Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

University of Wisconsin-Madison

An enantioselective, convergent, total synthesis of the antiviral marine natural product (-)-hennoxazole A has been completed in 17 steps, longest linear sequence, from serine methyl ester and in 9 steps from an achiral bisoxazole intermediate. Elaboration of a thiazolidinethione allowed for rapid assembly of the pyran-based ring system. Key late-stage coupling was effected by deprotonation of the bisoxazole methyl group, followed by alkylation with an allylic bromide side chain segment. [structure: see text]

Total synthesis of (-)-hennoxazole A.

Un &eacute;nantios&eacute;lective, synth&egrave;se convergente, totale du produit naturel marin antiviral (-)-hennoxazole A &eacute;t&eacute; termin&eacute;e en 17 &eacute;tapes, plus longue s&eacute;quence lin&eacute;aire, de l&#39;ester m&eacute;thylique de s&eacute;rine et en 9 &eacute;tapes d&#39;un bisoxazole achiraux interm&eacute;diaire. Elaboration d&#39;un thiazolidinethione autoris&eacute; pour un assemblage rapide du syst&egrave;me cyclique pyran-bas&eacute;. Principaux stades couplage s&#39;effectue par d&eacute;protonation du groupe m&eacute;thyle bisoxazole, suivi par alkylation avec un segment de cha&icirc;ne lat&eacute;rale bromure allylique. [structure : voir texte]

Total de synth&egrave;se de (-)-hennoxazole A.

Un enantioselettiva, sintesi convergente, totale del prodotto marino naturale antivirale (-)-hennoxazole A &egrave; stato completato in 17 passaggi pi&ugrave; lunga sequenza lineare, da Metilestere di Serina e in 9 passi da un bisoxazole achirali intermedi. Elaborazione di un permesso per il montaggio rapido del sistema base di Pirano anello di thiazolidinethione. Chiave accoppiamento di fase tardiva &egrave; stata effettuata dalla deprotonazione del gruppo metile bisoxazole, seguito da alchilazione con un segmento di catena laterale di bromuro allilici. [struttura: vedi testo]

Totale sintesi di (-)-hennoxazole A.

An enantioselective, convergent total synthesis of the antiviral marine natural product (-)-hennoxazole A is completed in 14 steps (longest linear sequence) from commercially available 4-methyloxazole-2-carboxylic acid. Synthesis of the C(1)-C(15) pyran/bisoxazole fragment takes advantage of an aldol-like coupling between a dimethyl acetal and an N-acetylthiazolidinethione for the direct, stereoselective installation of the C(8)-methoxy-bearing stereocenter. A one-pot acetoacetate acylation/decarboxylation/cyclodehydration of another elaborate thiazolidinethione allows for rapid assembly of the pyran-based ring system. Synthesis of the C(15)-C(25) skipped triene side chain fragment makes use of a [2,3]-Wittig-Still rearrangement for efficient installation of the trisubstituted Z-double bond. Key late-stage coupling of the two fragments is effected by deprotonation of the methyl group on the bisoxazole system using lithium diethylamide, followed by alkylation with an allylic bromide side chain segment to form the C(15)-C(16) bond.

Un &eacute;nantios&eacute;lective, synth&egrave;se totale convergente de l&#39;antiviral produit naturel marin (-)-hennoxazole A termin&eacute; en 14 &eacute;tapes (plus longue s&eacute;quence lin&eacute;aire) de l&#39;acide 4-methyloxazole-2-carboxylique disponible dans le commerce. Synth&egrave;se du fragment pyran/bisoxazole C(1)-C(15) tire parti d&#39;un couplage de type aldolique entre un dim&eacute;thylac&eacute;tal et un N-acetylthiazolidinethione pour le direct, installation st&eacute;r&eacute;os&eacute;lective de la 8-m&eacute;thoxy-roulement de C Centre st&eacute;r&eacute;og&egrave;ne. Une ac&eacute;toac&eacute;tate monotope acylation/d&eacute;carboxylation/cyclod&eacute;shydratation d&#39;un autre thiazolidinethione &eacute;labor&eacute;e permet un assemblage rapide du syst&egrave;me cyclique pyran-bas&eacute;. Synth&egrave;se de la C(15)-C(25) saut&eacute; tri&egrave;ne fragment de cha&icirc;ne lat&eacute;rale utilise un r&eacute;arrangement encore de [2,3] - Wittig - pour une installation efficace de la liaison trisubstitu&eacute;s Z-double. Principaux stades couplage des deux fragments est effectu&eacute; par d&eacute;protonation du groupe m&eacute;thyle sur le syst&egrave;me de bisoxazole &agrave; l&#39;aide de lithium lysergique, suivie par alkylation avec un segment de cha&icirc;ne bromure allylique c&ocirc;t&eacute; pour former la liaison C(15)-C(16).

