Mikrokoşa diziler, Nanofaze Malzeme Bilimleri Merkezi Kullanıcı Tesisleri Bölümü, Temel Enerji Bilimleri Ofisi, ABD Enerji Bakanlığı tarafından imal edildi ve karakterize edildi. Bu çalışma için maddi destek Oak Ridge Ulusal Laboratuar Direktörü Araştırma ve Geliştirme Fonu aracılığıyla sağlandı. Yazarlar ayrıca, bu araştırmalarda kullanılan P. aeruginosa suşlarının temini için J. Mougous Laboratuvarına (Washington Üniversitesi, Seattle, WA) teşekkür etmek istiyorlar .

<ol>
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Yazarların açıklayacak bir şeyi yok.

Bu makale, bakteri topluluğunun gelişiminin yüksek verimli ve yüksek içerikli canlı hücre görüntüleme tabanlı analizini mümkün kılmak için tasarlanmış bir mikrodalkaralı dizi cihazı ve deney protokollerini sundu. Buradaki gösterinin odağı, temas yoluyla aracılık edilen Tip VI sekresyonun topluluk gelişimine etkilerini incelemek iken, diziler esnek olacak şekilde tasarlandı ve geniş bir mikrobik topluluklar mikrobik-mikrop etkileşimleri çalışmasına uyacak şekilde tasarlandı. Buradaki ç...

