Questa ricerca è stata sostenuta da concedere R01NS062842 dal National Institute of Neurological Disorder and Stroke, National Institutes of Health (VVD) e dalle concessioni R21 NS064403 dal National Institute of Neurological Disorder and Stroke, National Institutes of Health con ARRA (VVD) e R21 EB006301 National Institute of Biomedical Imaging e Bioingegneria, National Institutes of Health (VVD).

Le sezioni per la macchina molecolare basata rilevamento deve essere preparato prima perché la loro preparazione richiede più tempo rispetto l&#39;assemblaggio del dispositivo molecolare. Il costrutto funziona bene con 5-6μm dello spessore di sezioni tagliate da paraformaldeide-fisso, inclusi in paraffina blocchi di tessuto. Utilizzare i marchi scivolo che mantengono sezioni e, come ProbeOn Inoltre diapositive carica e predepurata (Fisher Scientific) o simili. Si consiglia in un primo momento con un tessuto con un noto modello di danno al DNA che contiene sia I-II e DNasi DNasi tipo pause, come ad esempio trattati con desametasone ratto apoptotici timo 13,14. 1. La preparazione delle sezioni <ol><li> Posizionare le sezioni in un rack di diapositive e sparaffinatura in xilene per 15 minuti, trasferire in un bagno di xilene fresco per altri 5 minuti. </li><li> Reidratare passando attraverso le concentrazioni di etanolo classificato: 96% etanolo 2x5min; Etanolo 80% - 5min; acqua - min 2x5. </li><li> Digest sezione con Proteinase K. L&#39;uso 100μL di una soluzione 50μg/mL per sezione. Incubare 15 &#39;a temperatura ambiente (23 ° C) in una camera umidificata. Il tempo può essere necessario regolare a seconda del tipo di tessuto. Tessuti duri potrebbe richiedere più la digestione. Tempi di 15-25 minuti di solito vengono utilizzati. Digestione insufficiente può comportare il segnale più debole. Overdigestion sui risultati d&#39;altra parte nella scomparsa del segnale e la rottura sezione. </li><li> Sciacquare con acqua distillata per min 2x10. </li><li> Applicare 100 ml di sezione per il 2% BSA per preblocking. Incubare per 15 minuti a temperatura ambiente (23 ° C). Durante questo periodo assemblare la macchina molecolare. </li></ol> 2. Macchine per l&#39;assemblaggio molecolare Tutti i reagenti sono scalati per 25 microlitri di volume totale, che è sufficiente per un rilevamento singolo in una sezione di tessuto media dimensione (10x10mm). Il volume può essere aumentato se necessario. <ol><li> Combina in un piccolo tubo di plastica in questo ordine: </li></ol><table border="1"><tbody> &lt;tr&gt; <td><ol><li> Acqua bidistillata; </li><li> Polietilene glicole-8000 al 15%; </li><li> Soluzione di 66 mM Tris-HCl, pH 7,5, 5 mM MgCl 2, 0.1 mM dithioerythritol, 1 mM ATP, (cioè normale tampone T4 DNA ligasi); </li><li> 70 pmoli di Oligonucleotidi 1, </li><li> 215 pmoli (1,76-7,1 mg) VACC TOPO </li><li> 10 unità T4 DNA ligasi (500 U / mL) </li></ol></td></tr></tbody></table><ol start="2"><li> Mescolare delicatamente pipettaggio. Il costrutto molecolare quasi istantaneamente auto-assembla in questa soluzione e può essere utilizzato immediatamente a temperatura ambiente (23 ° C). L&#39;aumento della temperatura a 37 ° C blocca la T4 DNA ligasi componente basato su delle etichette. </li></ol> 3. L&#39;utilizzo di macchine molecolari in sezioni di tessuto per 5&#39;OH etichetta dual e 5&#39;PO4 rotture del DNA <ol><li> Aspirare la soluzione preblocking e applicare 25μL della miscela di reazione completo contenente 70 pmoli di Oligonucleotidi 1, 53-215 pmoli (1,76-7,1 mg) VACCTOPO e 10 unità T4 DNA ligasi (500 U / mL) in soluzione di 66 mM Tris-HCl, pH 7,5, 5mm MgCl 2, 0.1 mM dithioerythritol, 1 mM ATP, e il 15% di polietilene glicole-8000. L&#39;oligonucleotide stessa sequenza portando una sola etichetta FITC, oligonucleotidi 2, può essere usato al posto per il rilevamento di un singolo tipo di rotture del DNA. In tal caso omettere ligasi dalla reazione di marcatura. Anche in questa situazione una soluzione di 50 mM Tris-HCL, pH 7.4, 15% PEG-8000 può essere usato al posto di buffer di T4 DNA ligasi. </li><li> Incubare per 1 ora a temperatura ambiente (23 ° C) in una camera umidificata con un coprioggetto di plastica. Riparo dalla luce. Abbassamento della temperatura a 16 ° C riduce la ligasi a base di segnale, l&#39;aumento della temperatura a 37 ° C elimina completamente la ligasi-based del segnale. Un parziale inibizione di ligasi si verifica a volte nella miscela di reazione, forse a causa di contaminanti introdotti con preparazioni topoisomerasi. Pertanto, più di incubazione (2-4 ore) potrebbe essere necessario soprattutto quando snumeri ignificant di ligasi marcato break sono presenti. Sebbene entrambi i segnali e ligasi topo possono essere osservati in questa fase, in molti casi il segnale ligasi può essere ulteriormente migliorato ri-applicazione della miscela di reazione senza VACC TOPO e dual-etichettati oligonucleotidi, ma contenente un oligonucleotide a forma di gomito (oligonucleotidi 3) (35 mg / mL) e T4 DNA ligasi (250 U / mL). Per migliorare il segnale passare alla Fase 3, per vedere la reazione senza mezzo di passare al punto 5. </li><li> Togliere delicatamente coprioggetto immergendo i vetrini verticalmente in una vaschetta Coplin contenente acqua a temperatura ambiente. Aspirare l&#39;acqua in eccesso. </li><li> Potenziare segnale ligasi applicando 25 l di miscela di reazione senza VACC TOPO e oligonucleotidi 1, ma contenenti oligonucleotidi 3: 66 mM Tris-HCl, pH 7,5, 5 mM MgCl 2, 0.1 mM dithioerythritol, 1 mM ATP, 15% e il polietilene glicole 8000, 5 unità T4 DNA ligasi, 35 mg / mL Oligonucleotide 3 (a gomito smussato terminato). Il volume totale della soluzione di etichettatura can essere scalata fino a ospitare le sezioni più grandi. Incubare per 18 