Un enantioselettiva, convergente sintesi totale del prodotto marino naturale antivirale (-)-hennoxazole A &egrave; completato in 14 passi (pi&ugrave; lunga sequenza lineare) da acido 4-methyloxazole-2-carbossilico commercialmente disponibile. Sintesi del frammento di Pirano/bisoxazole C(1)-C(15) si avvale di un accoppiamento dell&#39;aldolo-come tra un dimetilacetale e una N-acetylthiazolidinethione per la diretta, installazione stereoselettive dei C 8-metossi-cuscinetto stereocentro. Un one-pot acetoacetato acilazione/decarbossilazione/cyclodehydration di thiazolidinethione un altro elaborato permette di assemblaggio rapido del sistema anello base di Pirano. Sintesi della C(15)-C(25) saltato triene frammento di catena laterale fa uso di un riarrangiamento di ancora - Wittig-[2,3] per installazione efficiente del trisubstituted Z-doppio legame. Chiave ritardo-fase accoppiamento dei due frammenti viene effettuata da deprotonazione del gruppo metile sul sistema bisoxazole utilizzando litio diethylamide, seguita da alchilazione con un segmento di catena laterale allilici bromuro per formare il legame C(15)-C(16).

Water-in-oil microdroplets in microfluidics are well-defined individual picoliter reaction compartments and, as such, have great potential for quantitative high-throughput biological screening. This, however, depends upon contents of the droplets not leaking out into the oil phase. To assess the mechanism of possible leaking, the retention of various fluorescein derivatives from droplets formed in mineral oil and stored for hours in a reservoir on chip was studied. Leaking into the oil phase was observed and was shown to be dependent on the nature of the compounds and on the concentration of the silicone-based polymeric surfactant Abil EM 90 used. In experiments in which droplets filled with fluorescein were mixed with droplets filled with only buffer, the rate of efflux from filled droplets to empty droplets was dependent on the number of neighboring droplets of different composition. Buffer droplets with five fluorescein-containing neighbors took up the fluorophore 4.5 times faster than buffer droplets without fluorescein neighbors. The addition of bovine serum albumin (BSA) substantially reduced leaking. A formulation with 5% BSA reduces leaking of the fluorophore from 45% to 3%. Inclusion of BSA enabled experiments to be carried out over periods up to 18 h, without substantial leaking (<5%). We demonstrate the utility of this additive by following the enzymatic activity of alkaline phosphatase expressed by Escherichia coli cells. The ability to reliably compartmentalize genotype (cell) and phenotype (reaction product) is the basis for using microdroplets in directed evolution studies, and the approaches described herein provide a test system for assessing emulsion formulations for such purposes.

Controlling the retention of small molecules in emulsion microdroplets for use in cell-based assays.

Using microfluidic techniques and a novel fluorous-tagged palladium catalyst, we generated droplet reactors with catalytically active walls and used these compartments for small molecule synthesis.

Suzuki-Miyaura coupling reactions in aqueous microdroplets with catalytically active fluorous interfaces.

&Agrave; l&#39;aide de techniques microfluidique et un catalyseur au palladium de fluor&eacute;-le tag roman, nous g&eacute;n&eacute;r&eacute; des r&eacute;acteurs de gouttelettes avec murs catalytiquement actives et utilis&eacute; ces compartiments pour la synth&egrave;se de petites mol&eacute;cules.

R&eacute;actions de couplage de Suzuki-Miyaura dans aqueux gouttelettes avec interfaces fluor&eacute;s catalytiquement actives.

Utilizzando tecniche di microfluidica e un catalizzatore al Palladio fluorous tagged romanzo, abbiamo generato reattori goccia con pareti cataliticamente attivi e usate questi comparti per la sintesi di piccole molecole.

Reazioni di accoppiamento di Suzuki-Miyaura in microgocce acquosa con interfacce fluorous cataliticamente attive.

