Martin Danner è riconosciuto per la sua assistenza nella preparazione di campioni e istituzione di procedure di coltura delle cellule. Gli autori vorrei ringraziare Biesterfeld Spezialchemie GmbH (Amburgo, Germania), soprattutto Robert Radsziwill per il continuo supporto e discussioni. La ricerca che porta a questi risultati ha ricevuto finanziamenti dal Consiglio europeo della ricerca nell'ambito settimo programma quadro (FP/2007-2013) convenzione di sovvenzione CER n. dell'Unione europea 340929.

<ol>
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La progettazione di strutture in composito consente la semplice regolazione delle proprietà dei materiali, come il modulo di Young o lo spessore dei campioni. Il modulo di Young di PDMS può essere cambiato in modo efficace in una vasta gamma alterando il rapporto di miscelazione tra i due componenti o fabbricazione di miscele utilizzando un diverso in silicone elastomero30,31. I metodi descritti non sono limitati al PDMS utilizzato nella presente inchiesta, ma soprattutto la prestazione adesiva dipende fortemente il tipo specifico utilizzato. Un passaggio fondamentale nell'ambito di questo protocollo è il processo di produzione dei film composito (Figura 1). È stato dimostrato che lo spessore dei film incide considerevolmente il comportamento di adesione dei film su diversi substrati, tra cui la pelle (Figura 5 e Figura 6). Oltre lo spessore del film, ora e temperatura durante il processo di polimerizzazione riguarda la proprietà del materiale32. Pertanto, parametri come lo spessore degli strati polimerici devono essere attentamente adattato e verificato.
Analisi delle proprietà adesive delle pellicole sottili è stata eseguita con misure di adesione di forza normale utilizzando due substrati di vetro con diverse rugosità fino a Ra = 0.338 µm (Figura 3). In generale, la rugosità influisce significativamente l'adesione delle superfici, soprattutto di materiali elastici33,34. La rugosità di vetro può essere facilmente variata di levigatura con carta vetrata di dimensioni diverse asperità, consentendo quindi la realizzazione di substrati esibendo i più alti valori di rugosità21. In aggiunta, altri materiali, ad esempio resina epossidica può essere utilizzata per la produzione di substrati15,35. Questo potrebbe essere una strategia importante modifica del protocollo presentato. Ad esempio, se substrati che esibiscono diverse superficie libera energie sono necessari o specifiche Topografie sono necessarie. Qui, pull-off stress e lavoro di separazione delle pellicole sottili prodotte di PDMS e SSA sono stati analizzati con un programma di installazione su misura (dispositivo di misurazione macroscopica adesione (MAD, Figura 4)). 36 l'allineamento ottico del substrato e penetratore è un passo fondamentale per l'analisi dei risultati delle misurazioni. Di conseguenza, regolazione dell'angolo di inclinazione deve essere eseguita con il goniometro, più preciso possibile. Ciò può essere ottenuto con sufficiente precisione manualmente portando il substrato a contatto con la superficie della pellicola fino a quando si ottiene un contatto orizzontale.
Nel protocollo attuale il tempo di attesa è stato mantenuto costante a un secondo (Figura 5 e Figura 7). Soprattutto per la ricerca delle prestazioni adesiva di un film elastico per una superficie di substrato grezzo, un'estensione del tempo di attesa fornisce informazioni aggiuntive. Ad esempio, un aumento nello sforzo di strappo con l'aumento del tempo di attesa è stato segnalato8. Oltre le misure eseguite nel protocollo attuale, altri metodi, ad esempio peel test potrebbe essere effettuato, permettendo una più vasta inchiesta di adesione prestazioni37.
Le proprietà adesive del film compositi che esibiscono differenti della pellicola sono stati determinati spessori dell'adesivo pelle morbida (Figura 7). I nostri risultati sono in linea con i dati pubblicati, che con un decremento di piombo di spessore di pellicola ad un aumento dello stress pull-off come il confinamento, cioè, il rapporto tra diametro e pellicola spessore del substrato, aumenti38,39 . Basato su questi risultati e i dati rappresentati in Figura 7, concludiamo che film compositi con uno spessore totale di circa 100 µm (spessore dello strato SSA di circa 60 µm applicato ad una pellicola PDMS con uno spessore di circa 40 µm) esibiscono favorevole adesione p roprietà su superfici ruvide.
Successivamente, sul film compositi incontaminate sono stati eseguiti esperimenti relazionati alla caratterizzazione biologica e trattate al plasma film compositi (Figura 8). Trattamento al plasma di elastomeri di silicone è una tecnica versatile, spesso applicata per aumentare le proprietà idrofile di superfici e promuovere attaccamento cellulare e cellulare diffusione40,41. Siliconi sono ben noti per la loro bassa tossicità e biostabilità alta ma possono contenere di monomeri residui o catalizzatori in grado di influenzare processi fisiologici, che conduce anche a citotossicità42,43. Nell'esperimenti condotti abbiamo osservato a meno di 5% citotossicità con rilascio di LDH come un indicatore e un test di esclusione del Trypan Blue. Nel protocollo presentato, l'intera popolazione cellulare, tra cui aggregati cellulari indipendente forma che la superficie è stata analizzata per analisi di proliferazione (figura 9B). Una modifica del protocollo potrebbe produrre risultati più differenziati. Per ciascun campione, il supernatante contenente aggregati cellulari indipendenti potrebbe essere trasferito in una provetta di reazione separata e non combinato con le cellule enzimaticamente rimosse dalla superficie del polimero. Ciò consente l'esatta determinazione delle cellule attaccate alla superficie e alla fine rivelano una più dettagliata valutazione dell'influenza dei polimeri sul processo di adesione cellulare. Oltre ai metodi di immunocytochemical ha presentati qui, le cellule potrebbero essere raccolte per indagine con metodi di immunoblot, permettendo una valutazione quantitativa dettagliata dell'espressione della proteina.
In sintesi, abbiamo stabilito le condizioni di produzione per la produzione di film sottili di compositi elastomerici per applicazioni in ricerca della coltura avanzata delle cellule. Inoltre, questi film sottili possiedono alta adattabilità alla pelle rugosità, consentendo il sofisticato design degli adesivi di pelle.