ore (una notte) a temperatura ambiente (23 ° C) in una camera umidificata con un coprioggetto di plastica. </li><li> Togliere delicatamente coprioggetto immergendo i vetrini verticalmente in vaschetta Coplin contenente acqua a temperatura ambiente. Quindi lavare min 3x10 sezione in acqua distillata. </li><li> Sciacquare con tampone bicarbonato di sodio. Soluzione alcalina lavare migliora FITC a fluorescenza, che è sensibile al pH e si riduce notevolmente al di sotto di pH 7. </li><li> Profilo di copertura con una soluzione antifading (Vectashield con DAPI), coprioggetto e analizzare il segnale utilizzando un microscopio a fluorescenza. Rotture del doppio filamento di DNA con 5&#39;OH sarà fluorescenza verde, 5&#39;PO 4 break - si fluorescenza rossa (Figura 2). </li></ol> 4. Rappresentante Risultati <img alt="Figura 2." src="/files/ftp_upload/3628/3628fig2.jpg" /> Figura 1. Semi-artificiale macchina molecolare individua due tipi diDanno al DNA in situ. La macchina fluorescente auto-assembla quando VACC TOPO si lega al doppio tornante 32-mer. La macchina inizia a funzionare dividendo stesso in due unità rilevatore di via topoisomerasi taglio a misura alla fine del 3 &#39;della sequenza di riconoscimento. Ciò si traduce in un processo ciclico in cui marcata con FITC unità separa in modo continuo e religates nuovo al rodamina marcato unità. Questo fino a quando persiste una rottura del DNA rilevabile si incontra. Quando ad esempio un accettore alternativo (5&#39;OH smussato rottura del DNA fine) è presente nella sezione di tessuto, la parte FITC sarà legare ad essa. La parte restante rodamina attribuirà a 5 &#39;PO 4 break finale del DNA smussato con l&#39;aiuto di DNA ligasi T4. Di conseguenza, entrambi i tipi di rotture del DNA sono rilevati simultaneamente. <img alt="Figura 1." src="/files/ftp_upload/3628/3628fig1.jpg" /> Figura 2. Etichette macchina molecolare doppio due tipi di rotture del DNAin una sezione di tessuto di desametasone trattati con timo. Blunt-ended rotture del DNA di DNasi I e II DNasi tipo vengono rilevati nelle aree corticali timici in fase di apoptosi. Fluorescenza verde citoplasmatica (5&#39;OH rotture del DNA), segna macrofagi corticali digerire materiale nucleare di timociti apoptotici. Questo segnale è prodotto da VACC TOPO, e localizza a phagolysosomes con il DNA contenente 5&#39;OH rotture del doppio filamento 11,12. Fluorescenza rossa (5&#39;PO 4 pause) nuclei etichette dei timociti apoptotici non inghiottito da parte dei macrofagi. Un massiccio numero di timociti contemporaneamente apoptosi accompagnata dalla generazione di 5&#39;PO 4 rotture del doppio filamento, visualizzati da ligasi a base di etichettatura. Queste interruzioni sono situati alla periferia nucleare, formando l&#39;anello a forma di modelli. Tutti i nuclei cellulari sono visualizzati da controcolorazione con DAPI (blu fluorescenza).