There has been an increasing drive toward miniaturizing and accelerating experiments with droplet-based microfluidics across the chemical disciplines. Current applications take advantage of the numerous techniques for manipulating nano- to femtoliter droplets within microfluidic devices. To expand the range of possible applications, we have developed a method for compartmentalizing pure compounds within droplets, at a gradient of concentrations, starting from chemical mixtures. In this technique, a mixture is injected into an ultra performance liquid chromatography (UPLC) system, and droplets are generated from the LC output at a frequency high enough to fraction each compound into approximately 10(5) droplets, compartmentalizing pure compounds into a sequence of droplets with a range of concentrations spanning 2-3 orders of magnitude. Here we used fluorescent dyes to quantify the concentration profile of the droplet collections, and to demonstrate the correspondence between the concentration profile of the droplets and the compound elution profile monitored with a UV absorbance detector, allowing the use of compounds that are not fluorescently labeled but show UV absorbance. Hence this technique is applicable to a wide variety of applications that require both compound purity and the ability to probe a variety of concentrations, such as drug screening and titrations.

Generation of picoliter droplets with defined contents and concentration gradients from the separation of chemical mixtures.

Il y a eu un lecteur de plus en plus vers la miniaturisation et acc&eacute;l&eacute;rant les exp&eacute;riences avec microfluidics ax&eacute;e sur les gouttelettes dans les disciplines chimiques. Les applications actuelles de profiter des nombreuses techniques de manipulation de nano-femtoliter gouttelettes au sein de dispositifs microfluidiques. Pour &eacute;largir la gamme des applications possibles, nous avons d&eacute;velopp&eacute; une m&eacute;thode pour compartimenter les compos&eacute;s purs dans les gouttelettes, &agrave; un gradient de concentrations, &agrave; partir de m&eacute;langes chimiques. Dans cette technique, un m&eacute;lange est inject&eacute; dans un syst&egrave;me de chromatographie en phase liquide (UPLC) ultra performant, et les gouttelettes sont g&eacute;n&eacute;r&eacute;s &agrave; partir de la LC sortie &agrave; une fr&eacute;quence assez &eacute;lev&eacute;e pour la fraction chaque compos&eacute; dans environ 10 5 gouttes, cloisonnant les compos&eacute;s purs en une s&eacute;quence de gouttelettes avec une gamme de concentrations allant de 2 &agrave; 3 ordres de grandeur. Ici nous avons utilis&eacute; des colorants fluorescents pour quantifier le profil de concentration des collections gouttelette et d&eacute;montrer la correspondance entre le profil de concentration des gouttelettes et le profil d&#39;&eacute;lution compos&eacute; surveill&eacute; avec un d&eacute;tecteur d&#39;absorbance UV, permettant l&#39;utilisation de compos&eacute;s qui ne sont pas &eacute;tiquet&eacute;s fluorescent mais montrer l&#39;absorbance UV. Par cons&eacute;quent, cette technique est applicable &agrave; un large &eacute;ventail d&#39;applications n&eacute;cessitant une puret&eacute; compos&eacute;e tant de la capacit&eacute; de sonder une vari&eacute;t&eacute; de concentrations, telles que le d&eacute;pistage des drogues et de titrages.

G&eacute;n&eacute;ration de gouttelettes picolitres avec contenu d&eacute;fini et des gradients de concentration de la s&eacute;paration des m&eacute;langes chimiques.

C&#39;&egrave; stato una crescente in auto verso la miniaturizzazione e accelerando esperimenti con microfluidics gocciolina-base attraverso le discipline chimiche. Applicazioni correnti di approfittare delle numerose tecniche per la manipolazione di nano-a femtoliter goccioline all&#39;interno di dispositivi microfluidici. Per espandere la gamma di possibili applicazioni, abbiamo sviluppato un metodo per la divisione in compartimenti puri composti all&#39;interno di goccioline, a un gradiente di concentrazione, a partire da miscele chimiche. In questa tecnica, una miscela &egrave; iniettata in un sistema di ultra performance liquid chromatography (UPLC) e le goccioline sono generate da LC uscita ad una frequenza sufficientemente elevata per frazione ogni composto in circa 5 gocce, divisione in compartimenti puri composti in una sequenza di goccioline con una gamma di concentrazioni che attraversa 2-3 ordini di grandezza. Qui abbiamo usato tinture fluorescenti per quantificare il profilo di concentrazione delle collezioni gocciolina e a dimostrare la corrispondenza tra il profilo di concentrazione delle gocce e il profilo di eluizione composti monitorati con un rivelatore di assorbanza UV, permettendo l&#39;uso di composti che non sono etichettati fluorescente, ma mostrano assorbimento UV. Quindi questa tecnica &egrave; applicabile a una vasta gamma di applicazioni che richiedono sia composto purezza e la capacit&agrave; di sondare una serie di concentrazioni, quali lo screening di stupefacenti e titolazioni.

