESC (mESC) wurden auf Electrospun Nanofasern hergestellt aus Polymethylglutarimide (PMGI), die ein synthetisches thermoplastisches Polymer ist stabil unter Kulturbedingungen kultiviert. Topologie und die Dichte der PMGI Nanofasern waren Schl&uuml;sselfaktoren. mESCs auf Nanofasern hatte eine Wachstumsrate, die vergleichbar mit denen konventionell kultiviert und behielten ihre Pluripotenz. Dar&uuml;ber hinaus differenziert self-renewed WSR in allen drei Keim-Schichten und bieten somit eine zuverl&auml;ssige M&ouml;glichkeit, mESCs ohne Zuf&uuml;hrung Zellen zu erweitern.

Chemisch definierter Ger&uuml;ste erstellt mit Electrospun synthetische Nanofasern, Maus embryonalen Stammzellen Kultur unter Zuf&uuml;hrung-freien Bedingungen beizubehalten.

Le cellule staminali embrionali (CES) sono risorse utili per la scoperta della droga, biologia dello sviluppo e gli studi della malattia. Cellulari spunti microenvironmental svolgono un ruolo critico nella regolazione funzioni ESC, ma &egrave; difficile controllarli con componenti sintetici. Nanofibre di tengono un potenziale per creare artificiali cellulari spunti per controllare le interazioni di adesione cellula-cellula e la cella. Mouse ESC (mescita) sono stati coltivati su nanofibre elettrofilate fatte da polymethylglutarimide (PMGI), che &egrave; un polimero sintetico termoplastico stabile in condizioni di coltura. Topologia sia la densit&agrave; di nanofibre PMGI sono stati fattori chiave. mESCs su nanofibre aveva un tasso di crescita paragonabile a quelli coltivati convenzionalmente e mantenuto la loro pluripotenza. Inoltre, CES self-renewed differenziato in tutte le tre strati germinali fornendo un modo affidabile per espandere mESCs senza cellule di alimentatore.

Ponteggi chimicamente definito creati con elettrofilate nanofibre sintetici per mantenere la coltura delle cellule staminali embrionali del mouse condizioni privo di alimentatore.

