Microwell Arrays wurden hergestellt und charakterisiert im Zentrum für Nanophase Materials Sciences User Facilities Division, Office of Basic Energy Sciences, US Department of Energy. Die finanzielle Unterstützung für diese Arbeit wurde durch den Forschungs- und Entwicklungsfonds des Oak Ridge National Laboratory Director Die Autoren danken auch dem J. Mougous Laboratory (University of Washington, Seattle, WA) für die Lieferung von P. aeruginosa- Stämmen, die in diesen Studien verwendet wurden.

<ol>
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Dieser Artikel präsentierte ein microwell-Array-Gerät und experimentelle Protokolle entwickelt, um High-Throughput-und High-Content-Live-Zelle Imaging-basierte Analyse der bakteriellen Community-Entwicklung zu ermöglichen. Während der Schwerpunkt der Demonstration hier war, um die Effekte der kontaktvermittelten Typ-VI-Sekretion auf die Entwicklung der Gemeinschaft zu untersuchen, wurden die Arrays so konzipiert, dass sie flexibel sind und die Untersuchung einer breiten Palette von mikrobiellen Gemeinschaften und Mi...

Die mikrobiellen Gemeinschaften sind sowohl durch deterministische Faktoren wie die Struktur der Umwelt und stochastische Prozesse geprägt, die mit dem Zelltod, der Teilung, der Proteinkonzentration, der Anzahl der Organellen und der Mutation verbunden sind. Innerhalb der natürlichen Umgebung kann es fast unmöglich sein, die individuellen Auswirkungen dieser Einflüsse auf die Zusammensetzung und die Aktivität der Gemeinschaft zu analysieren. Von natürlichen Strukturen verdeckt und in einem chemischen und biologischen Milieu begraben, die Mitglieder der Gemeinschaft zu identifizieren und ihre räumlich-zeitliche Verteilung in der natürlichen Umgebung zu lösen, ist äußerst anspruchsvoll. Dennoch haben die jüngsten Bemühungen die Bedeutung der räumlichen Organisation auf die Gemeinschaftsfunktion unterstrichen und auf die Notwendigkeit hingewiesen, sowohl die Mitgliedshäufigkeit als auch die Organisation in den laufenden Studien 2 , 3 , 4 zu berücksichtigen. EsIst klar, dass die lokale chemische Umgebung ( dh die Verfügbarkeit von Nährstoffen und sekundären Metaboliten), die physikalische Struktur ( zB Bodenarchitektur, Pflanzenwurzeln, Ozeanpartikel oder die intestinalen Mikrovilli), die Anwesenheit oder Abwesenheit von Sauerstoff und die Einführung von Pathogene Arten alle beeinflussen die Zusammensetzung, die Architektur und die Funktion der mikrobiellen Gemeinschaften 5 , 6 , 7 , 8 , 9 , 10 , 11 . Nichtsdestotrotz dominieren traditionelle Techniken für Kulturen, die diese Faktoren nicht erfassen. Gemeinschaftszusammensetzung ( z. B. das Vorhandensein von Co-abhängigen Spezies), die physikalische Bindung, die Signalmolekülkonzentration und der direkte Zellzellkontakt sind alle wichtigen Faktoren für die Gestaltung einer mikrobiellen Gemeinschaft und können in c verloren gehenOnventionelle Kulturbedingungen. Diese Eigenschaften sind in einer Bulk-Flüssigkultur oder auf einer Agarplatte schwer zu replizieren. Die Verfügbarkeit von mikrofluidischen, Mikrostrukturen und Nanofabrikationstechniken, die die Replikation der wichtigsten physikalischen und chemischen Eigenschaften natürlicher Umgebungen ermöglichen, hat es vielen Forschern ermöglicht, bakterielle Gemeinschaften zu bauen, um ihre Wechselwirkungen 12 , 13 , 14 zu untersuchen und synthetische Umgebungen zu entwickeln Imitieren natürliche Bedingungen 4 , 15 , 16 , 17 , 18 , 19 , 20 . Dieses Protokoll beschreibt eine Methode zur Herstellung einer Mikrowellen-Array-Vorrichtung und liefert detaillierte experimentelle Verfahren, die verwendet werden können, um Th zu funktionalisierenE Wells in der Array und Bakterien zu wachsen, sowohl als Single-Spezies-Kolonien und in Multi-Mitgliedsgemeinschaften. Diese Arbeit zeigt auch, wie Bakterien modifiziert, um fluoreszierende Reporter-Proteine ​​zu produzieren, können verwendet werden, um das Bakterienwachstum in den Brunnen im Laufe der Zeit zu überwachen. Ein ähnliches Array wurde zuvor vorgestellt und zeigte, dass es möglich ist, das Wachstum von Einzelkolonien von Pseudomonas aeruginosa ( P. aeruginosa) in Mikrovertiefungen zu verfolgen. Durch