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Nessun conflitto di interessi dichiarati.

In questo video mostriamo come assemblare e utilizzare un dual-etichettatura DNA sensore danni. Il sensore è una macchina molecolare guidata da bio-molecolari motore, un virus-encoded TOPO VACC proteina legata con componenti artificiali. Lo sviluppo ha presentato l&#39;esempio di un bio-enabled approccio che promuove l&#39;adattamento delle strutture biologiche, architetture e componenti effettivi e dei componenti delle cellule alla progettazione di atossici dispositivi su scala molecolare 12,15. Questo appr...

<ol><li> Xilene </li><li> 80 e 96% etanolo. </li><li> Soluzione al 2% di albumina di siero bovino (BSA) in acqua distillata. </li><li> Oligonucleotide 1: un&#39;arma a doppio tornante, doppio marcato con fluoresceina e tetrametilrodamina: </li></ol> 5&#39;-GGA AAG CCT GC F GCA GGT CCC TTA ACG R CAT IN GCG TT-3 &#39;; F - FITC-dT; R - tetrametilrodamina-dT. Altri rossi spostato fluorofori come BODIPY TR, rodamina o TAMRA può essere usato al posto di tetrametilrodamina come R. In alternativa puoi usare oligonucleotidi 2 -. Un&#39;arma a doppio tornante singolo marcato con fluoresceina (per il rilevamento di un singolo tipo di rotture del DNA (DNasi II-tipo solo) Il single-fluoroforo che trasporta la sonda è notevolmente meno costoso ed è comodo ogni volta che un singolo tipo di interruzione di DNA viene etichettato: 5&#39;-GGA AAG CCT GC F GCA TTA ACG GGT CCC ATG CGT CAT T-3 &#39;; F - FITC-dT </p&gt; <ol start="5"><li> Oligonucleotide 3. Blunt-ended rodamina marcato tornante per la valorizzazione del segnale legatura in situ con DNA ligasi T4: 5&#39;-GCG CTA GAC C R G GTC TAG CGC-3 &#39;; R - tetrametilrodamina-dT </li><li> Vaccinia DNA topoisomerasi l (VACC TOPO) - 3000 U / mL (Tecnologie Vivace). Negli esperimenti iniziali abbiamo usato 215 pmoli (7,1 mcg) del TOPO VACC per ogni microlitri 25 della miscela di reazione. Tuttavia, la concentrazione topoisomerasi può essere significativamente ridotto senza la perdita di sensibilità. Abbiamo poi usato una quantità quattro volte minore di VACC TOPO per sezione (1,76 mg in 25 ml di miscela di reazione per sezione) con risultati simili. Ridurre la quantità di enzima a 880 ng (in 25 microlitri della miscela di reazione) ha determinato un segnale più debole e 8,8 ng di VACC TOPO non ha prodotto alcun segnale rilevabile. </li><li> DNA ligasi T4 5 U / mL (Roche). Questa preparazione ligasi altamente concentrati dà il meglio di segnale nelle nostre condizioni sperimentali. </li&gt; <li> 10 x tampone di reazione per la DNA ligasi T4: 660 mM Tris-HCl, 50 mM MgCl 2, 10 dithioerythritol mm, 10 mm ATP, pH 7.5 (20 ° C) (Roche). ATP in tampone di reazione è facilmente distrutto in cicli ripetuti di congelamento-scongelamento. Aliquota del buffer in piccole porzioni 15-20 microlitri e conservare a - 20 ° C. Utilizzare una sola volta. </li><li> 30% (w / v) di PEG-8000 (Sigma) in acqua bidistillata. 15% PEG-8000 nella miscela di reazione stimola fortemente la rilevazione, aumentando la concentrazione efficace dei suoi componenti per esclusione del volume. </li><li> Proteinasi K (Roche) 20 mg / ml soluzione madre in acqua distillata. Conservare a - 20 ° C. Nella soluzione uso reazione 50mg/ml in PBS, prepara il. Non riutilizzare. </li><li> Vectashield con DAPI (Vector Laboratories). </li><li> Tampone bicarbonato di sodio: 50m M NaHCO 3, 15m M di NaCl, pH 8.2. </li><li> Vetro 22x22mm o 22x40mm o lamelle di plastica. Coprioggetto plastica sono preferibili durante la reazione, in quanto unore più facile da rimuovere dalla sezione. </li></ol>

in vitro assembly of semi-artificial molecular machine and its use for detection of dna damage