&#x80DA;&#x6027;&#x5E79;&#x7D30;&#x80DE; &#xFF08;Esc&#xFF09; &#x5275;&#x3001;&#x767A;&#x751F;&#x751F;&#x7269;&#x5B66;&#x3068;&#x75BE;&#x60A3;&#x7814;&#x7A76;&#x306E;&#x305F;&#x3081;&#x306E;&#x6709;&#x7528;&#x306A;&#x30EA;&#x30BD;&#x30FC;&#x30B9;&#x3067;&#x3059;&#x3002;Esc &#x30AD;&#x30FC;&#x306E;&#x6A5F;&#x80FD;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x7D30;&#x80DE;&#x30A2;&#x30A4;&#x30BD;&#x30EC;&#x30FC;&#x30BF;&#x30B1;&#x30FC;&#x30B8; &#x30AD;&#x30E5;&#x30FC;&#x3092;&#x518D;&#x751F;&#x304C;&#x305D;&#x308C;&#x3089;&#x3092;&#x5408;&#x6210;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x3068;&#x5236;&#x5FA1;&#x306B;&#x6311;&#x6226;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x30CA;&#x30CE;&#x30D5;&#x30A1;&#x30A4;&#x30D0;&#x30FC;&#x7D30;&#x80DE;&#x63A5;&#x7740;&#x3068;&#x7D30;&#x80DE;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3092;&#x5236;&#x5FA1;&#x3059;&#x308B;&#x4EBA;&#x5DE5;&#x7D30;&#x80DE;&#x306E;&#x30AD;&#x30E5;&#x30FC;&#x306E;&#x4F5C;&#x6210;&#x306B;&#x79D8;&#x3081;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x30DE;&#x30A6;&#x30B9; esc &#x30AD;&#x30FC; (mESC) &#x5408;&#x6210;&#x71B1;&#x53EF;&#x5851;&#x6027;&#x30DD;&#x30EA;&#x30DE;&#x30FC;&#x3067;&#x3042;&#x308B; polymethylglutarimide (PMGI) &#x304B;&#x3089;&#x306F;&#x57F9;&#x990A;&#x6761;&#x4EF6;&#x4E0B;&#x3067;&#x5B89;&#x5B9A;&#x3057;&#x305F;&#x30A8;&#x30EC;&#x30AF;&#x30C8;&#x30ED;&#x30B9;&#x30D4;&#x30CB;&#x30F3;&#x30B0; &#x30CA;&#x30CE;&#x30D5;&#x30A1;&#x30A4;&#x30D0;&#x30FC;&#x3092;&#x57F9;&#x990A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30C8;&#x30DD;&#x30ED;&#x30B8;&#x3068;&#x5BC6;&#x5EA6; PMGI &#x30CA;&#x30CE;&#x30D5;&#x30A1;&#x30A4;&#x30D0;&#x30FC;&#x306E;&#x4E3B;&#x8981;&#x56E0;&#x3068;&#x306A;&#x308A;&#x307E;&#x3057;&#x305F;&#x3002;&#x30CA;&#x30CE;&#x30D5;&#x30A1;&#x30A4;&#x30D0;&#x30FC;&#x306E; mESCs &#x8005;&#x5F93;&#x6765;&#x306E;&#x57F9;&#x990A;&#x3068;&#x540C;&#x6210;&#x9577;&#x7387;&#x3092;&#x6301;&#x3063;&#x3066;&#x3044;&#x305F;&#x3057;&#x3001;&#x5F7C;&#x3089;&#x306E;&#x591A;&#x80FD;&#x6027;&#x3092;&#x4FDD;&#x6301;&#x3057;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x81EA;&#x5DF1;&#x66F4;&#x65B0;&#x578B; Esc &#x306F;&#x305D;&#x308C;&#x306B;&#x3088;&#x3063;&#x3066;&#x30D5;&#x30A3;&#x30FC;&#x30C0;&#x30FC;&#x7D30;&#x80DE;&#x306A;&#x3057; mESCs &#x3092;&#x62E1;&#x5927;&#x3059;&#x308B;&#x4FE1;&#x983C;&#x6027;&#x306E;&#x9AD8;&#x3044;&#x65B9;&#x6CD5;&#x3092;&#x63D0;&#x4F9B;&#x3059;&#x308B;&#x3059;&#x3079;&#x3066;&#x306E; 3 &#x3064;&#x80DA;&#x82BD;&#x5C64;&#x306B;&#x5206;&#x5316;&#x3057;&#x305F;.

&#x5316;&#x5B66;&#x7684;&#x306B;&#x5B9A;&#x7FA9;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x8DB3;&#x5834;&#x30A8;&#x30EC;&#x30AF;&#x30C8;&#x30ED;&#x30B9;&#x30D4;&#x30CB;&#x30F3;&#x30B0;&#x5408;&#x6210;&#x30CA;&#x30CE;&#x30D5;&#x30A1;&#x30A4;&#x30D0;&#x30FC;&#x3092;&#x7121;&#x6599;&#x306E;&#x30D5;&#x30A3;&#x30FC;&#x30C0;&#x30FC;&#x306E;&#x6761;&#x4EF6;&#x4E0B;&#x3067;&#x306E;&#x30DE;&#x30A6;&#x30B9;&#x80DA;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x57F9;&#x990A;&#x3092;&#x7DAD;&#x6301;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F5C;&#x6210;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;

