high-throughput-identification-bacteria-repellent-polymers-for

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

University of Edinburgh

A quantitative and qualitative analysis of cellular adhesion, morphology and viability is essential in understanding and designing biomaterials such as those involved in implant surfaces or as tissue-engineering scaffolds. As a means to simultaneously perform these studies in a high-throughput (HT) manner, we report a normalized protocol which allows the rapid analysis of a large number of potential cell binding substrates using polymer microarrays and high-content fluorescence microscopy. The method was successfully applied to the discovery of optimal polymer substrates from a 214-member polyurethane library with mouse fibroblast cells (L929), as well as simultaneous evaluation of cell viability and cellular morphology. Analysis demonstrated high biocompatibility of the binding polymers and permitted the identification of several different cellular morphologies, showing that specific polymer interactions may provoke changes in cell shape. In addition, SAR studies showed a clear correspondence between cellular adhesion and polymer structure. The approach can be utilized to perform multiple experiments (up to 1024 single experiments per slide) in a highly reproducible manner, leading to the generation of vast amounts of data in a short time period (48-72 h) while reducing dramatically the quantities of polymers, reagents and cells used.

Deciphering cellular morphology and biocompatibility using polymer microarrays.

In this study, polymer microarrays were used for the rapid identification of polymer substrates upon which a suspension cell line would both adhere and proliferate giving a detailed and rapid understanding of cell-biomaterial interactions. Analysis demonstrated that suspension K562 human erythroleukemic cells, which normally grow in suspension, adhered and proliferated on several different polymers. Phenotypic and transcriptomic analysis techniques allowed examination of the interaction between cells and polymers permitting the elucidation of putative links between phenotypic responses to cell-biomaterial interactions and global gene expression.

A cooperative polymer-DNA microarray approach to biomaterial investigation.

Traumatic brain injury (TBI) is the one of the most common forms of head trauma, and it remains a leading cause of death and disability. It is known that the initial mechanical axonal injury triggers a complex cascade of neuroinflammatory and metabolic events, the understanding of which is essential for clinical, translational, and pharmacological research. These can occur even in mild TBI, and are associated with several post-concussion manifestations, including transiently heightened vulnerability to a second insult. Recent studies have challenged the tenet that ischemia is the ultimate modality of tissue damage following TBI, as metabolic dysfunction can develop in the presence of normal perfusion and before intracranial hypertension. In order to elucidate the cellular and molecular changes occurring in TBI as a direct result of neuronal injury and in the absence of ischemic damage, we performed a microarray analysis of expressed genes and molecular interaction pathways for different levels of severity of trauma using an in-vitro model. A stretch injury, equivalent to human diffuse axonal injury, was delivered to rat organotypic hippocampal slice cultures, and mRNA levels following a 10% (mild) and 50% (severe) stretch were compared with controls at 24 h. More genes were differentially expressed following 10% stretch than 50% stretch, indicating the early activation of complex cellular mechanisms. The data revealed remarkable differential gene expression following mTBI, even in the absence of cell damage. Pathway analysis revealed that molecular interactions in both levels of injury were similar, with IL-1beta playing a central role. Additional pathways of neurodegeneration involving RhoA (ras homolog gene family, member A) were found in 50% stretch.

Transcriptomics of traumatic brain injury: gene expression and molecular pathways of different grades of insult in a rat organotypic hippocampal culture model.

Amino functionalized cross-linked polystyrene microspheres of well defined sizes (0.2-2 mum) have been prepared and shown to be efficient and controllable delivery devices, capable of transporting anything from small dye molecules to bulky proteins into cells. However, the specific mechanism of cellular entry is largely unknown and widely variant from study to study. As such, chemical, biological and microscopic methods are used to elucidate the mechanism of cellular uptake for polystyrene microspheres of 0.2, 0.5 and 2 mum in mouse melanoma cells. Uptake is found to be wholly unreliant upon energetic processes, while lysosomal and endosomal tracking agents failed to show co-localisation with lysosomes/endosomes, suggesting a non-endocytic uptake pathway. To further explore the consequences of microsphere uptake, gene expression profiling is used to determine if there is a transcriptional response to "beadfection" in both murine and human cells. None of the common transcriptional responses to enhanced endocytosis are observed in beadfected cells, further supporting a non-endocytic uptake mechanism. Furthermore, the microspheres are noted to have a limited interaction with cells at a transcriptional level, supporting them as a non-toxic delivery vehicle.