Naturale bio-molecolari macchine lavorano in ogni cellula vivente e visualizzare una varietà di disegni 1-6. Eppure lo sviluppo di macchine molecolari artificiali centri su dispositivi in grado di movimento direzionale, cioè motori molecolari, e sulla loro parti meccaniche in scala ridotta (ruote, assali, ecc pendenti) 7-9. Questo imita la macro-macchine, anche se le proprietà fisiche fondamentali per questi dispositivi, come ad esempio inerzia e conservazione del momento, non sono utilizzabili in ambienti nanomondo 10. Progetti alternativi, che non seguono gli schemi meccanici macromachines e utilizzare meccanismi sviluppati per l&#39;evoluzione delle molecole biologiche, possono usufruire delle condizioni specifiche del nanomondo. Inoltre, adattando reale molecole biologiche ai fini della nano-design riduce i pericoli potenziali dei prodotti delle nanotecnologie potrebbero comportare. Qui mostriamo il montaggio e l&#39;applicazione di uno di questi bio-enabled costruire, comeemi-artificiale dispositivo molecolare che combina una macchina in natura molecolare con componenti artificiali. Dal punto di vista enzimologia, il nostro costruire è una fluorescente progettista enzima-substrato complesso mettere insieme per svolgere una specifica funzione utile. Questa assemblea è per definizione una macchina molecolare, in quanto contiene un 12. Eppure, la sua integrazione con le progettato parte - fluorescenti a doppio tornante - ha re-indirizza a una nuova attività di etichettatura danno al DNA 12. Il nostro costruire assembla da un 32-mer DNA e un enzima vaccino topoisomerasi I (VACC TOPO). La macchina utilizza poi il proprio materiale per fabbricare due unità rivelatore fluorescente (Figura 1). Una delle unità (fluorescenza verde) porta VACC TOPO covalentemente legata al suo 3&#39;end e un&#39;altra unità (fluorescenza rossa) è un tornante libero con un 3&#39;OH terminale. Le unità sono di breve durata e rimontare rapidamente nuovamente dentro il costrutto originale, che recleaves successivamente.In assenza di rotture del DNA queste due unità separate e in continuo religate in modo ciclico. In sezioni di tessuto con danno al DNA, la topoisomerasi che trasportano unità di rilevatore si attacca selettivamente a blunt-ended con 5&#39;OH rotture del DNA (DNasi II di tipo break) 11,12, fluorescente loro etichettatura. Il secondo, enzima senza tornante formata a seguito della scissione oligonucleotidi, sarà legare ad un 5&#39;PO 4 smussato-ended break (DNasi I di tipo break) 11,12, se il DNA ligasi T4 è presente nella soluzione 13,14. Quando il DNA ligasi T4 viene aggiunto a una sezione di tessuto o di una soluzione contenente DNA con 5&#39;PO 4 blunt-ended pause, la ligasi reagisce con 5&#39;PO 4 estremità del DNA, formando semi-stabile enzima-DNA complessi. I tornanti smussato conclusa potranno interagire con questi complessi rilasciando tornanti ligasi e il collegamento covalente al DNA, così etichettatura 5&#39;PO 4 blunt-ended rotture del DNA. Questo sviluppo esemplifica un nuovo approccio pratico °progettazione e di macchine molecolari e fornisce un utile sensore per il rilevamento di apoptosi e danno al DNA nelle cellule e nei tessuti fissi.

Watch this Scientific Journal Video about In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage at JoVE.com

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Dimostriamo il montaggio e l&#39;applicazione di una scala molecolare dispositivo alimentato da una proteina topoisomerasi. Il costrutto è un bio-sensore molecolare che le etichette due tipi principali di rotture del DNA nelle sezioni di tessuto allegando due fluorofori diversi per i loro fini.

In Assemblea in vitro di semi-artificiale della macchina molecolare e il suo utilizzo per il rilevamento del danno al DNA

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

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Bioengineering

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

11.5K Views.  Baylor College of Medicine. Dimostriamo il montaggio e l'applicazione di una scala molecolare dispositivo alimentato da una proteina topoisomerasi. Il costrutto è un bio-sensore molecolare che le etichette due tipi principali di rotture del DNA nelle sezioni di tessuto allegando due fluorofori diversi per i loro fini.

Video: In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

Fabbricazione di elettrochimica-DNA biosensori per il rilevamento Reagentless di acidi nucleici, proteine ​​e piccole molecole

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

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Bioingegneria

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

CometChip: A High-throughput di 96-Well Piattaforma per la misurazione dei danni cagionati DNA in cellule umane Microarrayed

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

L&#39;uso di un high-throughput<em&gt; In Vitro</em&gt; Microfluidic sistema per sviluppare orale biofilm Multi-specie

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Oculare Test (EIT) per identificazione del pericolo di sostanze chimiche irritanti oculari utilizzando Ricostruito umana Cornea come epiteliale (RHCE) Tessuto Modello

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning e montaggio dei microvasi 3D

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Automatizzato robot Liquid Handling Assembly di dispositivi modulari del DNA

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Sintesi di cationizzata Magnetoferritin per la magnetizzazione ultra-veloce di celle

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation.

Costruzione e l'uso di una camera di stimolazione elettrica per migliorare la differenziazione osteogenica in cellule mesenchimali staminali/Stromal In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

Microarray di bioparticelle per l'analisi chemiotattica e molecolare dello sciame neutrofilo umano in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Modelli avanzati di fegato 3D per test di genotossicità in vitro a seguito dell'esposizione a lungo termine ai nanomateriali

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...