die Modulation von Well-Size und Seeding-Dichte können die Ausgangsbedingungen von Tausenden von Wachstumsexperimenten parallel variiert werden, um zu bestimmen, wie die anfänglichen Inokulationsbedingungen die Fähigkeit der Bakterien beeinflussen, 21 zu wachsen. Die aktuelle Arbeit verwendet eine leicht modifizierte Version des microwell-Arrays, die auf der vorherigen Arbeit aufbaut, indem sie den gleichzeitigen Vergleich von mehreren Arrays ermöglicht und ein robusteres experimentelles Protokoll verwendet. Das Array, das in dieser Arbeit verwendet wird, enthält mehrere Subarrays oder Array ensemBles, die Brunnen unterschiedlicher Größe enthalten, die von 15 - 100 μm im Durchmesser reichen, die in drei verschiedenen Tonhöhen angeordnet sind ( dh 2x, 3x und 4x der Bohrlochdurchmesser). Die Arrays werden in Silizium geätzt, und das Wachstum der in den Silizium-Arrays ausgesetzten Bakterien wird durch Versiegeln der Arrays mit einem Deckglas ermöglicht, das mit einem Medium-infundierten Agarosegel beschichtet worden ist. P. aeruginosa- Mutanten, die entworfen wurden, um das Typ-VI-Sekretionssystem zu studieren, werden in dieser Demonstration verwendet. Die hier vorgestellten Ergebnisse bauen auf das ultimative Ziel, Multimember-Communities innerhalb von Mikrowellen-Arrays zu analysieren, so dass Forscher die Fülle und Organisation von Bakterien in situ überwachen können, während sie die chemische Umgebung kontrollieren und untersuchen. Dies sollte letztlich Einblicke in die &quot;Regeln&quot; geben, die die Gemeinschaftsentwicklung und die Nachfolge bestimmen.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parylene N</td>
			<td>Specialty Coating Systems</td>
			<td>CAS NO.:1633-22-3</td>
		</tr>
		<tr>
			<td>Parylene coater</td>
			<td>Specialty Coating Systems</td>
			<td>Labcoter 2 Parylene Deposition Unit PDS2010</td>
		</tr>
		<tr>
			<td>Silicon Wafer</td>
			<td>WRS Materials</td>
			<td>100mm diameter, 500-550&#956;m thickness, Prime, 10-20 resistivity, N/Phos&#60;100&#62;,</td>
		</tr>
		<tr>
			<td>adhesion promoter</td>
			<td>Shin-Etsu Microsci</td>
			<td>MicroPrime P20 adhesion promoter</td>
		</tr>
		<tr>
			<td>postive tone photoresist</td>
			<td>Rohm and Haas Electronics Materials LLC (Owned by Dow)</td>
			<td>Microposit S1818 Positive Photoresist (code 10018357)</td>
		</tr>
		<tr>
			<td>Quintel Contact Aligner</td>
			<td>Neutronix Quintel Corp</td>
			<td>NXQ 7500 Mask Aligner</td>
		</tr>
		<tr>
			<td>Reactive Ion Etching Tool</td>
			<td>Oxford Instruments</td>
			<td>Plasmalab System 100 Reactive Ion Etcher</td>
		</tr>
		<tr>
			<td>R2A Broth</td>
			<td>TEKnova</td>
			<td>R0005</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A9647</td>
		</tr>
		<tr>
			<td>Multimode Plate Reader</td>
			<td>Perkin Elmer</td>
			<td>Enspire, 2300-0000</td>
		</tr>
		<tr>
			<td>Fluorescent Microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti-U</td>
		</tr>
		<tr>
			<td>Automated Stage</td>
			<td>Prior</td>
			<td>ProScan III</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Nikon</td>
			<td>DS-QiMc</td>
		</tr>
		<tr>
			<td>Stage-top environmental control chamber</td>
			<td>In Vivo Scientific</td>
			<td>STEV ECU-HOC</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>ThermoFisher Scientific</td>
			<td>14190144</td>
		</tr>
		<tr>
			<td>UltraPure Agarose</td>
			<td>ThermoFisher Scientific</td>
			<td>16500500</td>
		</tr>
		<tr>
			<td>25 x 75 mm No. 1.5 coverslip</td>
			<td>Nexterion</td>
			<td>High performance #1.5H coverslips</td>
		</tr>
		<tr>
			<td>Fluorescence Reference Slides</td>
			<td>Ted Pella</td>
			<td>2273</td>
		</tr>
		<tr>
			<td>Physical Stylus Profilometer</td>
			<td>KLA Tencor</td>
			<td>P-6</td>
		</tr>
		<tr>
			<td>lab wipes</td>
			<td>Kimberly Clark</td>
			<td>Kimipe KIMTECH SCIENCE Brand, 34155</td>
		</tr>
		<tr>
			<td>commercial software</td>
			<td>Nikon</td>
			<td>NIS Elements</td>
		</tr>
		<tr>
			<td>Zeiss 710 Confocal Microscope</td>
			<td>Zeiss</td>
			<td></td>
		</tr>
		<tr>
			<td>filter cubes</td>
			<td>Nikon</td>
			<td>Nikon FITC (96311), Nikon Texas Red(96313)</td>
		</tr>
	</tbody>
</table>