&#xBC30;&#xC544; &#xC904;&#xAE30; &#xC138;&#xD3EC; (ESCs)&#xB294; &#xC758;&#xC57D;&#xD488;&#xC758; &#xBC1C;&#xACAC;, &#xBC1C;&#xB2EC; &#xC0DD;&#xBB3C;&#xD559; &#xBC0F; &#xC9C8;&#xBCD1; &#xC5F0;&#xAD6C;&#xB97C; &#xC704;&#xD55C; &#xC720;&#xC6A9;&#xD55C; &#xB9AC;&#xC18C;&#xC2A4;&#xC785;&#xB2C8;&#xB2E4;. &#xC140;&#xB8F0;&#xB7EC; microenvironmental &#xC2E0;&#xD638; ESC &#xAE30;&#xB2A5; &#xC870;&#xC808;&#xC5D0; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xC7AC;&#xC0DD; &#xD558;&#xC9C0;&#xB9CC; &#xD569;&#xC131; &#xAD6C;&#xC131; &#xC694;&#xC18C;&#xC640; &#xADF8;&#xB4E4;&#xC744; &#xD1B5;&#xC81C; &#xD558;&#xB294; &#xAC83;&#xC744; &#xB3C4;&#xC804;. Nanofibers &#xC138;&#xD3EC; &#xC811;&#xCC29; &#xBC0F; &#xC140; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC744; &#xC81C;&#xC5B4; &#xD558;&#xAE30; &#xC704;&#xD55C; &#xC778;&#xACF5; &#xC138;&#xD3EC; &#xC2E0;&#xD638;&#xB97C; &#xB9CC;&#xB4DC;&#xB294; &#xC7A0;&#xC7AC;&#xB825;&#xC744; &#xBCF4;&#xC720;. Esc &#xD0A4; &#xB9C8;&#xC6B0;&#xC2A4; (mESC) electrospun nanofibers &#xBB38;&#xD654; &#xC870;&#xAC74; &#xD558;&#xC5D0;&#xC11C; &#xC548;&#xC815; &#xB41C; &#xD569;&#xC131; &#xC5F4;&#xAC00; &#xC18C;&#xC131; &#xD3F4;&#xB9AC;&#xBA38; polymethylglutarimide (PMGI)&#xC5D0;&#xC11C; &#xB9CC;&#xB4E0;&#xC5D0; &#xACBD;&#xC791; &#xD588;&#xB2E4;. &#xD1A0;&#xD3F4;&#xB85C;&#xC9C0; &#xBC0F; PMGI nanofibers&#xC758; &#xBC00;&#xB3C4; &#xC8FC;&#xC694; &#xC694;&#xC778; &#xD588;&#xB2E4;. mESCs nanofibers&#xC5D0; &#xC804;&#xD1B5;&#xC801;&#xC73C;&#xB85C; &#xAD50;&#xC591; &#xADF8; &#xBE44;&#xAD50; &#xC131;&#xC7A5;&#xB960; &#xC84C;&#xACE0; &#xADF8;&#xB4E4;&#xC758; pluripotency &#xC720;&#xC9C0;. &#xB610;&#xD55C;, self-renewed ESCs &#xD53C;&#xB354; &#xC138;&#xD3EC; &#xC5C6;&#xC774; Mescs&#xB97C; &#xD655;&#xC7A5; &#xD558;&#xB294; &#xC2E0;&#xB8B0;&#xD560; &#xC218; &#xC788;&#xB294; &#xBC29;&#xBC95;&#xC744; &#xC81C;&#xACF5; &#xD558;&#xB294; &#xBAA8;&#xB4E0; &#xC138; &#xC138;&#xADE0; &#xCE35;&#xC73C;&#xB85C; &#xBD84;&#xD654;.