Investigation of microsphere-mediated cellular delivery by chemical, microscopic and gene expression analysis.

Polymer microarrays as platforms for cell-based assays are presented, offering a unique approach to high-throughput cellular analysis. These high-throughput (HT) platforms are used for the screening of new materials with the purpose of first finding substrates upon which a specific cell line would adhere and second to gain a rapid understanding of the interactions between cells and biomaterials. Arrays presented here are fabricated using pre-synthesised polymers by contact printing via a robotic microarrayer. These large arrays of polymers are then incubated with cell cultures and the results obtained are used to significantly help the design of synthetic biomaterials, implant surfaces and tissue-engineering scaffolds by finding correlations between their chemical structure and their biological performance. The flexibility of polymer microarrays analysis not only greatly refines our knowledge of multitude of cell-biomaterial interactions but could also be used in biocompatibility assessments as novel biomarkers.

Polymer microarrays for cellular high-content screening.

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Maintaining stable differentiated somatic cell function in culture is essential to a range of biological endeavors. However, current technologies, employing, for example, primary hepatic cell culture (essential to the development of a bio-artificial liver and improved drug and toxicology testing), are limited by supply, expense, and functional instability even on biological cell culture substrata. As such, novel biologically active substrates manufacturable to GMP standards have the potential to improve cell culture-based assay applications. Currently hepatic endoderm (HE) generated from pluripotent stem cells is a genotypically diverse, cheap, and stable source of "hepatocytes"; however, HE routine applications are limited due to phenotypic instability in culture. Therefore a manufacturable subcellular matrix capable of supporting long-term differentiated cell function would represent a step forward in developing scalable and phenotypically stable hESC-derived hepatocytes. Adopting an unbiased approach we screened polymer microarrays and identified a polyurethane matrix which promoted HE viability, hepatocellular gene expression, drug-inducible metabolism, and function. Moreover, the polyurethane supported, when coated on a clinically approved bio-artificial liver matrix, long-term hepatocyte function and growth. In conclusion, our data suggest that an unbiased screening approach can identify cell culture substrate(s) that enhance the phenotypic stability of primary and stem cell-derived cell resources.

Unbiased screening of polymer libraries to define novel substrates for functional hepatocytes with inducible drug metabolism.

Le maintien stable fonction diff&#xE9;renci&#xE9;e de cellules somatiques dans la culture est essentielle &#xE0; une s&#xE9;rie d&#x26;#39;activit&#xE9;s biologiques. Cependant, les technologies actuelles, en employant, par exemple, des cultures primaires de cellules h&#xE9;patiques (indispensable pour le d&#xE9;veloppement d&#x26;#39;un foie bio-artificiel et de m&#xE9;dicaments am&#xE9;lior&#xE9;s et des analyses toxicologiques), sont limit&#xE9;es par l&#x26;#39;offre, les frais, et de l&#x26;#39;instabilit&#xE9; fonctionnelle, m&#xEA;me sur des substrats de culture de cellules biologiques. Ainsi, les nouveaux substrats biologiquement actifs manufacturables aux normes GMP ont le potentiel d&#x26;#39;am&#xE9;liorer les applications cellulaires essai de culture &#xE0; base de. Endoderme h&#xE9;patique Actuellement (HE) g&#xE9;n&#xE9;r&#xE9; &#xE0; partir de cellules souches pluripotentes est une source g&#xE9;notypiquement diversifi&#xE9;, pas cher, et stable de &#xAB;h&#xE9;patocytes&#xBB;; cependant, il les applications de routine sont limit&#xE9;es en raison de l&#x26;#39;instabilit&#xE9; ph&#xE9;notypique dans la culture. Par cons&#xE9;quent, une matrice de manufacturable subcellulaire capable de supporter &#xE0; long terme la fonction des cellules diff&#xE9;renci&#xE9;es repr&#xE9;senterait un pas en avant dans le d&#xE9;veloppement &#xE9;volutives et ph&#xE9;notypiquement stables CSEh d&#xE9;riv&#xE9;es h&#xE9;patocytes. L&#x26;#39;adoption d&#x26;#39;une approche impartiale nous avons cribl&#xE9; puces polym&#xE8;res et identifi&#xE9; une matrice de polyur&#xE9;thane qui a favoris&#xE9; la viabilit&#xE9; SE, l&#x26;#39;expression des g&#xE8;nes h&#xE9;patocellulaire, la drogue inductible du m&#xE9;tabolisme, et la fonction. En outre, le polyur&#xE9;thane pris en charge, lorsqu&#x26;#39;elle est appliqu&#xE9;e sur une matrice de foie bio-artificiel approuv&#xE9; cliniquement, &#xE0; long terme la fonction des h&#xE9;patocytes et la croissance. En conclusion, nos donn&#xE9;es sugg&#xE8;rent qu&#x26;#39;une approche impartiale de d&#xE9;pistage permet d&#x26;#39;identifier substrat de culture cellulaire (s) qui am&#xE9;liorent la stabilit&#xE9; ph&#xE9;notypique des primaires et de la tige des ressources cellulaires d&#xE9;riv&#xE9;es de cellules.

