Bu araştırma, Ulusal Nörolojik Bozukluklar Enstitüsü ve İnme, Ulusal Sağlık Enstitüleri (VVD) ve National Institute of Nörolojik Bozukluklar ve İnme, Ulusal arra yoluyla Institutes of Health (VVD) ve R21 R21 NS064403 hibe hibe R01NS062842 tarafından desteklenen Biyomedikal Görüntüleme ve Biyomühendislik EB006301 National Institute, National Institutes of Health (VVD).

Hazırlanması moleküler cihaz montajı daha fazla zaman alır, çünkü moleküler makine tabanlı tespiti için bölümler hazırlanmalıdır. Inşa paraformaldehid sabit, parafine gömülü doku blokları kesilmiş 5-6μm kalınlığında bölümleri ile iyi çalışır. ProbeOn Plus ücret ve precleaned slaytlar (Fisher Scientific) veya benzeri gibi iyi bölümlerde muhafaza slayt markalar kullanın. Biz deksametazon ile tedavi edilen apoptotik sıçan timus 13,14 DNaz I-ve DNaz II tipi molaları içeren DNA hasarı bilinen bir desen, bir doku kullanarak önerilir. 1. Kesit hazırlanması <ol><li> 15 dakika ksilen bir slayt raf ve mumunu gidermek bölümleri Place, ek bir 5 dakika boyunca taze bir ksilen banyo aktarın. </li><li> Kademeli etanol konsantrasyonları geçerek rehidrate:% 96,% 80 Etanol-2x5min Etanol - 5min; su - 2x5 dk. </li><li> Özet bölümü ile Proteinase K. Bölüm başına bir 50μg/mL çözüm 100μL kullanın. Nemlendirilmiş bir odasında oda sıcaklığında (23 ° C) inkübe 15 &#39;. Zaman doku tipine bağlı olarak ayarlanması gerekebilir. Sert dokular uzun sindirim gerektirebilir. 15-25 dk Times genellikle kullanılır. Yetersiz sindirim zayıf sinyal neden olabilir. Öte yandan sonuçlarına ilişkin sinyal ortadan kalkması ve bölüm kesintisi Overdigestion. </li><li> 2x10 dakika süreyle distile su ile durulayın. </li><li> Preblocking için% 2 BSA 100 mcL Bölüm başına uygulayın. 15 dakika oda sıcaklığında (23 ° C) inkübe edin. Bu süre zarfında moleküler makine monte edin. </li></ol> 2. Moleküler makineler montaj Tüm reaktifler ortalama büyüklüğü doku bölümü (10x10mm) tek bir algılama için yeterli olan 25 mcL toplam hacmi, ölçeklenir. Gerektiği kadar hacmi kadar ölçeklendirilebilir. <ol><li> Bu sırada küçük bir plastik tüp birleştirin: </li></ol><table border="1"><tbody> &lt;tr&gt; <td><ol><li> Bidistile su; </li><li> % 15 polietilen glikol-8000; </li><li> Çözüm 66 mM Tris HCl, pH 7.5, 5 mM MgCl 2, 0.1 mM dithioerythritol, 1 mM ATP (yani normal T4 DNA ligaz tampon); </li><li> Oligonükleotid 1 70 pmoles, </li><li> 215 pmoles (1.76 - 7,1 mcg) VACC TOPO. </li><li> 10 birimleri T4 DNA ligaz (500 U / ml) </li></ol></td></tr></tbody></table><ol start="2"><li> Pipetleme yavaşça karıştırın. Moleküler neredeyse anında bu çözüm kendi kendine bir araya inşa etmek ve oda sıcaklığında (23 ° C) hemen kullanılabilir. Sıcaklığının artmasıyla 37 ° C blokları etiketleme T4 DNA ligaz tabanlı bir bileşen. </li></ol> 3. Moleküler makineler kullanarak çift etiket 5&#39;OH ve 5&#39;PO4 DNA kırıkları doku kesitlerinde <ol><li> Preblocking çözüm aspire ve 70 pmoles Oligonükleotid 1, 53 içeren tam bir reaksiyon karışımı 25μL geçerli 215 pmoles (1.76 - 7,1 mcg) VACC66 mM-Tris HCl, pH 7.5, 5mM MgCl 2, 0.1 mM dithioerythritol, 1 mM ATP, ve% 15 polietilen glikol-8000 çözümü TOPO ve 10 adet T4 DNA ligaz (500 U / ml). Tek bir FITC etiket, 2 Oligonükleotid taşıyan aynı sıra oligonükleotid, tek bir DNA sonlarının türü tespiti için yerine kullanılabilir. Böyle bir durumda etiketleme reaksiyon ligaz atlayabilirsiniz. Ayrıca bu duruma bir çözüm 50mm Tris-HCL, pH 