Mikrobiyal topluluklar, hem hücre ölümü, bölünme, protein konsantrasyonu, organel sayısı ve mutasyon 1 ile ilişkili olan ortamın yapısı ve stokastik süreçler gibi belirleyici faktörler tarafından şekillendirilir 1 . Doğal ortamda, bu etkilerin toplumdaki bileşimi ve etkinliği üzerindeki bireysel etkisini çözümlemek neredeyse imkansız olabilir. Doğal yapılar tarafından örtülmüş ve kimyasal ve biyolojik bir ortamda gömülmüş, topluluk üyelerinin tanımlanması ve doğal çevre içerisinde zaman-zaman dağılımının daha da çözülmesi son derece zorlu bir konudur. Bununla birlikte, son zamanlardaki çabalar, mekânsal organizasyonun topluluk işlevi üzerindeki önemini vurgulamış ve devam eden çalışmalarda hem üye bolluğunu hem de örgütlenmeyi hesaba katma ihtiyacına işaret etmektedir 2 , 3 , 4 . O, Yerel kimyasal çevrenin (besin maddelerinin ve sekonder metabolitlerin mevcudiyeti), fiziksel yapının ( örneğin toprak yapısı, bitki kökleri, okyanus parçacıkları veya bağırsak mikrovillikleri), oksijenin varlığı veya yokluğunun ve Patojenik türlerin hepsi mikrobiyal toplulukların kompozisyonu, mimarisi ve fonksiyonunu etkiler 5 , 6 , 7 , 8 , 9 , 10 , 11 . Bununla birlikte, kültürler için bu faktörleri yakalamayı ihmal eden geleneksel teknikler hakim olmaya devam etmektedir. Topluluk bileşimi ( örneğin birlikte bağımlı türlerin varlığı), fiziksel bağlanma, sinyal molekül konsantrasyonu ve direkt hücre-hücre teması, mikrobiyal bir topluluğun şekillenmesi için önemli faktörlerdir ve cÖnceki kültür koşulları. Bu özelliklerin bir toplu sıvı kültüründe veya bir agar plakasında kopyalanması zordur. Bununla birlikte, doğal ortamların önemli fiziksel ve kimyasal özelliklerinin çoğaltılmasına izin veren mikroakışkan, mikro-modelleme ve nanofabrikasyon teknikleri, birçok araştırmacıya, 12 , 13 , 14 arasındaki etkileşimleri incelemek için bakteriyel topluluklar inşa ettirdi ve Doğal koşulları 4 , 15 , 16 , 17 , 18 , 19 , 20&#39;yi taklit edin. Bu protokol, bir mikrodellek dizilimi cihazı imal etmek için bir yöntem açıklamaktadır ve bu yöntemin işlevselleştirilmesi için kullanılabilecek ayrıntılı deneysel prosedürler sağlamaktadır.Hem kuyuları hem de tek tür kolonileri olarak ve çok üyeli topluluklarda bakteri yetiştirmek için kullanıldı. Bu çalışma aynı zamanda, floresan muhabir proteinleri üretmek üzere modifiye edilen bakterilerin, zaman içinde kuyu içerisindeki bakterilerin büyümesini izlemek için nasıl kullanılabileceğini göstermektedir. Benzer bir dizi daha önce sunulmuş ve mikrowelllerde Pseudomonas aeruginosa ( P. aeruginosa) tek tür kolonilerin büyümesini izlemek mümkün olduğunu göstermiştir. Kuyu boyutu ve ekim yoğunluğunu modüle ederek, binlerce büyüme deneyinin başlangıç ​​koşulları, ilk inokülasyon koşullarının bakterilerin yetişme yeteneğini nasıl etkilediğini belirlemek için paralel olarak değiştirilebilir. Mevcut çalışma, çoklu dizilerin eşzamanlı karşılaştırılmasını sağlayarak ve daha sağlam bir deney protokolü kullanarak önceki çalışmaya dayanan mikrodevre dizisinin biraz değiştirilmiş bir sürümünü kullanıyor. Bu çalışmada kullanılan dizi, çoklu alt sıralar veya dizi ensem içerirÇapları 15 - 100 μm arasında değişen, üç farklı aralıklarla ( yani , kuyu çapında 2x, 3x ve 4x) düzenlenmiş farklı boyutlarda oyuklar içeren oyuklar. Diziler silikona kazınır ve silikon dizilerde tohumlanan bakterilerin büyümesi, orta-enfüze bir agaroz jeli ile kaplanmış bir lamel ile dizileri sızdırmaz hale getirerek etkinleştirilir. Bu demonstrasyonda Tip VI sekresyon sistemini incelemek üzere tasarlanmış P. aeruginosa mutantları kullanılır. Burada sunulan sonuçlar, mikrowell dizilerdeki çok elemanlı toplulukların analizinin nihai hedefine doğru inşa ederek, araştırmacıların kimyasal çevrenin kontrol edilmesi ve araştırılması sırasında yerinde bakterilerin bolluğunu ve organizasyonunu izlemesini sağlıyor. Bu, nihayetinde topluluk gelişimini ve halefiyetini yöneten &quot;kurallar&quot; hakkında fikir verir.

<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

Mikrobiyal toplulukların gelişimi, topluluk üyelerinin mekansal dağılımını ve faaliyetlerini önemli ölçüde değiştirebilen karmaşık deterministik ve stokastik faktörlerin bir kombinasyonuna bağlıdır. Binlerce bakteri topluluğunu paralel olarak hızlı bir şekilde bir araya getirmek ve izlemek için kullanılabilecek bir mikro kollu dizi platformu geliştirdik. Bu protokol, platformun kullanımını vurgular ve platform içindeki dizilerin bir topluluğunda basit, iki üyeli toplulukların gelişimini optik olarak izlemek için kullanımını açıklar. Bu gösteri, Tip VI salgı patojenisitesini incelemek üzere geliştirilen bir dizi mutantın bir parçası olan Pseudomonas aeruginosa&#39;nın iki mutantını kullanıyor. MCherry veya GFP genlerinin kromozomal girdileri, her bir mikrowell içindeki topluluk üyelerinin bolluğunu ve konumunu izlemek için kullanılabilen farklı emisyon dalga boylarına sahip floresan proteinlerin yapılı ifadesini kolaylaştırır. Bu protokol ayrıntılı bir metodu açıklamaktadır.D, bakteri karışımlarını dizinin kuyularına monte etmek ve zaman içindeki tüm üye popülasyonun göreli büyümesini ölçmek için zaman atlamalı floresans görüntüleme ve niceliksel görüntü analizi kullanmak için kullanılmıştır. Mikro platformun tohumlanması ve montajı, dizi içindeki mikrobik toplulukların kantitatif analizi için gerekli görüntüleme prosedürleri ve mikrobik tür alanları arasındaki etkileşimleri ortaya çıkarmak için kullanılabilecek yöntemlerin hepsi tartışılmıştır.

