La financiación de la Wellcome Trust es amablemente reconocido (número de concesión 085245/Z/08/Z). La nanotecnología Nottingham y el Centro de Nanociencia es amablemente reconocido por dar acceso al sistema Raman y de la Agencia de Desarrollo de East Midlands para la financiación de este equipo.

<ol>
	<li>Hook, A. L., Anderson, D. G., Langer, R., Williams, P., Davies, M. C., Alexander, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+methods+applied+in+biomaterial+development+and+discovery.">High throughput methods applied in biomaterial development and discovery.</a> Biomaterials. 31, 187-198 (2010).</li><li>Anderson, D. G., Levenberg, S., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoliter-scale+synthesis+of+arrayed+biomaterials+and+application+to+human+embryonic+stem+cells.">Nanoliter-scale synthesis of arrayed biomaterials and application to human embryonic stem cells.</a> Nature Biotechnology. 22, 863-866 (2004).</li><li>Mei, Y., Saha, K., Bogatyrev, S. R., Yang, J., Hook, A. L., Kalcioglu, Z. I., Cho, S. W., Mitalipova, M., Pyzocha, N., Rojas, F., Van Vliet, K. J., Davies, M. C., Alexander, M. R., Langer, R., Jaenisch, R., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+development+of+biomaterials+for+clonal+growth+of+human+pluripotent+stem+cells.">Combinatorial development of biomaterials for clonal growth of human pluripotent stem cells.</a> Nature Materials. 9, 768-778 (2010).</li><li>Pernagallo, S., Wu, M., Gallagher, M. P., Bradley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonising+new+frontiers-microarrays+reveal+biofilm+modulating+polymers.">Colonising new frontiers-microarrays reveal biofilm modulating polymers.</a> Journal of Materials Chemistry. 21, 96-101 (2011).</li><li>Yang, J., Mei, Y., Hook, A. L., Taylor, M., Urquhart, A. J., Bogatyrev, S. R., Langer, R., Anderson, D. G., Davies, M. C., Alexander, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+surface+functionalities+that+control+human+embryoid+body+cell+adhesion+revealed+by+high+throughput+surface+characterization+of+combinatorial+material+microarrays.">Polymer surface functionalities that control human embryoid body cell adhesion revealed by high throughput surface characterization of combinatorial material microarrays.</a> Biomaterials. 31, 8827-8838 (2010).</li><li>Urquhart, A. J., Anderson, D. G., Taylor, M., Alexander, M. R., Langer, R., Davies, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+surface+characterisation+of+a+combinatorial+material+library.">High throughput surface characterisation of a combinatorial material library.</a> Advanced Materials. 19, 2486-2491 (2007).</li><li>Taylor, M., Urquhart, A. J., Zelzer, M., Davies, M. C., Alexander, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picoliter+water+contact+angle+measurement+on+polymers.">Picoliter water contact angle measurement on polymers.</a> Langmuir. 23, 6875-6878 (2007).</li><li>Urquhart, A. J., Taylor, M., Anderson, D. G., Langer, R., Davies, M. C., Alexander, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TOF-SIMS+analysis+of+a+576+micropatterned+copolymer+array+to+reveal+surface+moieties+that+control+wettability.">TOF-SIMS analysis of a 576 micropatterned copolymer array to reveal surface moieties that control wettability.</a> Analytical Chemistry. 80, 135-142 (2008).</li><li>Hook, A. L., Yang, J., Chen, X., Roberts, C. J., Mei, Y., Anderson, D. G., Langer, R., Alexander, M. R., Davies, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acrylate+polymers+with+hydro-responsive+topography.">Acrylate polymers with hydro-responsive topography.</a> Soft Matter. 7, 7194-9197 (2011).</li><li>Pernagallo, S., Unciti-Broceta, A., Diaz-Mochon, J. J., Bradley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+cellular+morphology+and+biocompatibility+using+polymer+microarrays.">Deciphering cellular morphology and biocompatibility using polymer microarrays.</a> Biomedical Materials. 3, (2008).</li></ol>

No tenemos nada que revelar.