D&#xE9;pistage impartiale des biblioth&#xE8;ques de polym&#xE8;res pour d&#xE9;finir de nouveaux substrats pour les h&#xE9;patocytes fonctionnels avec le m&#xE9;tabolisme des m&#xE9;dicaments inductible

Aufrechterhaltung stabiler differenzierte somatische Funktion in Kultur ist wichtig, eine Reihe von biologischen Unternehmungen. Aktuelle Technologien, Einsatz, z. B. prim&auml;re hepatische Zellkultur (wesentlich f&uuml;r die Entwicklung eines Bio-k&uuml;nstliche Leber und verbesserte Drogen-und Toxikologie Tests), sind jedoch durch Lieferung, Kosten und funktionelle Instabilit&auml;t auch auf biologische Zelle Kultur Substraten beschr&auml;nkt. Als solche haben neuartige biologisch aktive Substraten herstellbarer nach GMP-Standards, die Zelle Kultur-gegr&uuml;ndeten Probe Anwendungen zu verbessern. Derzeit ist Leber Endodermis (HE) aus pluripotenten Stammzellen erzeugt eine genotypisch verschiedene, g&uuml;nstige und stabile &quot;Hepatozyten&quot;; HE Routineanwendungen sind jedoch begrenzt durch die ph&auml;notypische Instabilit&auml;t in Kultur. Daher w&uuml;rde eine bearbeitbare subzellulare Matrix unterst&uuml;tzt langfristige differenzierte Zelle-Funktion einen Schritt nach vorn bei der Entwicklung von skalierbarer und ph&auml;notypisch stabiler hESC gewonnenen Hepatozyten darstellen. Eine unvoreingenommene Ansatz wir geschirmt Polymer-Microarrays und identifiziert eine Polyurethan-Matrix die er Lebensf&auml;higkeit, hepatozellul&auml;res Genexpression, Droge-inducible Stoffwechsel und Funktion bef&ouml;rdert. Dar&uuml;ber hinaus unterst&uuml;tzt das Polyurethan, wenn eine klinisch zugelassenen Bio-k&uuml;nstliche Leber Matrix, langfristige Hepatozyten-Funktion und Wachstum beschichtet. Abschlie&szlig;end zufolge unsere Daten ein unvoreingenommene Pr&uuml;fung Ansatz Zelle Kultur Substrate(s) identifizieren kann, die die ph&auml;notypische Stabilit&auml;t der Prim&auml;r- und Stammzellen Cell-derived Zelle Ressourcen zu verbessern.

Unvoreingenommene Screening von Polymer-Bibliotheken, um neuartige Substrate f&uuml;r funktionale Hepatozyten mit Induktion Medikamentenstoffwechsel definieren.