Burada sunulan deney platformu, bakteri topluluklarının yüksek verimli ve yüksek içerikli çalışmaları için tasarlanmıştır. Tasarım, çeşitli büyüklükteki kuyulardan büyüyen binlerce topluluğun aynı anda analiz edilmesini sağlıyor. Bu mikrodellek dizilimi tasarımı ile, nihai topluluk bileşiminin ilk ekim yoğunlukları, iyi boyut ve kimyasal çevre üzerindeki bağımlılığı belirlenebilir. Bu çalışma, mikrodalgalı dizide iki üyeli bir toplumun büyümes...

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

Mikro Kuyu Dizisi Platformunda Mikrobiyal Topluluk Gelişiminin Birleştirilmesi ve Takibi | Biyomühendislik | Video

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

Mikrobiyal toplulukların gelişimi, çevresel mimari, üye bolluğu, özellikleri ve etkileşimleri de içeren faktörlerin bir kombinasyonuna bağlıdır. Bu protokol, femtolitör kuyularda bulunan binlerce topluluğun eşzamanlı olarak izlenebilmesi için sentetik, mikrofabrik bir ortam tanımlamakta olup niş boyut ve sınırlama gibi anahtar faktörler yaklaşık olarak belirlenebilmektedir.

Bir Mikro Kuyruklu Dizi Platformunda Mikrobik Topluluk Geliştirmenin Montajı ve Takibi

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

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8.9K Görüntüleme.  Oak Ridge National Laboratory.  Mikrobiyal toplulukların gelişimi, çevresel mimari, üye bolluğu, özellikleri ve etkileşimleri de içeren faktörlerin bir kombinasyonuna bağlıdır. Bu protokol, femtolitör kuyularda bulunan binlerce topluluğun eşzamanlı olarak izlenebilmesi için sentetik, mikrofabrik bir ortam tanımlamakta olup niş boyut ve sınırlama gibi anahtar faktörler yaklaşık olarak belirlenebilmektedir. 

Video: 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.

Encapsulated Hücre Agregalar içinde Takip Hipoksik Sinyalizasyonu

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

Biyomühendislik

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.

Için Mekanik-adaptif Polimer Nanokompozitler bir çevre kontrollü Mikrogerilim Test<em&gt; Ex vivo</em&gt; Karakterizasyonu

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.

Bir Intein aracılı yapay protein Hidrojel sentezi

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.

Bir Gelişimi<em&gt; İn Vitro</em&gt; Göz Platformu Lensler 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.

Micropatterning ve 3D mikrodamarlar Meclisi

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.

Parçacıkların Mekansal Takip için Çoklanmış Elektronik Algılama ile mikroakışkan Platformu

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.

Kemirgen fonksiyonel açıkları sıçan Motor korteks elektrot implantasyon nedeniyle değerlendirmek için sınama davranış

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.

Metaorganizma Araştırma ve Koruma için Biyoremediasyon ve Probiyotikler Gelişimi için Mikrobiyal Suşların Prospektileri

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.

Referanssız Çekiş Kuvveti Mikroskopi Platformunun İmalatı ve Uygulanması

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.

Mikroakışkan Karıştırma Platformu Kullanarak Gen Teslimatı için Lipid Nanopartiküllerinin Formülei ve Karakterizasyonsu

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