Microarrays de polímero han sido utilizados con éxito para el descubrimiento de nuevos materiales por cientos de detección de la novela de polímero en un ensayo biológico e identificar &#39;hit&#39; materiales que posteriormente pueden ampliarse a dispositivos útiles. En este caso, la caracterización de la superficie se describe se puede emplear con posterioridad a la prueba biológica y exclusivamente en el &#39;golpe&#39; materiales para estudiar los materiales con mayor detalle. Esta estrategia puede ser de interés si HTSC no está disponible para el experimentador emplear este enfoque. Sin embargo, para utilizar completamente microarrays de polímero para estudiar las interacciones de material biológico-toda la gama de cientos de materiales deben ser analizados antes de los ensayos biológicos usando metodologías HTSC, que posteriormente se pueden utilizar para observar generales estructura-función de las tendencias. La impresión por contacto se apoya en el perno de metal se deslice hacia arriba y hacia abajo libremente dentro del soporte de pasador. Limpieza y Pin pin titular es lo más importante es asegurarse de printing se produce con éxito y debe llevarse a cabo con rigor. Antes de comenzar una impresión ejecutar el movimiento apropiado de la clavija dentro del soporte de pasador se puede probar mediante la realización de una marcha en seco, sin monómeros presentes. La etapa de limpieza debe continuar hasta que el movimiento se consigue pin reproduciblemente. Pensamiento considerable debe ir en el diseño de la mezcla de monómeros. Con el fin de producir fácilmente una biblioteca combinatoria de polímeros, cientos de copolímeros se forman mezclando una monómeros pocos en diferentes proporciones. Típicamente producir 576 bibliotecas miembros ya que esto forma una matriz 24 x 24, que es adecuada para la geometría de un portaobjetos de vidrio. Con el fin de producir una biblioteca combinatoria que explora el espacio más combinatoria el método más sencillo es mezclar 24 monómeros pairwise en la relación 2:1. Alternativamente, la inclusión de los gradientes de composición dentro de la matriz son útiles para permitir las observaciones de las tendencias, lo que permite composiciones óptimas de monómero a ser determined. Como un ejemplo de este 22 monómeros se pueden utilizar como primer componente en una mezcla de co-monómero que se diluyó secuencialmente con 1 de 6 segundos componentes. Si 5 diluciones se utilizan, por ejemplo mezclando los componentes primero y segundo en proporciones de 90:10, 75:25, 50:50, 25:75 y 10:90, esto resultaría en 488 soluciones de copolímero únicas. Para llevar el total a 576, repeticiones de los homopolímeros de los monómeros usados ​​pueden ser introducidos, que a menudo es una muestra de referencia importante. 576 soluciones de monómero debe ser dispensado en 2 384 placas de pocillos. Para la programación del robot que es más fácil tener dos placas idénticas en términos de la posición de los monómeros, por lo tanto, las soluciones de monómero se debe dividir de manera uniforme entre las dos placas. Una cantidad significativa de tiempo se pueden guardar en la preparación de las placas de código mediante el uso de pipetas multicanal, y el diseño de placas de origen debe ser determinado con el fin de explotar el uso de las pipetas multicanal. &lt;/ P&gt; Para lograr HTSC automatizado de las matrices de la posición de la mancha debe ser correctamente alineado con el aparato de caracterización. Normalmente, el paso de una serie de acrilato es 500-1000 micras y el diámetro del punto de polímero es de 300 m. La mayoría de las etapas XY tienen una resolución por debajo de 10 micras, con lo que la tolerancia es adecuada para el aparato de caracterización de la superficie de forma fiable acceder a las posiciones de la matriz una vez que las dimensiones correctas se han introducido para el software de posicionamiento de la muestra. La limitación para el posicionamiento automatizado es, de hecho, la precisión de la impresión de la matriz. Para garantizar la precisión de la impresión es importante para evitar el movimiento del sustrato en la etapa de impresión ya sea mediante una succión o abrazaderas de resorte, junto con las dimensiones apropiadas de diapositivas (nota que tanto un tamaño de EE.UU. y UE portaobjetos estándar existen). ToF-SIMS es una técnica extremadamente sensible superficie que se observó ninguna contaminación en muestras. Así, máximo cuidado debe tomarsepara evitar el contacto con la superficie. Las muestras sólo deben ser manipulados, pero la superficie de interés no en contacto con, con guantes limpios (preferiblemente polietileno) y con pinzas recién limpiadas. Por lo general se lava con cloroformo y hexano. Almacenamiento de las muestras antes de las mediciones se realiza mejor en un soporte de muestras que contiene las diapositivas, aparte, por ejemplo la. 5 soporte de diapositivas o 20 soporte de diapositivas Las matrices están diseñados específicamente para ser compatible con muchos formatos de ensayos biológicos y lecturas, es decir, el sustrato utilizado es un portaobjetos de microscopio ideal para escáneres de fluorescencia y microscopios de luz. Esto significa que el formato es muy adecuado para explorar muchos materiales biológicos interacciones. Además, el formato permite a cientos de materiales que se proyectarán en paralelo. Esto permite que muchos materiales que se proyectarán más que los métodos convencionales mediante el cual es monitoreada cada nuevo material química individualmente. El mayor alcance de las interacciones biológicas materiales permitens para la elucidación de los mecanismos de las interacciones superficiales biológicas, así como encontrar el material óptimo para una aplicación dada.