&#xAC74;&#xC124; &#xAE30;&#xACC4; &#xD654;&#xD559;&#xC801;&#xC73C;&#xB85C; &#xC815;&#xC758; &#xB41C; &#xC784;&#xB300; &#xAE09;&#xC9C0;&#xB300; &#xC5C6;&#xB294; &#xC870;&#xAC74; &#xD558;&#xC5D0;&#xC11C; &#xB9C8;&#xC6B0;&#xC2A4; &#xBBF8; &#xBC1C;&#xB2EC; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xBB38;&#xD654;&#xB97C; &#xC720;&#xC9C0; &#xD558;&#xAE30; &#xC704;&#xD574; &#xD569;&#xC131; nanofibers electrospun &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xB9CC;&#xB4E0;.

Las c&eacute;lulas madre embrionarias (CES) son recursos &uacute;tiles para el descubrimiento de f&aacute;rmacos, biolog&iacute;a del desarrollo y estudios de la enfermedad. Celulares microambiental se&ntilde;ales desempe&ntilde;an un papel cr&iacute;tico en la regulaci&oacute;n de las funciones de la ESC, pero es un reto para controlarlos con componentes sint&eacute;ticos. Nanofibras tienen un potencial para crear se&ntilde;ales celulares artificiales para el control de las interacciones de adherencia y c&eacute;lula c&eacute;lula. Rat&oacute;n ESC (mESC) fueron cultivadas en nanofibras de electrospun hecho de polymethylglutarimide (PMGI), que es un pol&iacute;mero termopl&aacute;stico sint&eacute;tico estable bajo condiciones de cultivo. Topolog&iacute;a tanto la densidad de nanofibras PMGI fueron factores clave. Troncales en nanofibras ten&iacute;an una tasa de crecimiento comparable a las cultivadas convencionalmente y conservan su pluripotencia. Adem&aacute;s, la CES self-renewed diferenciado en todos tres capas germinales, proporcionando as&iacute; una manera confiable de ampliar troncales sin fibroblastos.

Andamios qu&iacute;micamente definidos creados con nanofibras sint&eacute;tico electrospun para mantener la cultura de c&eacute;lulas madre embrionarias de rat&oacute;n en condiciones libres de alimentador.

Phenotypic and transcriptional modulation of human pluripotent stem cells induced by nano/microfabrication materials.

Besides conventional approaches for regulating in-coming molecules for gas storage, separation, or molecular sensing, the control of molecular release from the pores is a prerequisite for extending the range of their application, such as drug delivery. Herein, we report the fabrication of a new porous coordination polymer (PCP)-based composite consisting of a gold nanorod (GNR) used as an optical switch and PCP crystals for controlled molecular release using light irradiation as an external trigger. The delicate core-shell structures of this new platform, composed of an individual GNR core and an aluminum-based PCP shell, were achieved by the selective deposition of an aluminum precursor onto the surface of GNR followed by the replication of the precursor into aluminum-based PCPs. The mesoscopic structure was characterized by electron microscopy, energy dispersive X-ray elemental mapping, and sorption experiments. Combination at the nanoscale of the high storage capacity of PCPs with the photothermal properties of GNRs resulted in the implementation of unique motion-induced molecular release, triggered by the highly efficient conversion of optical energy into heat that occurs when the GNRs are irradiated into their plasmon band. Temporal control of the molecular release was demonstrated with anthracene as a guest molecule and fluorescent probe by means of fluorescence spectroscopy. 

Integration of porous coordination polymers and gold nanorods into core-shell mesoscopic composites toward light-induced molecular release.

Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells (hESCs and hiPSCs, respectively), continue to attract researchers and clinicians as well as patients worldwide because of their applicability in regenerative medicine, cell-based therapies, and drug discovery. However, conventional biomedical approaches for stem cell research provide only limited insights regarding how stem cells are regulated in our body and how we could control them. To accelerate the progress of stem cell research and their applications, interdisciplinary research needs to be carried out to enable access of or mimic in vivo physiological conditions and to investigate their functions. In the past few decades, progress in microfabrication engineering has had a tremendous impact in both academia and industry (electronics, mechanics, chemicals, and environments), and many people have realized that microfabricated tools hold great potential for applications in stem cell research because they allow precise manipulation of a wide range of biological materials, including small molecules, DNA/RNA, proteins, cells, and tissues. In this review, I describe advanced microfabricated tools for studying the mechanisms of in vivo stem cell regulation and precise manipulation of stem cells, notably hPSCs, and concerns of using such tools for stem cell research. 