Generazione di goccioline picolitri con contenuti definiti e gradienti di concentrazione dalla separazione di miscele chimiche.

Microdroplets in microfluidics offer a great number of opportunities in chemical and biological research. They provide a compartment in which species or reactions can be isolated, they are monodisperse and therefore suitable for quantitative studies, they offer the possibility to work with extremely small volumes, single cells, or single molecules, and are suitable for high-throughput experiments. The aim of this Review is to show the importance of these features in enabling new experiments in biology and chemistry. The recent advances in device fabrication are highlighted as are the remaining technological challenges. Examples are presented to show how compartmentalization, monodispersity, single-molecule sensitivity, and high throughput have been exploited in experiments that would have been extremely difficult outside the microfluidics platform.

Microdroplets in microfluidics: an evolving platform for discoveries in chemistry and biology.

Gouttelettes en microfluidique offrent un grand nombre d&#39;opportunit&eacute;s dans la recherche chimique et biologique. Ils fournissent un compartiment dans lequel les esp&egrave;ces ou les r&eacute;actions peuvent &ecirc;tre isol&eacute;es, ils sont monodisperses et donc adapt&eacute; &agrave; des &eacute;tudes quantitatives, ils offrent la possibilit&eacute; de travailler avec tr&egrave;s petits volumes, cellules individuelles ou des mol&eacute;cules simples et conviennent pour des exp&eacute;riences de haut d&eacute;bit. L&#39;objectif de cette revue est de montrer l&#39;importance de ces fonctionnalit&eacute;s en permettant de nouvelles exp&eacute;riences en biologie et en chimie. Les progr&egrave;s r&eacute;cents dans la fabrication de dispositifs sont mis en &eacute;vidence comme le sont les autres d&eacute;fis technologiques. Des exemples sont pr&eacute;sent&eacute;s pour montrer comment compartimentation monodispersion, sensibilit&eacute; de mol&eacute;cules simples et &agrave; haut d&eacute;bit ont &eacute;t&eacute; exploit&eacute;s dans des exp&eacute;riences qui auraient &eacute;t&eacute; extr&ecirc;mement difficiles &agrave; l&#39;ext&eacute;rieur de la plate-forme de la microfluidique.

Gouttelettes en microfluidique : une plateforme en constante &eacute;volution pour les d&eacute;couvertes en chimie et en biologie.

Microgocce di microfluidics offre un gran numero di opportunit&agrave; nella ricerca chimica e biologica. Essi forniscono un vano in cui specie o reazioni possono essere isolati, sono monodisperse e quindi adatti per studi quantitativi, offrono la possibilit&agrave; di lavorare con volumi estremamente piccoli, le cellule singole o singole molecole e sono adatti per gli esperimenti ad alta velocit&agrave;. Lo scopo di questa recensione &egrave; per mostrare l&#39;importanza di queste funzionalit&agrave; di attivazione di nuovi esperimenti di biologia e chimica. I recenti progressi nella fabbricazione del dispositivo sono evidenziati come sono le restanti sfide tecnologiche. Vengono presentati esempi per mostrare come la compartimentazione, monodispersity, sensibilit&agrave; singola molecola e ad alto rendimento sono state sfruttate in esperimenti che sarebbero stato estremamente difficili di fuori della piattaforma microfluidica.

Microgocce di microfluidica: una piattaforma in continua evoluzione per le scoperte nella chimica e biologia.