Mantenendo stabile di cellule somatiche differenziate funzione nella cultura &egrave; essenziale per una vasta gamma di attivit&agrave; biologiche. Tuttavia, tecnologie attuali, che impiegano, per esempio, coltura di cellule epatiche primario (essenziale per lo sviluppo di un fegato bio-artificiale e droga migliorato e tossicologia test), sono limitati dalla fornitura, spese e instabilit&agrave; funzionale anche su substrati di coltura biologica delle cellule. Come tale, romanzo substrati biologicamente attivi manufacturable agli standard GMP hanno il potenziale per migliorare applicazioni dosaggio basato su colture delle cellule. Endoderma epatica (HE) generato da cellule staminali pluripotenti &egrave; attualmente una fonte genotipicamente diversa, economico e stabile di &quot;epatociti&quot;; Tuttavia, egli sistematiche applicazioni sono limitate a causa di instabilit&agrave; fenotipica nella cultura. Quindi una matrice sottocellulare manufacturable capace di supportare la funzione delle cellule differenziate a lungo termine rappresenterebbe un passo avanti nello sviluppo di epatociti derivato da hESC fenotipicamente stabili e scalabili. Adottando un approccio imparziale abbiamo proiettato microarrays del polimero e identificato una matrice poliuretanica che ha promosso vitalit&agrave;, espressione genica epatocellulare, droga-inducible metabolismo e funzione. Inoltre, il poliuretano supportati, quando rivestiti su una matrice fegato bio-artificiale clinicamente, funzione degli epatociti a lungo termine e crescita. In conclusione, i nostri dati suggeriscono che un approccio imparziale lo screening pu&ograve; identificare la cella cultura substrate(s) che migliorano la stabilit&agrave; fenotipica delle risorse cella primario e derivato da cellule staminali.

Selezione imparziale delle librerie di polimero per definire nuovi substrati per epatociti funzionali con metabolismo del farmaco inducibile.