Cutting-edge microfabricated biomedical tools for human pluripotent stem cell research.

Functional cellular substrates for localized cell stimulation by small molecules provide an opportunity to control and monitor cell signalling networks chemically in time and space. However, despite improvements in the controlled delivery of bioactive compounds, the precise localization of gaseous biomolecules at the single-cell level remains challenging. Here we target nitric oxide, a crucial signalling molecule with site-specific and concentration-dependent activities, and we report a synthetic strategy for developing spatiotemporally controllable nitric oxide-releasing platforms based on photoactive porous coordination polymers. By organizing molecules with poor reactivity into polymer structures, we observe increased photoreactivity and adjustable release using light irradiation. We embed photoactive polymer crystals in a biocompatible matrix and achieve precisely controlled nitric oxide delivery at the cellular level via localized two-photon laser activation. The biological relevance of the exogenous nitric oxide produced by this strategy is evidenced by an intracellular change in calcium concentration, mediated by nitric oxide-responsive plasma membrane channel proteins. 

Localized cell stimulation by nitric oxide using a photoactive porous coordination polymer platform.

Nanofibrous gelatin substrates are suited for long-term expansion of human pluripotent stem cells (hPSCs) under feeder- and serum-free culture conditions. A combinatorial library with different sets of processing parameters was established to assess the culture performance of hPSCs on nanofibrous substrates in terms of cell adhesion and growth rate, using Matrigel as control. Then, the optimal conditions were applied to long-term expansion of hPSCs with several cell lines, showing a maintained pluripotency over more than 20 passages without introducing any abnormal chromosome. In addition, this approach allowed us to avoid enzymatic disassociation and mechanic cutting during passages, thereby promoting a better hPSC culture and long-term expansion. 

Nanofibrous gelatin substrates for long-term expansion of human pluripotent stem cells.

Three-dimensional (3D) printing is advantageous over conventional technologies for the fabrication of sophisticated structures such as 3D micro-channels for future applications in tissue engineering and drug screening. We aimed to apply this technology to cell-based assays using polydimethylsiloxane (PDMS), the most commonly used material for fabrication of micro-channels used for cell culture experiments. Useful properties of PDMS include biocompatibility, gas permeability and transparency. We developed a simple and robust protocol to generate PDMS-based devices using a soft lithography mold produced by 3D printing. 3D chemical gradients were then generated to stimulate cells confined to a micro-channel. We demonstrate that concentration gradients of growth factors, important regulators of cell/tissue functions in vivo, influence the survival and growth of human embryonic stem cells. Thus, this approach for generation of 3D concentration gradients could have strong implications for tissue engineering and drug screening. 

3D printing of soft lithography mold for rapid production of polydimethylsiloxane-based microfluidic devices for cell stimulation with concentration gradients.

Noncontact manipulation of nano/micromaterials presents a great challenge in fields ranging from biotechnology to nanotechnology. In this study we developed a new strategy for the manipulation of molecules and cells based on diffusiophoresis driven by a concentration gradient of a polymer solute. By using laser focusing in a microfluidic device, we created a sharp concentration gradient of poly(ethylene glycol) (PEG) in a solution of this polymer. Because diffusiophoresis essentially depends on solute gradients alone, PEG solute contrast resulted in trapping of DNA and eukaryotic cells with little material dependence. Furthermore, quantitative analysis revealed that the motility of migrating cells was enhanced with the PEG concentration, consistent with a theoretical model of boosted cell migration. Our results support that a solute contrast of polymer can exert an interfacial force gradient that physically propels objects and may have application for the manipulation of soft materials. 