This paper presents a droplet-based microfluidic platform for miniaturized combinatorial synthesis. As a proof of concept, a library of small molecules for early stage drug screening was produced. We present an efficient strategy for producing a 7 Ã— 3 library of potential thrombin inhibitors that can be utilized for other combinatorial synthesis applications. Picolitre droplets containing the first type of reagent (reagents A(1), A(2), â€¦, A(m)) were formed individually in identical microfluidic chips and then stored off chip with the aid of stabilizing surfactants. These droplets were then mixed to form a library of droplets containing reagents A(1-m), each individually compartmentalized, which was reinjected into a second microfluidic chip and combinatorially fused with picolitre droplets containing the second reagent (reagents B(1), B(2), â€¦, B(n)) that were formed on chip. The concept was demonstrated with a three-component Ugi-type reaction involving an amine (reagents A(1-3)), an aldehyde (reagents B(1-7)), and an isocyanide (held constant), to synthesize a library of small molecules with potential thrombin inhibitory activity. Our technique produced 10(6) droplets of each reaction at a rate of 2.3 kHz. Each droplet had a reaction volume of 3.1 pL, at least six orders of magnitude lower than conventional techniques. The droplets can then be divided into aliquots for different downstream screening applications. In addition to medicinal chemistry applications, this combinatorial droplet-based approach holds great potential for other applications that involve sampling large areas of chemical parameter space with minimal reagent consumption; such an approach could be beneficial when optimizing reaction conditions or performing combinatorial reactions aimed at producing novel materials.

Microfluidic platform for combinatorial synthesis in picolitre droplets.

Cet article pr&eacute;sente une plateforme ax&eacute;e sur la gouttelette microfluidiques pour synth&egrave;se combinatoire miniaturis&eacute;e. Comme une preuve de concept, une biblioth&egrave;que de petites mol&eacute;cules pour le criblage de drogue de stade pr&eacute;coce a &eacute;t&eacute; produite. Nous pr&eacute;sentons une strat&eacute;gie efficace pour la production d&#39;une biblioth&egrave;que de 7 &times; 3 des inhibiteurs potentiels de la thrombine qui peuvent &ecirc;tre utilis&eacute;s pour d&#39;autres applications de synth&egrave;se combinatoire. Picolitre gouttelettes contenant le premier type de r&eacute;actif (r&eacute;actifs a.1, a.2,..., A(m)) ont &eacute;t&eacute; form&eacute; individuellement en puces microfluidiques identiques et ensuite stock&eacute; au large de la puce &agrave; l&#39;aide d&#39;agents tensio-actifs de stabilisation. Ces gouttelettes ont &eacute;t&eacute; ensuite m&eacute;lang&eacute;s pour former une biblioth&egrave;que des gouttelettes contenant des r&eacute;actifs A(1-m), chacun individuellement compartiment&eacute;, qui a &eacute;t&eacute; r&eacute;inject&eacute;e dans une deuxi&egrave;me puce microfluidique et combinatoirement fusionn&eacute;e avec picolitre gouttelettes contenant le deuxi&egrave;me r&eacute;actif (r&eacute;actifs b (1), b (2),..., B(n)) qui se sont form&eacute;es sur la puce. Le concept a &eacute;t&eacute; d&eacute;montr&eacute; avec une r&eacute;action de type Ugi trois composantes impliquant une amine (r&eacute;actifs A(1-3)), un ald&eacute;hyde (r&eacute;actifs B(1-7)) et un isocyanure (tenue constante), pour synth&eacute;tiser une biblioth&egrave;que de petites mol&eacute;cules avec potentiel activit&eacute; inhibitrice de la thrombine. Notre technique a produit des gouttelettes de 10 (6) de chaque r&eacute;action &agrave; un taux de 2,3 kHz. Chaque gouttelette avait un volume r&eacute;actionnel de 3.1 pL, au moins six ordres de grandeur plus faible que les techniques conventionnelles. Les gouttelettes peuvent &ecirc;tre divis&eacute;s en parties aliquotes pour diff&eacute;rents choix en aval des demandes. En plus des applications de la chimie m&eacute;dicinale, cette approche combinatoire goutte a un grand potentiel pour d&#39;autres applications qui impliquent l&#39;&eacute;chantillonnage de vastes zones de l&#39;espace de param&egrave;tre chimique avec une consommation minimale de r&eacute;actif ; une telle approche pourrait &ecirc;tre b&eacute;n&eacute;fique lors de l&#39;optimisation des conditions de r&eacute;action ou d&#39;effectuer des r&eacute;actions combinatoires visant &agrave; produire de nouveaux mat&eacute;riaux.

Plate-forme microfluidique pour synth&egrave;se combinatoire en gouttelettes picolitres.