<table border="1"><tbody><tr><td> Nombre del reactivo / equipo </td><td> Empresa </td><td> Catálogo / Número de modelo </td></tr><tr><td> Diapositivas epoxi </td><td> Genetix </td><td> K2652 </td></tr><tr><td> Póngase en contacto con impresora </td><td> BioDot </td><td> XYZ3060 Plataforma </td></tr><tr><td> Perno del metal </td><td> Arrayit </td><td> 946MP6B </td></tr><tr><td> TOF-SIMS instrumento </td><td> ION-TOF </td><td></td></tr><tr><td> XPS instrumento </td><td> Kratos </td><td></td></tr><tr><td> WCA aparato </td><td> Krüss </td><td> DSA 100 </td></tr><tr><td> AFM </td><td> Bruker </td><td> Dimension Icono </td></tr><tr><td> RPMI-1640 medio de cultivo celular </td><td> Sigma-Aldrich </td><td> R0883 </td></tr><tr><td> SYTO17 </td><td> Invitrogen </td><td> S-7579 </td></tr></tbody></table>

polymer microarrays for high throughput discovery of biomaterials

La mezcla es una operación unitaria que combina dos o más componentes en una mezcla homogénea. Este trabajo consiste en mezclar dos corrientes viscosas líquidas utilizando un mezclador estático en línea. El mezclador es un diseño en dos y se recombinan-que emplea flujo de cizallamiento y extensionales para aumentar el contacto interfacial entre los componentes. Un prototipo dividida y se recombinan-(SAR) mezclador se construyó mediante la alineación de una serie de finas cortadas con láser de poli (metacrilato de metilo) (PMMA) placas mantiene en su lugar en un tubo de PVC. Mezcla en este dispositivo se ilustra en la fotografía en la figura. 1. Colorante rojo se añadió a una porción del fluido de prueba y se utiliza como el componente minoritario que se mezcla con el mayor (sin teñir) componente. En la entrada de la mezcladora, la capa de inyección de líquido trazador se divide en dos capas a medida que fluye a través de la sección de mezcla. En cada sección de mezcla posterior, el número de capas horizontales se duplica. En última instancia, la única corriente de colorante se dispersa uniformemente Throughout la sección transversal del dispositivo. El uso de un fluido de prueba no newtoniano de 0,2% de Carbopol y un fluido trazador dopado de composición similar, la mezcla en la unidad se visualizaron usando imágenes por resonancia magnética (MRI). La RM es una sonda experimental muy potente de química molecular y el medio ambiente físico, así como la estructura de la muestra en las escalas de longitud de micras a centímetros. Esta sensibilidad ha dado lugar a una amplia aplicación de estas técnicas para caracterizar propiedades físicas, químicas y / o biológicas de materiales que van desde humanos a los alimentos a los medios de comunicación poroso 1, 2. El equipo y las condiciones utilizadas aquí son adecuadas para líquidos de formación de imágenes que contienen cantidades sustanciales de RMN 1 H móviles tales como agua ordinaria y líquidos orgánicos incluyendo aceites. Tradicionalmente MRI ha utilizado súper imanes conductores que no son adecuados para ambientes industriales y no portátil dentro de un laboratorio (Fig. 2). Los recientes avances in la tecnología de imanes han permitido la construcción de grandes volúmenes imanes industrialmente compatibles adecuados para flujos de proceso de imagen. Aquí, MRI proporciona espacialmente resueltas concentraciones de los componentes en diferentes posiciones axiales durante el proceso de mezcla. Este documento de trabajo en tiempo real de la mezcla de fluidos altamente viscosos vía distributiva mezcla con una aplicación para productos de cuidado personal.