Directing and Boosting of Cell Migration by the Entropic Force Gradient in Polymer Solution.

A microfluidic human pluripotent stem cell (hPSC) array has been developed for robust and reproducible hPSC culture methods to assess chemically defined serum- and feeder-free culture conditions. This microfluidic platform, combined with image cytometry, enables the systematic analysis of multiple simultaneously detected marker expression in individual cells, for screening of various chemically defined media across hPSC lines, and the study of phenotypic responses. 

Microfluidic Image Cytometry for Single-Cell Phenotyping of Human Pluripotent Stem Cells.

Human pluripotent stem cells hold great promise for applications in drug discovery and regenerative medicine. Microfluidic technology is a promising approach for creating artificial microenvironments; however, although a proper 3D microenvironment is required to achieve robust control of cellular phenotypes, most current microfluidic devices provide only 2D cell culture and do not allow tuning of physical and chemical environmental cues simultaneously. Here, the authors report a 3D cellular microenvironment plate (3D-CEP), which consists of a microfluidic device filled with thermoresponsive poly(N-isopropylacrylamide)-β-poly(ethylene glycol) hydrogel (HG), which enables systematic tuning of both chemical and physical environmental cues as well as in situ cell monitoring. The authors show that H9 human embryonic stem cells (hESCs) and 253G1 human induced pluripotent stem cells in the HG/3D-CEP system maintain their pluripotent marker expression under HG/3D-CEP self-renewing conditions. Additionally, global gene expression analyses are used to elucidate small variations among different test environments. Interestingly, the authors find that treatment of H9 hESCs under HG/3D-CEP self-renewing conditions results in initiation of entry into the neural differentiation process by induction of PAX3 and OTX1 expression. The authors believe that this HG/3D-CEP system will serve as a versatile platform for developing targeted functional cell lines and facilitate advances in drug screening and regenerative medicine.

Characterization of Phenotypic and Transcriptional Differences in Human Pluripotent Stem Cells under 2D and 3D Culture Conditions.

Human pluripotent stem cells (hPSCs) hold great potential for industrial and clinical applications. Clinical-grade scaffolds and high-quality hPSCs are required for cell expansion as well as easy handling and manipulation of the products. Current hPSC culture methods do not fulfill these requirements because of a lack of proper extracellular matrices (ECMs) and cell culture wares. We developed a layered nano-on-micro fibrous cellular matrix mimicking ECM, named "fiber-on-fiber (FF)" matrix, which enables easy handling and manipulation of cultured cells. While non-woven sheets of cellulose and polyglycolic acid were used as a microfiber layer facilitating mechanical stability, electrospun gelatin nanofibers were crosslinked on the microfiber layer, generating a mesh structure with connected nanofibers facilitating cell adhesion and growth. Our results showed that the FF matrix supports effective hPSC culture with maintenance of their pluripotency and normal chromosomes over two months, as well as effective scaled-up expansion, with fold increases of 54.1 ± 15.6 and 40.4 ± 8.4 in cell number per week for H1 human embryonic stem cells and 253G1 human induced pluripotent stem cells, respectively. This simple approach to mimick the ECM may have important implications after further optimization to generate lineage-specific products.

Nano-on-micro fibrous extracellular matrices for scalable expansion of human ES/iPS cells.

Cellular microenvironments are generally sophisticated, but crucial for regulating the functions of human pluripotent stem cells (hPSCs). Despite tremendous effort in this field, the correlation between the environmental factors-especially the extracellular matrix and soluble cell factors-and the desired cellular functions remains largely unknown because of the lack of appropriate tools to recapitulate in vivo conditions and/or simultaneously evaluate the interplay of different environment factors. Here, a combinatorial platform is developed with integrated microfluidic channels and nanofibers, associated with a method of high-content single-cell analysis, to study the effects of environmental factors on stem cell phenotype. Particular attention is paid to the dependence of hPSC short-term self-renewal on the density and composition of extracellular matrices and initial cell seeding densities. Thus, this combinatorial approach provides insights into the underlying chemical and physical mechanisms that govern stem cell fate decisions.