Questo libro presenta una piattaforma basata su goccia microfluidica per sintesi combinatoria miniaturizzato. Come un proof of concept, una biblioteca di piccole molecole per lo screening di stupefacenti fase precoce &egrave; stata prodotta. Vi presentiamo una strategia efficace per produrre una libreria 7 &times; 3 di potenziali inibitori della trombina che pu&ograve; essere utilizzata per altre applicazioni di sintesi combinatoria. Picolitre goccioline contenenti il primo tipo di reagente (reagenti a (1), a (2),..., A(m)) erano formata individualmente nel chip microfluidici identici e poi memorizzati off chip con l&#39;aiuto di stabilizzare i tensioattivi. Queste goccioline sono stati poi mescolate per formare una libreria di goccioline contenenti reagenti A(1-m), ciascuno individualmente compartimentato, che &egrave; stata reinserita in un secondo chip microfluidici e combinatoriamente si fuse con la picolitre goccioline contenenti il secondo reattivo (reagenti B(1), precisati,..., B(n)) che si sono formate sul chip. Il concetto &egrave; stato dimostrato con una reazione di Ugi-tipo di tre componenti che comportano un&#39;ammina (reagenti A(1-3)), un&#39;aldeide (reagenti B(1-7)) e un isocyanide (tenuto costante), per sintetizzare una libreria di piccole molecole con attivit&agrave; inibitoria di potenziale trombina. La nostra tecnica ha prodotto 10 gocce di ogni reazione ad un tasso di 2,3 kHz. Ogni goccia aveva un volume di reazione di 3.1 pL, almeno sei ordini di grandezza inferiori rispetto alle tecniche convenzionali. Le goccioline possono poi essere suddivisi in aliquote per lo screening a valle diverse applicazioni. Oltre alle applicazioni di chimica farmaceutica, questo approccio basato su goccia combinatorio detiene un potenziale grande per altre applicazioni che coinvolgono il campionamento grandi aree dello spazio di parametro chimico con consumo di reagente minima; tale approccio potrebbe essere utile quando l&#39;ottimizzazione delle condizioni di reazione o eseguire reazioni combinatoriale finalizzato alla produzione di nuovi materiali.

Piattaforma microfluidica per sintesi combinatoria in goccioline picolitre.

Investigation of "on water" conditions using a biphasic fluidic platform.

Enqu&ecirc;te &laquo; sur l&#39;eau &raquo; des conditions &agrave; l&#39;aide d&#39;une plate-forme fluidique biphasique.

Inchiesta di &quot;sull&#39;acqua&quot; condizioni utilizzando una piattaforma fluidici bifasico.

Microfluidics is emerging as a promising platform for cell culture, enabling increased microenvironment control and potential for integrated analysis compared to conventional macroculture systems such as well plates and Petri dishes. To advance the use of microfluidic devices for cell culture, it is necessary to better understand how miniaturization affects cell behavior. In particular, microfluidic devices have significantly higher surface-area-to-volume ratios than conventional platforms, resulting in lower volumes of media per cell, which can lead to cell stress. We investigated cell stress under a variety of culture conditions using three cell lines: parental HEK (human embryonic kidney) cells and transfected HEK cells that stably express wild-type (WT) and mutant (G601S) human ether-a-go-go related gene (hERG) potassium channel protein. These three cell lines provide a unique model system through which to study cell-type-specific responses in microculture because mutant hERG is known to be sensitive to environmental conditions, making its expression a particularly sensitive readout through which to compare macro- and microculture. While expression of WT-hERG was similar in microchannel and well culture, the expression of mutant G601S-hERG was reduced in microchannels. Expression of the endoplasmic reticulum (ER) stress marker immunoglobulin binding protein (BiP) was upregulated in all three cell lines in microculture. Using BiP expression, glucose consumption, and lactate accumulation as readouts we developed methods for reducing ER stress including properly increasing the frequency of media replacement, reducing cell seeding density, and adjusting the serum concentration and buffering capacity of culture medium. Indeed, increasing the buffering capacity of culture medium or frequency of media replacement partially restored the expression of the G601S-hERG in microculture. This work illuminates how biochemical properties of cells differ in macro- and microculture and suggests strategies that can be used to modify cell culture protocols for future studies involving miniaturized culture platforms.

Effect of microculture on cell metabolism and biochemistry: do cells get stressed in microchannels?