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&#xBB38;&#xD654;&#xC5D0;&#xC11C; &#xC720;&#xC9C0; &#xC548;&#xC815;&#xC801;&#xC778; &#xCC28;&#xBCC4;&#xD654; &#xB41C; &#xCCB4;&#xC138;&#xD3EC; &#xAE30;&#xB2A5; &#xB2E4;&#xC591; &#xD55C; &#xC0DD;&#xBB3C; &#xD559;&#xC801; &#xB178;&#xB825;&#xC5D0; &#xADFC;&#xBCF8;&#xC801; &#xC774;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xD604;&#xC7AC; &#xAE30;&#xC220;, &#xACE0;&#xC6A9;, &#xC608;&#xB97C; &#xB4E4;&#xC5B4;, &#xAE30;&#xBCF8; &#xAC04;&#xC7A5; &#xC138;&#xD3EC; &#xBC30;&#xC591; (&#xBC14;&#xC774;&#xC624; &#xC778;&#xACF5; &#xAC04; &#xBC0F; &#xD5A5;&#xC0C1; &#xB41C; &#xC57D;&#xBB3C; &#xBC0F; &#xB3C5;&#xADF9;&#xBB3C; &#xD14C;&#xC2A4;&#xD2B8;&#xC758; &#xAC1C;&#xBC1C;&#xC5D0; &#xD544;&#xC218;&#xC801;&#xC778;) &#xACF5;&#xAE09;, &#xBE44;&#xC6A9;, &#xBC0F; &#xC0DD;&#xBB3C; &#xD559;&#xC801; &#xC140; &#xBB38;&#xD654; substrata&#xC5D0;&#xB3C4; &#xAE30;&#xB2A5;&#xC801; &#xBD88;&#xC548;&#xC815;&#xC131;&#xC5D0; &#xC758;&#xD574; &#xC81C;&#xD55C; &#xB429;&#xB2C8;&#xB2E4;. &#xC774;&#xC640; &#xAC19;&#xC774; &#xC18C;&#xC124; &#xC0DD;&#xBB3C;&#xD559;&#xC801;&#xC73C;&#xB85C; &#xD65C;&#xC131; &#xAE30;&#xD310; &#xC81C;&#xC791; GMP &#xD45C;&#xC900; &#xC140; &#xBB38;&#xD654; &#xAE30;&#xBC18; &#xBD84;&#xC11D; &#xACB0;&#xACFC; &#xC751;&#xC6A9; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC744; &#xD5A5;&#xC0C1; &#xC2DC;&#xD0AC; &#xAC00;&#xB2A5;&#xC131;&#xC774; &#xC788;&#xB2E4;. &#xD604;&#xC7AC; &#xAC04;&#xC7A5; endoderm (&#xADF8;&#xB294;) pluripotent &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xC0DD;&#xC131; &#xB418;&#xB294; &quot;hepatocytes&quot;;&#xC758; genotypically &#xC800;&#xB834;, &#xB2E4;&#xC591; &#xD558; &#xACE0; &#xC548;&#xC815;&#xC801;&#xC778; &#xC18C;&#xC2A4; &#xADF8;&#xB7EC;&#xB098; &#xADF8;&#xB294; &#xC77C;&#xC0C1;&#xC801;&#xC778; &#xC751;&#xC6A9; &#xD504;&#xB85C;&#xADF8;&#xB7A8; &#xBB38;&#xD654; phenotypic &#xBD88;&#xC548;&#xC815;&#xC73C;&#xB85C; &#xC778;&#xD574; &#xC81C;&#xD55C; &#xB429;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C; &#xC81C;&#xC791; subcellular &#xB9E4;&#xD2B8;&#xB9AD;&#xC2A4; &#xC7A5;&#xAE30; &#xCC28;&#xBCC4;&#xD654; &#xB41C; &#xC138;&#xD3EC; &#xAE30;&#xB2A5;&#xC744; &#xC9C0;&#xC6D0;&#xD560; &#xC218; &#xC788;&#xB294; &#xD655;&#xC7A5; &#xAC00;&#xB2A5; &#xD558; &#xACE0; &#xC548;&#xC815;&#xC801;&#xC778; phenotypically hESC &#xD30C;&#xC0DD; hepatocytes &#xAC1C;&#xBC1C;&#xC5D0; &#xC55E;&#xC73C;&#xB85C; &#xB2E8;&#xACC4;&#xB97C; &#xB098;&#xD0C0;&#xB0B4;&#xB294; &#xAC83; &#xC774;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xD3F4;&#xB9AC;&#xBA38; microarrays &#xC0C1;&#xC601; &#xD558; &#xACE0; &#xC0DD;&#xC874;, &#xAC04;&#xC138;&#xD3EC; &#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604;, &#xB9C8;&#xC57D;&#xC744; &#xC720;&#xB3C4;&#xD560; &#xC218; &#xC788;&#xB294; &#xC2E0;&#xC9C4; &#xB300;&#xC0AC;&#xC640; &#xAE30;&#xB2A5; &#xADF8;&#xB294; &#xC2B9;&#xC9C4; &#xD3F4;&#xB9AC;&#xC6B0;&#xB808;&#xD0C4; &#xB9E4;&#xD2B8;&#xB9AD;&#xC2A4;&#xB97C; &#xC2DD;&#xBCC4; &#xACF5;&#xD3C9;&#xD55C; &#xC811;&#xADFC; &#xBC29;&#xC2DD;&#xC744; &#xCC44;&#xD0DD;. &#xB610;&#xD55C;,&#xB294; &#xD3F4;&#xB9AC;&#xC6B0;&#xB808;&#xD0C4; &#xC9C0;&#xC6D0; &#xC784;&#xC0C1; &#xC2B9;&#xC778; &#xB41C; &#xBC14;&#xC774;&#xC624; &#xC778;&#xACF5; &#xAC04; &#xB9E4;&#xD2B8;&#xB9AD;&#xC2A4;, &#xC7A5;&#xAE30; hepatocyte &#xAE30;&#xB2A5; &#xBC0F; &#xC131;&#xC7A5;&#xC5D0; &#xCF54;&#xD305; &#xD558;&#xB294; &#xACBD;&#xC6B0;. &#xACB0;&#xB860;&#xC801;&#xC73C;&#xB85C;, &#xC6B0;&#xB9AC;&#xC758; &#xB370;&#xC774;&#xD130; &#xACF5;&#xD3C9;&#xD55C; &#xC2EC;&#xC0AC; &#xBC29;&#xC2DD; &#xBC0F; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xC720;&#xB798; &#xC138;&#xD3EC; &#xC790;&#xC6D0;&#xC758; phenotypic &#xC548;&#xC815;&#xC131;&#xC744; &#xD5A5;&#xC0C1; &#xC2DC;&#xD0A4;&#xB294; &#xC138;&#xD3EC; &#xBB38;&#xD654; substrate(s) &#xC2DD;&#xBCC4;&#xD560; &#xC218; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xD3F4;&#xB9AC;&#xBA38; &#xB77C;&#xC774;&#xBE0C;&#xB7EC;&#xB9AC;&#xB97C; &#xC720;&#xB3C4;&#xD560; &#xC218; &#xC788;&#xB294; &#xC57D;&#xBB3C; &#xB300;&#xC0AC;&#xC640; &#xAE30;&#xB2A5;&#xC801; hepatocytes &#xC18C;&#xC124; &#xAE30;&#xD310; &#xC815;&#xC758;&#xC758; &#xACF5;&#xD3C9;&#xD55C; &#xC2EC;&#xC0AC;.