In questo protocollo, sono presentati metodi dettagliati per la produzione di film sottile elastomero. La tecnica di lama medico ampiamente disponibile è stata utilizzata per la produzione di film sottili di composito. La tecnica di fabbricazione è stata effettuata su fogli di polietilentereftalato (PET), che consente la produzione successiva di questi film in larga scala. L'enfasi di questo protocollo è la valutazione della riproducibilità, precisa produzione di diversi strati di film compositi e determinazione delle proprietà biologiche e adesione della patch composito finale. Il poly-(dimethylsiloxane) di elastomero di silicone (PDMS) è ampiamente utilizzato nella tecnologia biomedica, compresa la produzione di pelle adesivi, applicazioni di microfluidica e ulteriori ricerche campi1,2,3 ,4. Recentemente, un'altra sottoclasse di PDMS, i cosiddetti Soft pelle adesivi (SSAs) sono stati introdotti, in particolare per la pelle delicata di legame e de-bonding.
SSAs in silicone sono elastomeri vinile funzionalizzato, differiscono dai polimeri analoghe dall'assenza di silice5di rinforzo. Simile a altri PDMS, SSA modulo di Young può essere adattato in una vasta gamma di concentrazione cross-linker modulante o polimerizzazione tempo6,7,8. Questo cambiamento di modulo di Young di elastomeri di silicone influisce in modo significativo le proprietà adesive del materiale e ha anche profonde conseguenze sulle cellule procariote ed eucariote coltivate sulla superficie9,10 , 11. il livello biologico cellulare, è stato dimostrato, che le cellule eucariotiche rispondono sul livello di trasduzione del segnale per una modulazione dell'elasticità matrice o spessore della superficie9,10,12 ,13,14. Di conseguenza, esiste un vasto interesse nelle applicazioni della coltura delle cellule di polimeri con proprietà meccaniche sintonizzabile. D'importanza, l'intrinsecamente bassa energia superficiale degli elastomeri a base di silicone non fornisce le condizioni ottimali per la coltura cellulare delle cellule eucariotiche. Trattamento al plasma di ossigeno è una tecnica ampiamente utilizzata per aumentare PDMS bassa energia superficiale temporaneamente, che porta ad un potenziamento della sua forza di strappo, diminuita superficie adsorbimento di molecole, mentre in parallelo promuovere attaccamento, diffusione e proliferazione di cellule eucariotiche15,16,17,18.
Oltre alle proprietà dei materiali, la topografia di superficie influenza in modo significativo di adesione cellulare e l'interazione adesivo tra due materiali19,20,21,22. Rugosità superficiale ha parecchi effetti sulla formazione contatto tra due superfici: riduzione della zona di contatto, alta memorizzati energia elastica che circonda asperità così come influenza sulla propagazione di crepa può alterare la resistenza adesiva23, 24. Adesione di pellicole autoadesive per la pelle umana è un settore emergente di applicazione, ad esempio, medicazioni, fissazione di elettrodi ECG o altri dispositivi elettronici indossabili25,26,27, 28. Per misurare la prestazione adesiva di autoadesivi in relazione alla topografia superficiale, substrati di vetro con diversi gradi di rugosità possono essere utilizzati nella normale adesione misure8,21. Qui, i due substrati di vetro sono stati selezionati per indagare le proprietà adesive delle pellicole polimeriche. Composito, primo film con un strato di rinforzo PDMS in un rapporto di 10 a 1 peso parti coperte da PDMS con diverso rapporto di miscelazione miscelazione sono state caratterizzate. In una seconda fase uno strato adesivo di SSA è stato preparato con una quantità di peso uguale di entrambi i componenti e con vario spessore di pellicola sopra un supporto film PDMS.