Microfluidic-Nanofiber Hybrid Array for Screening of Cellular Microenvironments.

In mammalian evolutionary history, Cetacea (whales, dolphins and porpoises) achieved astonishing success by adapting to an aquatic environment. One unique characteristic of cetaceans, contributing to this adaptive success, is efficient lipid utilization. Here, we report a comparative genetic analysis of five aquatic and five terrestrial Cetartiodactyla species using 144 genes associated with lipid metabolism. Mutation ratio (d /d ), amino acid substitution in functional domains and metabolic pathways were evaluated using branch-site model in PAML, Pfam and KEGG, respectively. Our tests detected 20 positively selected genes in Cetacea compared to 11 in Bovidae with little overlap between the lineages. We identified lineage-specific patterns of amino acid substitutions and functional domains that were mutually exclusive between cetaceans and bovids, supporting divergent evolution of lipid metabolism since the divergence of these taxa from a common ancestor. Moreover, a pathway analysis showed that the identified genes in cetaceans were associated with lipid digestion, lipid storage and energy-producing pathways. This study emphasizes the evolutionary context of lipid metabolism modification of cetaceans and provides a foundation for future studies of elucidating the adapted biological mechanisms of cetacean lipid metabolism and a framework for incorporating ecological context into studies aimed at investigating adaptive evolution.

Article	Year
Microfluidic image cytometry. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 21104065	2011
Delivery of intact transcription factor by using self-assembled supramolecular nanoparticles. Angewandte Chemie (International ed. in English)\| PubMed ID: 21370360	2011
Chemically-defined scaffolds created with electrospun synthetic nanofibers to maintain mouse embryonic stem cell culture under feeder-free conditions. Biotechnology letters\| PubMed ID: 22714273	2012
Phenotypic and transcriptional modulation of human pluripotent stem cells induced by nano/microfabrication materials. Advanced healthcare materials\| PubMed ID: 23184815	2013
Integration of porous coordination polymers and gold nanorods into core-shell mesoscopic composites toward light-induced molecular release. Journal of the American Chemical Society\| PubMed ID: 23672307	2013
Cutting-edge microfabricated biomedical tools for human pluripotent stem cell research. Journal of laboratory automation\| PubMed ID: 23850864	2013
Localized cell stimulation by nitric oxide using a photoactive porous coordination polymer platform. Nature communications\| PubMed ID: 24158008	2013
Nanofibrous gelatin substrates for long-term expansion of human pluripotent stem cells. Biomaterials\| PubMed ID: 24811263	2014
3D printing of soft lithography mold for rapid production of polydimethylsiloxane-based microfluidic devices for cell stimulation with concentration gradients. Biomedical microdevices\| PubMed ID: 25686903	2015
Directing and Boosting of Cell Migration by the Entropic Force Gradient in Polymer Solution. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 26496637	2015
Microfluidic Image Cytometry for Single-Cell Phenotyping of Human Pluripotent Stem Cells. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 26542717	2015
Characterization of Phenotypic and Transcriptional Differences in Human Pluripotent Stem Cells under 2D and 3D Culture Conditions. Advanced healthcare materials\| PubMed ID: 27775225	2016
Nano-on-micro fibrous extracellular matrices for scalable expansion of human ES/iPS cells. Biomaterials\| PubMed ID: 28187394	2017
Microfluidic-Nanofiber Hybrid Array for Screening of Cellular Microenvironments. Small (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 28272774	2017
Genetic signatures of lipid metabolism evolution in Cetacea since the divergence from terrestrial ancestor. Journal of evolutionary biology\| PubMed ID: 30074670	2018

Ken-ichiro Kamei

Biography

JoVE Journal Publications

Other Publications