Mantener estable la funci&#xF3;n de c&#xE9;lula som&#xE1;tica diferenciada de la cultura es esencial para una amplia gama de esfuerzos biol&#xF3;gicos. Sin embargo, las tecnolog&#xED;as actuales, empleando, por ejemplo, cultivo primario de c&#xE9;lulas hep&#xE1;ticas (esencial para el desarrollo de un h&#xED;gado bioartificial y f&#xE1;rmaco mejorada y pruebas toxicol&#xF3;gicas), est&#xE1;n limitadas por la oferta, los gastos y la inestabilidad funcional incluso en biol&#xF3;gicos sustratos de cultivo celular. Como dichos sustratos, novela biol&#xF3;gicamente activos manufacturable a las normas GMP tiene el potencial de mejorar la cultura de c&#xE9;lulas basadas en las aplicaciones de ensayo. Actualmente endodermo hep&#xE1;tica (EH) generado a partir de c&#xE9;lulas madre pluripotentes es una fuente genot&#xED;picamente diversa, barata y estable de &#x26;quot;hepatocitos&#x26;quot;, sin embargo, el Excmo aplicaciones de rutina son limitadas debido a la inestabilidad fenot&#xED;pica en la cultura. Por lo tanto una matriz manufacturable subcelular capaz de soportar a largo plazo la funci&#xF3;n de c&#xE9;lulas diferenciadas que representan un paso adelante en el desarrollo escalables y estables fenot&#xED;picamente derivados hESC-hepatocitos. La adopci&#xF3;n de un enfoque imparcial, se revisaron las micromatrices de pol&#xED;mero y se identificaron una matriz de poliuretano que promovi&#xF3; el Excmo viabilidad, la expresi&#xF3;n de genes hepatocelular, el metabolismo de las drogas-inducible, y la funci&#xF3;n. Adem&#xE1;s, el poliuretano apoyo, cuando est&#xE1;n cubiertas en una matriz de h&#xED;gado cl&#xED;nicamente aprobado bioartificial, a largo plazo de la funci&#xF3;n del hepatocito y el crecimiento. En conclusi&#xF3;n, nuestros datos sugieren que un enfoque exclusivamente de cribado pueden identificar sesgos sustrato de cultivo celular (s) que mejoran la estabilidad fenot&#xED;pica de los recursos primarios de c&#xE9;lulas y c&#xE9;lulas madre derivadas.

Evaluaci&#xF3;n objetiva de las Bibliotecas de pol&#xED;mero para definir nuevos sustratos de hepatocitos funcionales, con metabolismo inducible de Drogas

This manuscript describes the use of a novel biochip platform for the rapid analysis/identification of nucleic acids, including DNA and microRNAs, with very high specificity. This approach combines a unique dynamic chemistry approach for nucleic acid testing and analysis developed by DestiNA Genomics with the STMicroelectronics In-Check platform, which comprises two microfluidic optimized and independent PCR reaction chambers, and a sequential microarray area for nucleic acid capture and identification by fluorescence. With its compact bench-top "footprint" requiring only a single technician to operate, the biochip system promises to transform and expand routine clinical diagnostic testing and screening for genetic diseases, cancers, drug toxicology and heart disease, as well as employment in the emerging companion diagnostics market.

Novel biochip platform for nucleic acid analysis.

Rapid endothelisation is of critical importance in the prevention of adverse remodelling after device implantation. Currently, there is a need for alternative strategies to promote re-endothelialisation for intravascular stents and vascular grafts. Using polymer microarray technology 345 polymers are comprehensively assessed and a matrix is identified that specifically supports both progenitor and mature endothelial cell activity in vitro and in vivo while minimising platelet attachment.

Novel biopolymers to enhance endothelialisation of intra-vascular devices.