<table><tbody><tr><td>2-Propanol, 97%</td><td>Stockmeier Chemie</td><td>1000452610000</td><td>Isopropanol</td></tr><tr><td>Abrasive diamnod hand pad</td><td>Bohle</td><td>MO 5007522</td><td>Grit: 220</td></tr><tr><td>Accutase</td><td>Capricorn Scientific</td><td>ACC-1B</td><td></td></tr><tr><td>Albumin Fraktion V</td><td>Roth</td><td>0163.2</td><td>BSA</td></tr><tr><td>Alexa Fluor 488 Phalloidin</td><td>ThermoFischer Scientific</td><td>A12379</td><td>highly toxic</td></tr><tr><td>Aquamount</td><td>Polysciences</td><td>18606-20</td><td>water soluble mounting medium</td></tr><tr><td>CytoTox-ONE Homogeneous Membrane Integrity Assay</td><td>Promega</td><td>G7890</td><td></td></tr><tr><td>DPBS, without Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>ThermoFischer Scientific</td><td>14190094</td><td></td></tr><tr><td>Fetal bovine serum gold</td><td>GE Health Care Life Science</td><td>A15-151</td><td>FBS</td></tr><tr><td>Goniometer OCA35</td><td>Dataphysics</td><td></td><td>for the determination of the static water contact angle</td></tr><tr><td>Hoechst Dye 33342</td><td>Sigma-Aldrich</td><td>B1155-100MG</td><td>bisBenzimide H 33342 trihydrochloride, highly toxic</td></tr><tr><td>Microscope Axiovert 25</td><td>Zeiss</td><td></td><td>Microscope used for cell culture documentation</td></tr><tr><td>Microscope Eclipse LV100ND</td><td>Nikon</td><td></td><td>Microscope used for film thickness determination</td></tr><tr><td>Paraformaldehyde, aqueous solution 16%</td><td>Electron Microscopy Sciences</td><td>RT 15710</td><td>electron microscopy grade</td></tr><tr><td>penicillin und streptomycin solution</td><td>Sigma-Aldrich</td><td>P4333-100ML</td><td></td></tr><tr><td>Phenom XL Scanning Electron Microscope (SEM)</td><td>Phenom</td><td></td><td></td></tr><tr><td>Poly-(vinyl alcohol) 4-88, MW 31000</td><td>Sigma-Aldrich</td><td>81381-1KG</td><td>Mowiol 4-88</td></tr><tr><td>Poly-dimethyl siloxanes, Sylgard 184</td><td>Dow Corning</td><td>(400)000108351397</td><td>PDMS</td></tr><tr><td>RPMI 1640 basal medium</td><td>ThermoFischer Scientific</td><td>21875034</td><td></td></tr><tr><td>soft skin adhesive (SSA)</td><td>Dow Corning</td><td>(400)000108251792</td><td>MG 7-9800 Soft Skin Adhesive (SSA)</td></tr><tr><td>speed mixer DAC 600.2 VAC-P</td><td>Hauschild</td><td></td><td></td></tr><tr><td>stylus profilomter</td><td>Zeiss</td><td></td><td>Model: SURFCOM 1500SD3</td></tr><tr><td>Tecan Infinite M200 pro</td><td>Tecan</td><td></td><td>fluorescence plate reader</td></tr><tr><td>Triton X 100</td><td>Calbiochem</td><td>648466</td><td></td></tr><tr><td>Trypan Blue solution</td><td>Sigma-Aldrich</td><td>T8154-100ML</td><td>highly toxic</td></tr><tr><td>Trypsin/EDTA solution</td><td>PAN-Biotech</td><td>P10-023500</td><td>0.05% Trypsin, 0.02% EDTA in PBS</td></tr><tr><td>UV glue</td><td>Bohle</td><td>BO MV76002</td><td>medium viscosity</td></tr></tbody></table>