assembly and tracking of microbial community development within a microwell array platform

Die Entwicklung der mikrobiellen Gemeinschaften hängt von einer Kombination von komplexen deterministischen und stochastischen Faktoren ab, die die räumliche Verteilung und die Aktivitäten der Gemeinschaftsmitglieder drastisch verändern können. Wir haben eine microwell-Array-Plattform entwickelt, mit der Tausende von Bakteriengemeinschaften parallel versammelt und verfolgt werden können. Dieses Protokoll unterstreicht den Nutzen der Plattform und beschreibt seine Verwendung zur optischen Überwachung der Entwicklung einfacher, zweiköpfiger Gemeinschaften innerhalb eines Ensembles von Arrays innerhalb der Plattform. Diese Demonstration verwendet zwei Mutanten von Pseudomonas aeruginosa , Teil einer Reihe von Mutanten entwickelt, um Typ VI Sekretion Pathogenität zu studieren. Chromosomale Insertionen von entweder mCherry- oder GFP-Genen erleichtern die konstitutive Expression von fluoreszierenden Proteinen mit unterschiedlichen Emissionswellenlängen, die verwendet werden können, um die Häufigkeit und die Lage des Gemeinschaftsmitglieds innerhalb jeder Mikrovertiefung zu überwachen. Dieses Protokoll beschreibt ein detailliertes methoD zum Zusammenbau von Bakteriengemischen in die Vertiefungen des Arrays und unter Verwendung von zeitraffer Fluoreszenzbildgebung und quantitativer Bildanalyse, um das relative Wachstum jeder Mitgliedspopulation über die Zeit zu messen. Die Aussaat und Montage der Mikrotiterplattform, die bildgebenden Verfahren, die für die quantitative Analyse von mikrobiellen Gemeinschaften innerhalb des Arrays notwendig sind, und die Methoden, die verwendet werden können, um Wechselwirkungen zwischen mikrobiellen Speziesbereichen zu diskutieren, die alle diskutiert werden.

Die hier vorgestellte experimentelle Plattform ist für Hochdurchsatz- und Hochinhaltensstudien von Bakteriengemeinschaften ausgelegt. Das Design ermöglicht es Tausende von Gemeinden, die in Brunnen verschiedener Größen wachsen, gleichzeitig analysiert werden. Mit diesem Microwell-Array-Design kann die Abhängigkeit der endgültigen Gemeinschaftszusammensetzung auf den anfänglichen Seeding-Dichten, der Well-Größe und der chemischen Umgebung bestimmt werden. Diese Arbeit zeigt das W...