Microfluidique se profile comme une plate-forme prometteuse pour la culture cellulaire, permettant le contr&ocirc;le accru microenvironnement et potentiel d&#39;analyse int&eacute;gr&eacute;e par rapport aux syst&egrave;mes conventionnels macroculture tels que des plats et des bo&icirc;tes de P&eacute;tri. Pour faire progresser l&#39;utilisation de dispositifs microfluidiques pour culture cellulaire, il est n&eacute;cessaire de mieux comprendre comment la miniaturisation affecte le comportement de la cellule. En particulier, des dispositifs microfluidiques ont des rapports de surface-aire-volume significativement plus &eacute;lev&eacute;es que les plateformes classiques, r&eacute;sultant en une baisse des volumes de m&eacute;dias par cellule, ce qui peut conduire au stress cellulaire. Nous avons &eacute;tudi&eacute; le stress cellulaire sous diverses conditions de culture &agrave; l&#39;aide de trois lign&eacute;es cellulaires : parentales cellules HEK (rein embryonnaire humain) et cellules HEK transfect&eacute;es exprimant stablement sauvage (WT) et potassium g&egrave;ne connexes humain &eacute;ther-a-go-go (hERG) mutant (G601S) cha&icirc;ne de prot&eacute;ine. Ces trois lign&eacute;es cellulaires d&#39;un syst&egrave;me de mod&egrave;le unique gr&acirc;ce &agrave; l&#39;&eacute;tude des r&eacute;ponses sp&eacute;cifiques des cellules-type en microculture car hERG mutant est connu pour &ecirc;tre sensibles aux conditions environnementales, rendant son expression une lecture particuli&egrave;rement sensible permettant de comparer les macro - et microculture. Alors que l&#39;expression de WT-hERG ressemblait &agrave; microcanaux et de la culture bien, l&#39;expression de mutant G601S-hERG a &eacute;t&eacute; r&eacute;duite &agrave; microcanaux. Expression de la prot&eacute;ine de r&eacute;ticulum endoplasmique (re) stress marqueur immunoglobuline liaison (BiP) a augment&eacute; dans tous les trois lign&eacute;es cellulaires en microculture. En utilisant l&#39;expression BiP, la consommation de glucose et accumulation de lactate comme lectures nous avons d&eacute;velopp&eacute; des m&eacute;thodes pour r&eacute;duire le stress du re y compris correctement augmente la fr&eacute;quence de remplacement de m&eacute;dias, r&eacute;duire la densit&eacute; de semis de cellules et ajuster la concentration en s&eacute;rum et pouvoir tampon du milieu de culture. En effet, augmentant la capacit&eacute; tampon du milieu de culture ou la fr&eacute;quence de remplacement de m&eacute;dias partiellement restaur&eacute; l&#39;expression de la G601S-hERG dans microculture. Cet ouvrage &eacute;claire les propri&eacute;t&eacute;s comment biochimiques des cellules diff&egrave;rent en macro - et microculture et propose des strat&eacute;gies qui peuvent servir &agrave; modifier les protocoles de culture de cellules pour l&#39;avenir des &eacute;tudes impliquant des plates-formes de culture miniaturis&eacute;s.

Effet de microculture sur le m&eacute;tabolisme cellulaire et biochimie : faire les cellules obtenir a soulign&eacute; en MICROCANAUX ?

Although the field of microfluidics has made significant progress in bringing new tools to address biological questions, the accessibility and adoption of microfluidics within the life sciences are still limited. Open microfluidic systems have the potential to lower the barriers to adoption, but the absence of robust design rules has hindered their use. Here, we present an open microfluidic platform, suspended microfluidics, that uses surface tension to fill and maintain a fluid in microscale structures devoid of a ceiling and floor. We developed a simple and ubiquitous model predicting fluid flow in suspended microfluidic systems and show that it encompasses many known capillary phenomena. Suspended microfluidics was used to create arrays of collagen membranes, mico Dots (Î¼Dots), in a horizontal plane separating two fluidic chambers, demonstrating a transwell platform able to discern collective or individual cellular invasion. Further, we demonstrated that Î¼Dots can also be used as a simple multiplexed 3D cellular growth platform. Using the Î¼Dot array, we probed the combined effects of soluble factors and matrix components, finding that laminin mitigates the growth suppression properties of the matrix metalloproteinase inhibitor GM6001. Based on the same fluidic principles, we created a suspended microfluidic metabolite extraction platform using a multilayer biphasic system that leverages the accessibility of open microchannels to retrieve steroids and other metabolites readily from cell culture. Suspended microfluidics brings the high degree of fluidic control and unique functionality of closed microfluidics into the highly accessible and robust platform of open microfluidics.