thin film composite silicon elastomers for cell culture and skin applications: manufacturing and characterization

In questo protocollo, presentiamo metodi per fabbricare film composito sottile in elastomero per applicazioni della coltura avanzata delle cellule e per lo sviluppo di adesivi di pelle. Due diversi poli-(dimethyl siloxanes) (PDMS e morbida pelle adesivo (SSA)), sono stati utilizzati per indagini approfondite di effetti biologici e caratteristiche adesive. Il film compositi sono costituiti da uno strato di supporto flessibile e un rivestimento superiore adesivo. Entrambi gli strati sono stati fabbricati dalla tecnica di applicazione lama medico. Nella ricerca attuale, il comportamento adesivo dei film composito è stato studiato come una funzione di spessore dello strato o una variazione del modulo di Young del livello superiore. Il modulo di Young di PDMS è stato modificato variando la base per il rapporto di miscelazione di reticolante. Inoltre, lo spessore del film di SSA è stato variato da circa 16 µm a circa 320 µm. scansione microscopia elettronica (SEM) e microscopia ottica sono stati utilizzati per misure di spessore. Le proprietà adesive del film elastomero dipendono fortemente lo spessore del film, il modulo di Young dei polimeri e le caratteristiche superficiali. Di conseguenza, è stato studiato normale adesione di questi film su substrati di vetro che esibiscono le superfici lisce e ruvide. Il rapporto di miscelazione di elastomeri di silicone dipendono strappo stress e lavoro di separazione.
Inoltre, lo spessore dell'adesivo pelle morbida posizionato sopra uno strato di appoggio solidale è stato variato per produrre le patch per le applicazioni della pelle. Citotossicità, proliferazione e adesione cellulare dei fibroblasti murini L929 su pellicole di PDMS (rapporto di miscelazione 10:1) e SSA (miscelazione rapporto 50: 50) sono stati condotti. Ci hanno mostrato qui, per la prima volta, il confronto fianco a fianco di sottili pellicole compositi prodotte di entrambi polimeri e presentare lo studio delle loro proprietà biologiche - e adesivo.

Watch this Scientific Journal Video about Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization at JoVE.com

Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization

Un protocollo per il processo di fabbricazione di strutture in composito polimerico pellicola sottile che possiede di diversi giovani moduli o spessori è presentato. Film sono prodotti per studi su colture cellulari avanzati o come adesivi di pelle.