Watch this Scientific Journal Video about Assembly and Tracking of Microbial Community Development within a Microwell Array Platform at JoVE.com

Aufbau und Verfolgung der Entwicklung mikrobieller Gemeinschaften innerhalb einer Microwell-Array-Plattform | Bioingenieurwesen | Video

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

Die Entwicklung der mikrobiellen Gemeinschaften hängt von einer Kombination von Faktoren ab, einschließlich Umweltarchitektur, Mitgliedshäufigkeit, Züge und Wechselwirkungen. Dieses Protokoll beschreibt eine synthetische, mikrogefertigte Umgebung für die gleichzeitige Verfolgung von Tausenden von Gemeinschaften, die in Femtoliter-Vertiefungen enthalten sind, wo Schlüsselfaktoren wie Nischengröße und Einschluss angenähert werden können.

Zusammenbau und Verfolgung der mikrobiellen Gemeinschaftsentwicklung innerhalb einer Microwell Array Plattform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

assembly-tracking-microbial-community-development-within-microwell

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8.9K Aufrufe.  Oak Ridge National Laboratory.  Die Entwicklung der mikrobiellen Gemeinschaften hängt von einer Kombination von Faktoren ab, einschließlich Umweltarchitektur, Mitgliedshäufigkeit, Züge und Wechselwirkungen. Dieses Protokoll beschreibt eine synthetische, mikrogefertigte Umgebung für die gleichzeitige Verfolgung von Tausenden von Gemeinschaften, die in Femtoliter-Vertiefungen enthalten sind, wo Schlüsselfaktoren wie Nischengröße und Einschluss angenähert werden können. 

Serie: Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact &beta;-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80&deg; C as many 150 &mu;L aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. 
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1&alpha; for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.

A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.

Tracking Hypoxische Signaling in Encapsulated Zellaggregate

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

Forschung

Biotechnik

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Umweltfreundliche kontrollierten Microtensile Testing von mechanisch-adaptive Polymer-Nanokomposite für<em&gt; Ex vivo</em&gt; Charakterisierung

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

Synthese einer Intein-vermittelten Protein-Hydrogel Künstliche

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

Development of an In Vitro Ocular Platform to Test Contact Lenses

Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs.

Entwicklung eines<em&gt; In Vitro</em&gt; Ocular Platform Kontaktlinsen zu Test

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

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.

Mikrostrukturierung und Montage von 3D-Micro-Ader

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

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Mikrofluidik-Plattform mit Multiplexed elektronische Sensoren für Raumverfolgung von Partikeln

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.

Nagetier Verhaltens Tests, um funktionelle Defizite verursacht durch Mikroelektrode Implantation im motorischen Kortex Ratte bewerten

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes ...

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Globally, 4.5 billion people are economically dependent on the sea, where most of their livelihood is provided by coral reefs. Corals are of great importance and therefore their extinction leads to catastrophic consequences. There are several possible solutions to remediate marine pollutants and local contamination, including bioremediation. Bioremediation is the capacity of organisms to degrade contaminants. The approach presents several advantages, such as sustainability, relatively low cost, and the fact that it can be applied in different ecosystems, causing minimal impacts to the environment. As an extra advantage, the manipulation of endogenous microbiomes, including putative beneficial microorganisms for corals (pBMCs), may have probiotic effects for marine animals. In this context, the use of the two approaches, bioremediation and pBMC inoculation combined, could be promising. This strategy would promote the degradation of specific pollutants that can be harmful to corals and other metaorganisms while also increasing host resistance and resilience to deal with pollution and other threats. This method focuses on the selection of pBMCs to degrade two contaminants: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Both have been reported to negatively impact marine animals, including corals, and humans. The protocol describes how to isolate and test bacteria capable of degrading the specific contaminants, followed by a description of how to detect some putative beneficial characteristics of these associated microbes to their coral host. The methodologies described here are relatively cheap, easy to perform, and highly adaptable. Almost any kind of soluble target compound can be used instead of EE2 and oil.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

Prospektion mikrobielle Stämme für Bioremediation und Probiotika Entwicklung für Metaorganismus Forschung und Konservierung

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. ...

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

Herstellung und Implementierung einer referenzfreien Traktionskraftmikroskopie-Plattform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

Formulierung und Charakterisierung von Lipid-Nanopartikeln für die Genabgabe mit einer mikrofluidischen Mischplattform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...

Zusammenbau und Verfolgung der mikrobiellen Gemeinschaftsentwicklung innerhalb einer Microwell Array Plattform

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