The liver is a highly resilient organ that possesses enormous regenerative capacity. This is mediated mainly through the most abundant cell type found in the liver, the hepatocyte. When the regenerative capacity of the hepatocyte is compromised, during chronic or acute liver injury, hepatic progenitor cells (HPCs) are activated to replace the damaged tissue. The HPC resides in a laminin-rich environment; as HPCs differentiate toward a hepatic or biliary fate, the extracellular matrix (ECM) composition changes, influencing cell behavior. To assess the impact that the biological ECM and the synthetic ECM have on the maintenance of hepatic stem cell gene expression, a murine hepatic stem cell line was employed. We demonstrate that hepatic stem cell gene expression could be maintained using a biological or synthetic substratum, but not on plastic alone.

Maintaining hepatic stem cell gene expression on biological and synthetic substrata.

Faithfully recapitulating human physiology "in a dish" from a renewable source remains a holy grail for medicine and pharma. Many procedures have been described that, to a limited extent, exhibit human tissue-specific function in vitro. In particular, incomplete cellular differentiation and/or the loss of cell phenotype postdifferentiation play a major part in this void. We have developed an interdisciplinary approach to address this problem, using skill sets in cell biology, materials chemistry, and pharmacology. Pluripotent stem cells were differentiated to hepatocytes before being replated onto a synthetic surface. Our approach yielded metabolically active hepatocyte populations that displayed stable function for more than 2 weeks in vitro. Although metabolic activity was an important indication of cell utility, the accurate prediction of cellular toxicity in response to specific pharmacological compounds represented our goal. Therefore, detailed analysis of hepatocellular toxicity was performed in response to a custom-built and well-defined compound set and compared with primary human hepatocytes. Importantly, stem cell-derived hepatocytes displayed equivalence to primary human material. Moreover, we demonstrated that our approach was capable of modeling metabolic differences observed in the population. In conclusion, we report that pluripotent stem cell-derived hepatocytes will model toxicity predictably and in a manner comparable to current gold standard assays, representing a major advance in the field.

Developing high-fidelity hepatotoxicity models from pluripotent stem cells.

The class V POU domain transcription factor Oct4 (Pou5f1) is a pivotal regulator of embryonic stem cell (ESC) self-renewal and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. Oct4 is also an important evolutionarily conserved regulator of progenitor cell differentiation during embryonic development.

A conserved Oct4/POUV-dependent network links adhesion and migration to progenitor maintenance.

Nosocomial infections due to bacteria have serious implications on the health and recovery of patients in a variety of medical scenarios. Since bacterial contamination on medical devices contributes to the majority of nosocomical infections, there is a need for redesigning the surfaces of medical devices, such as catheters and tracheal tubes, to resist the binding of bacteria. In this work, polyurethanes and polyacrylates/acrylamides, which resist binding by the major bacterial pathogens underpinning implant-associated infections, were identified using high-throughput polymer microarrays. Subsequently, two 'hit' polymers, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)) and PA515 (poly(methoxyethylmethacrylate-co-diethylaminoethylacrylate-co-methylmethacrylate)), were used to coat catheters and substantially shown to decrease binding of a variety of bacteria (including isolates from infected endotracheal tubes and heart valves from intensive care unit patients). Catheters coated with polymer PA13 showed up to 96% reduction in bacteria binding in comparison to uncoated catheters.

Bacteria repelling poly(methylmethacrylate-co-dimethylacrylamide) coatings for biomedical devices†Electronic supplementary information (ESI) available: Polymer microarray screening, including analysis of bacterial adhesion by fluorescence microscopy and SEM, and chemical composition of bacteria repelling polymers identified in the screen; polymer synthesis and characterisation; preparation of catheter pieces and solvent studies, and details for confocal imaging/analysis. See DOI: 10.1039/c4tb01129eClick here for additional data file.

Engineered nanoparticles (eNPs) for biological and biomedical applications are produced from functionalised nanoparticles (NPs) after undergoing multiple handling steps, giving rise to an inevitable loss of NPs. Herein we present a practical method to quantify nanoparticles (NPs) number per volume in an aqueous suspension using standard spectrophotometers and minute amounts of the suspensions (up to 1 μL). This method allows, for the first time, to analyse cellular uptake by reporting NPs number added per cell, as opposed to current methods which are related to solid content (w/V) of NPs. In analogy to the parameter used in viral infective assays (multiplicity of infection), we propose to name this novel parameter as multiplicity of nanofection.