Thin Film composito silicone elastomeri per colture cellulari e applicazioni della pelle: produzione e caratterizzazione

In this protocol, we present methods to fabricate thin elastomer composite films for advanced cell culture applications and for the development of skin ...

thin-film-composite-silicon-elastomers-for-cell-culture-skin

Research

JoVE Journal

Developmental Biology

10.6K Views.  INM - Leibniz Institute for New Materials. Un protocollo per il processo di fabbricazione di strutture in composito polimerico pellicola sottile che possiede di diversi giovani moduli o spessori è presentato. Film sono prodotti per studi su colture cellulari avanzati o come adesivi di pelle.

Video: Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

Piattaforme microfabbricazione per coltura cellulare meccanicamente dinamica

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Ricerca

Bioingegneria

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Diretto Cellular Self-Assembly di fabbricare Cell-Derived Anelli tessuto per l&#39;analisi biomeccanica e di ingegneria dei tessuti

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

Seta Film per la cultura del sistema In vitro Analisi e Progettazione Biomaterial

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

Silicon Microchips for Manipulating Cell-cell Interaction

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from development to malignant transformation.&nbsp; In particular, interaction with neighboring stroma has been implicated in a plethora of biological phenomena; however, conventional techniques limit the ability to interrogate the spatial and dynamic elements of such interactions.
In Micromechanical Reconfigurable Culture (RC), we employ a micromachined silicon substrate with moving parts to dynamically control cell-cell interactions through mechanical repositioning. Previously, this method has been applied to investigate intercellular communication in co-cultures of hepatocytes and non-parenchymal cells, demonstrating time-dependent interactions and a limited range for soluble signaling 1.
Here, we describe in detail the preparation and use of the RC system. We begin by demonstrating the handling of the device parts using tweezers, including actuating between the gap and contact configurations (cell populations separated by a narrow 80-&micro;m gap, or in direct intimate contact). Next, we detail the process of preparing the substrates for culture, and the multi-step cell seeding process required for obtaining confluent cell monolayers. Using live microscopy, we then illustrate real-time manipulation of cells between the different possible experimental configurations. Finally, we demonstrate the steps required in order to regenerate the device surface for reuse: toluene and piranha cleaning, polystyrene coating, and oxygen plasma treatment.

This article describes an experimental approach for dynamic regulation of cell-cell interactions between adherent cells on a micrometer scale. Manipulation of intercellular communication between hepatocytes and stromal cell is demonstrated. The developed platform enables investigation of cell-cell interactions in a variety of biological processes, including development and pathogenesis.

I microchip di silicio per la manipolazione di interazione cellula-cellula

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from ...

Biologia

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

Studio della Migrazione cellulare in canali microfabbricati

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Stretching celle micropatterned su una membrana PDMS

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of cutaneous wound healing on the skin. Existing methods, however, still rely on visual inspections through various microscopic tools and devices that normally include high-cost, sophisticated systems and require well trained personnel for operation and data analysis. Here, we describe methods and protocols to fabricate a conformal, skin-like electronics system that enables conformal lamination to the skin surface near the wound tissues, which provides recording of high fidelity electrical signals such as skin temperature and thermal conductivity. The methods of device fabrication provide details of step-by-step preparation of the microelectronic system that is completely enclosed with elastomeric silicone materials to offer electrical isolation. The experimental study presents multifunctional, biocompatible, waterproof, reusable, and flexible/stretchable characteristics of the device for clinical applications. Protocols of clinical testing provide an overview and sequential process of cleaning, testing setup, system operation, and data acquisition with the skin-like electronics, gently mounted on hypersensitive, cutaneous wound and contralateral tissues on patients.

This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management.

Fabbricazione e la caratterizzazione di un Conformal pelle simile sistema elettronico di gestione quantitativa, cutanea Ferita

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Sintesi di biocompatibili a cristalli liquidi in elastomero Schiume come impalcature cellulari per 3D spaziali colture cellulari

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

Coltura di cellule di idrogeli ultrasottile Innova elastico come una membrana dello scantinato biomimetici per Dual

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

Generazione di un modello skin-on-a-chip tridimensionale semplificato in una piattaforma microfluidica microlavorazione

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...