Suspended microfluidics.

Bien que le domaine de la microfluidique a beaucoup progress&eacute; en apportant de nouveaux outils pour r&eacute;pondre &agrave; des questions biologiques, l&#39;accessibilit&eacute; et l&#39;adoption de microfluidics dans les sciences de la vie sont encore limit&eacute;es. Les syst&egrave;mes microfluidiques ouvert ont le potentiel d&#39;abaisser les obstacles &agrave; l&#39;adoption, mais l&#39;absence de r&egrave;gles de conception robuste a emp&ecirc;ch&eacute; leur utilisation. Nous pr&eacute;sentons ici une plateforme ouverte microfluidique, suspension microfluidique, qui utilise la tension superficielle &agrave; remplir et &agrave; entretenir un fluide dans des structures de micro-&eacute;chelle d&eacute;pourvu d&#39;un plafond et le plancher. Nous avons d&eacute;velopp&eacute; un mod&egrave;le simple et omnipr&eacute;sent, pr&eacute;dire l&#39;&eacute;coulement du fluide dans les syst&egrave;mes microfluidiques suspendu et montrent qu&#39;elle englobe de nombreux ph&eacute;nom&egrave;nes connus de capillaires. Microfluidique suspendue a &eacute;t&eacute; utilis&eacute;e pour cr&eacute;er des tableaux de membranes de collag&egrave;ne, mico points (&mu;Dots), dans un plan horizontal qui s&eacute;pare deux chambres fluidiques, d&eacute;montrant une plate-forme transwell capable de discerner l&#39;invasion cellulaire collectif ou individuelle. De plus, nous avons d&eacute;montr&eacute; que &mu;Dots peut aussi servir comme un simple multiplex&eacute;s plate-forme 3D de croissance cellulaire. En utilisant le tableau de &mu;Dot, nous avons sond&eacute; les effets combin&eacute;s de facteurs solubles et des constituants de la matrice, trouver que cette laminine att&eacute;nue les propri&eacute;t&eacute;s de la suppression de la croissance de l&#39;inhibiteur de m&eacute;talloprot&eacute;inase de matrice GM6001. Bas&eacute; sur les m&ecirc;mes principes fluidiques, nous avons cr&eacute;&eacute; une plateforme d&#39;extraction de m&eacute;tabolite microfluidique suspendu en utilisant un syst&egrave;me biphasique multicouche qui s&#39;appuie sur l&#39;accessibilit&eacute; des MICROCANAUX ouverte pour r&eacute;cup&eacute;rer facilement les st&eacute;ro&iuml;des et autres m&eacute;tabolites de culture cellulaire. Suspension microfluidique apporte le degr&eacute; &eacute;lev&eacute; de contr&ocirc;le fluidique et des fonctionnalit&eacute;s uniques de microfluidique ferm&eacute;e dans la plate-forme tr&egrave;s accessible et robuste de microfluidique ouverte.

Suspension microfluidique.

Nucleic acid (NA) extraction and purification has become a common technique in both research and clinical laboratories. Current methods require repetitive wash steps with a pipette that is laborious and time consuming making it inefficient for clinical settings. We present here a simple technique that relies on spontaneous biphasic plug flow inside a capillary to achieve sample preparation. By filling the sample with oil, aqueous contaminants were displaced from the captured NA without pipetting wash buffers or use of external force and equipment. mRNA from mammalian cell culture was purified and PCR amplification showed similar threshold cycle values as those obtained from a commercially available kit. HIV viral like particles were spiked into serum and a 6-fold increase in viral RNA extraction yield was achieved compared to the conventional wash method. In addition, viral RNA was successfully purified from human whole blood, and a limit of detection of approximately 14 copies of RNA extracted per sample. The results demonstrate the utility of the current technique for nucleic acid purification for clinical purposes, and the overall approach provides a potential method to implement nucleic acid testing in low resource settings.

Ashleigh B. Theberge

Department of Urology,
Molecular and Environmental Toxicology Center

University of Wisconsin-Madison

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

Ashleigh B. Theberge

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Urology,Molecular and Environmental Toxicology Center

University of Wisconsin-Madison

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

Department of Urology,
Molecular and Environmental Toxicology Center