Number of Nanoparticles per Cell through a Spectrophotometric Method - A key parameter to Assess Nanoparticle-based Cellular Assays.

Hyaluronidases (Hyals) are broadly used in medical applications to facilitate the dispersion and/or absorption of fluids or medications. This study reports the isolation, cloning, and industrial-scale recombinant production, purification and full characterization, including X-ray structure determination at 1.45 Å, of an extracellular Hyal from the nonpathogenic bacterium Streptomyces koganeiensis. The recombinant S. koganeiensis Hyal (rHyal_Sk) has a novel bacterial catalytic domain with high enzymatic activity, compared with commercially available Hyals, and is more thermostable and presents higher proteolytic resistance, with activity over a broad pH range. Moreover, rHyal_Sk exhibits remarkable substrate specificity for hyaluronic acid (HA) and poses no risk of animal cross-infection.

Identification and characterization of a bacterial hyaluronidase and its production in recombinant form.

Over the last decade, circulating microRNAs have received attention as diagnostic and prognostic biomarkers. In particular, microRNA122 has been demonstrated to be an early and more sensitive indicator of drug-induced liver injury than the widely used biomarkers such as alanine aminotransferase and aspartate aminotransferase. Recently, microRNA122 has been used in vitro to assess the cellular toxicity of new drugs and as a biomarker for the development of a rapid test for drug overdose/liver damage. In this proof-of-concept study, we report a PCR-free and label-free detection method that has a limit of detection (3 standard deviations) of 15 fmoles of microRNA122, by integrating a dynamic chemical approach for "Single Nucleobase Labelling" with a bead-based platform (Luminex(®)) thereby, in principle, demonstrating the exciting prospect of rapid and accurate profiling of any microRNAs related to diseases and toxicology.

Article	Year
Deciphering cellular morphology and biocompatibility using polymer microarrays. Biomedical materials (Bristol, England)\| PubMed ID: 18708702	2008
A cooperative polymer-DNA microarray approach to biomaterial investigation. Lab on a chip\| PubMed ID: 19156288	2009
Transcriptomics of traumatic brain injury: gene expression and molecular pathways of different grades of insult in a rat organotypic hippocampal culture model. Journal of neurotrauma\| PubMed ID: 19903084	2010
Investigation of microsphere-mediated cellular delivery by chemical, microscopic and gene expression analysis. Molecular bioSystems\| PubMed ID: 20094660	2010
Polymer microarrays for cellular high-content screening. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 21104063	2011
Unbiased screening of polymer libraries to define novel substrates for functional hepatocytes with inducible drug metabolism. Stem cell research\| PubMed ID: 21277274	2011
Novel biochip platform for nucleic acid analysis. Sensors (Basel, Switzerland)\| PubMed ID: 22969389	2012
Novel biopolymers to enhance endothelialisation of intra-vascular devices. Advanced healthcare materials\| PubMed ID: 23184801	2012
Maintaining hepatic stem cell gene expression on biological and synthetic substrata. BioResearch open access\| PubMed ID: 23515003	2012
Developing high-fidelity hepatotoxicity models from pluripotent stem cells. Stem cells translational medicine\| PubMed ID: 23757504	2013
A conserved Oct4/POUV-dependent network links adhesion and migration to progenitor maintenance. Current biology : CB\| PubMed ID: 24210613	2013
Bacteria repelling poly(methylmethacrylate-co-dimethylacrylamide) coatings for biomedical devices†Electronic supplementary information (ESI) available: Polymer microarray screening, includin... Journal of materials chemistry. B, Materials for biology and medicine\| PubMed ID: 25580245	2014
Number of Nanoparticles per Cell through a Spectrophotometric Method - A key parameter to Assess Nanoparticle-based Cellular Assays. Scientific reports\| PubMed ID: 25976173	2015
Identification and characterization of a bacterial hyaluronidase and its production in recombinant form. FEBS letters\| PubMed ID: 27311405	2016
Novel bead-based platform for direct detection of unlabelled nucleic acids through Single Nucleobase Labelling. Talanta\| PubMed ID: 27769437	2016

Salvatore Pernagallo

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