Watch this Scientific Journal Video about Polymer Microarrays for High Throughput Discovery of Biomaterials at JoVE.com

Polymer Microarrays for High Throughput Discovery of Biomaterials

Una descripción de la formación de un microarray de polímero utilizando una técnica de fotopolimerización en el chip. La caracterización rendimiento de alta superficie mediante microscopía de fuerza atómica, las mediciones de ángulo de contacto del agua, de rayos X y espectroscopía de fotoelectrones de tiempo de vuelo espectrometría de masa iónica secundaria y un ensayo de adhesión celular también se describe.

Microarrays de polímeros de alto rendimiento para el descubrimiento de Biomateriales

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

polymer-microarrays-for-high-throughput-discovery-of-biomaterials

Research

JoVE Journal

Bioengineering

14.4K vistas.  University of Nottingham. Una descripción de la formación de un microarray de polímero utilizando una técnica de fotopolimerización en el chip. La caracterización rendimiento de alta superficie mediante microscopía de fuerza atómica, las mediciones de ángulo de contacto del agua, de rayos X y espectroscopía de fotoelectrones de tiempo de vuelo espectrometría de masa iónica secundaria y un ensayo de adhesión celular también se describe.

Video: Polymer Microarrays for High Throughput Discovery of Biomaterials

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida albicans biofilm Chip (Ca BChip) para la selección de alto rendimiento antifúngico

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Investigación

Bioingeniería

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

Síntesis combinatoria y de alto rendimiento liberación de proteínas de película de polímero y nanopartículas Bibliotecas

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

De alto rendimiento de síntesis de carbohidratos y funcionalización de nanopartículas polianhidrido

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

Evaluación de Biomateriales para el aumento de la vejiga usando análisis cistométricos en modelos de roedores Varios

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Una piel llena de defectos modelo para evaluar la vascularización de Biomateriales<em&gt; En Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

A Coupled Experiment-finite Element Modeling Methodology for Assessing High Strain Rate Mechanical Response of Soft Biomaterials

This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g. brain, liver, tendon, fat, etc.) when exposed to high strain rates. This study utilized a Split-Hopkinson Pressure Bar (SHPB) to generate strain rates of 100-1,500 sec-1. The SHPB employed a striker bar consisting of a viscoelastic material (polycarbonate). A sample of the biomaterial was obtained shortly postmortem and prepared for SHPB testing. The specimen was interposed between the incident and transmitted bars, and the pneumatic components of the SHPB were activated to drive the striker bar toward the incident bar. The resulting impact generated a compressive stress wave (i.e. incident wave) that traveled through the incident bar. When the compressive stress wave reached the end of the incident bar, a portion continued forward through the sample and transmitted bar (i.e. transmitted wave) while another portion reversed through the incident bar as a tensile wave (i.e. reflected wave). These waves were measured using strain gages mounted on the incident and transmitted bars. The true stress-strain behavior of the sample was determined from equations based on wave propagation and dynamic force equilibrium. The experimental stress-strain response was three dimensional in nature because the specimen bulged. As such, the hydrostatic stress (first invariant) was used to generate the stress-strain response. In order to extract the uniaxial (one-dimensional) mechanical response of the tissue, an iterative coupled optimization was performed using experimental results and Finite Element Analysis (FEA), which contained an Internal State Variable (ISV) material model used for the tissue. The ISV material model used in the FE simulations of the experimental setup was iteratively calibrated (i.e. optimized) to the experimental data such that the experiment and FEA strain gage values and first invariant of stresses were in good agreement.

The current study prescribes a coupled experiment-finite element simulation methodology to obtain the uniaxial dynamic mechanical response of soft biomaterials (brain, liver, tendon, fat, etc.). The multiaxial experimental results that arose because of specimen bulging obtained from Split-Hopkinson Pressure Bar testing were rendered to a uniaxial true stress-strain behavior when simulated through iterative optimization of the finite element analysis of the biomaterial.

Un elemento de modelado Metodología Junto Experimento-finito para la evaluación de alta Strain Tasa de respuesta mecánica de Biomateriales Soft

This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g. ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Usando Tomoauto: Un protocolo para la de alto rendimiento automatizado tomografía crioelectrónica

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

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

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

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

Una plataforma microfluídica para el aislamiento de gran procesamiento de celda única y Cultura

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

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 contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

De alto rendimiento de identificación de bacterias polímeros repelentes para dispositivos médicos

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

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.