7.4,% 15 PEG-8000 T4 DNA ligaz tamponu yerine kullanılan olabilir. </li><li> Plastik lamel ile nemlendirilmiş bir odasında oda sıcaklığında (23 ° C) 1 saat süreyle inkübe edin. Işıktan koruyun. Düşürülmesi sıcaklık 16 ° C ligaz tabanlı sinyal azaltır; 37 ° C sıcaklık artışı ligaz tabanlı sinyal tamamen ortadan kaldırır. Ligaz bir kısmi inhibisyonu bazen topoizomeraz preparatları ile tanıtıldı kirleticiler nedeniyle muhtemelen, reaksiyon karışımı oluşur. Bu nedenle, uzun bir inkübasyon (2-4 saat) gerekli olabilir, özellikle sligaz etiketli sonlarının ignificant numaraları mevcuttur. Ligaz sinyali ligaz ve topo sinyalleri hem de birçok durumda, bu aşamada uyulması rağmen VACC TOPO ve çift etiketli Oligonükleotid olmadan reaksiyon karışımı yeniden uygulama tarafından daha da geliştirilerek, ancak bir saç tokası şeklindeki oligonükleotid (Oligonükleotid 3) içeren (35 mg / ml) ve T4 DNA ligaz (250 U / ml). Artırmak için sinyal Adım 5 gitmek reaksiyon geliştirme olmadan görmek için, Adım 3&#39;e geçin. </li><li> Yavaşça Coplin kavanoza oda sıcaklığında su içeren bir slaytları dikey çeker lamelleri çıkarın. Aşırı su aspire. </li><li> VACC TOPO ve Oligonükleotid 1 olmadan reaksiyon karışımı 25 mcL uygulanması, ancak Oligonükleotid 3 içeren ligaz sinyal geliştirin: 66 mM Tris HCl, pH 7.5, 5 mM MgCl 2, 0.1 mM dithioerythritol, 1 mM ATP, ve% 15 polietilen glikol- 8000, 5 adet T4 DNA ligaz, 35 mg / ml Oligonükleotid 3 (künt sona erdi saç tokası). Etiketleme çözümü ca toplam hacmin büyük bölümleri barındıracak kadar ölçeklendirilebilir. Plastik lamel ile nemlendirilmiş bir odasında, oda sıcaklığında (23 ° C) 18 saat (gece) inkübe edin. </li><li> Yavaşça Coplin kavanozu oda sıcaklığında su içeren dikey slaytlar çeker lamelleri çıkarın. Sonra bölüm 3x10 dakika distile su ile yıkayın. </li><li> Sodyum bikarbonat tamponu ile durulayın. Alkali çözelti pH duyarlıdır ve pH 7&#39;nin altındaki önemli ölçüde azalır FITC floresanı, geliştirir durulama. </li><li> Antifading bir çözüm (DAPI ile Vectashield), lamel Cover bölüm ve floresan mikroskop kullanılarak sinyal analiz. 5&#39;OH ile çift iplikli DNA kırıkları yeşil, 5&#39;PO 4 tatili floresan-kırmızı (Şekil 2) floresan olacak. </li></ol> 4. Temsilcisi Sonuçlar <img alt="Şekil 2." src="/files/ftp_upload/3628/3628fig2.jpg" /> Şekil 1. Yarı yapay moleküler makine iki tür algılarIn situ DNA hasarı. VACC TOPO çift saç tokası 32-mer bağlanır floresan makine kendini-bir araya. Bu makine ile iki dedektör birimler halinde kendisi bölme işlemi başlar topoizomeraz-3 &#39;tanıma dizisi sonunda kesilir. Bu sonuç FITC etiketli ünitesi sürekli olarak ayırır ve rodamin etiketli ünitesine geri religates döngüsel bir süreç. Bu saptanabilir DNA sonu karşılaştı kadar devam eder. Böyle bir alternatif alıcısı (5&#39;OH künt uçlu DNA break) doku bölümünde olduğunda, FITC parçası Arter. Kalan rodamin parçası T4 DNA ligaz yardımıyla 5 &#39;PO 4 DNA künt sonu tatili eklenecektir. Sonuç olarak, her iki tür DNA kırıkları aynı anda tespit edilir. <img alt="Şekil 1." src="/files/ftp_upload/3628/3628fig1.jpg" /> Şekil 2. Moleküler makine çift etiketleri iki tür DNA kırıklarıdeksametazon ile tedavi edilen timus bir doku bölümünde. DNaz künt uçlu DNA kırıkları ve DNaz II tipi apoptozis geçiren timik kortikal alanlarda algılanır. Yeşil sitoplazmik floresans (5&#39;OH DNA kırıkları), apoptotik timositleri nükleer malzeme sindirerek kortikal makrofajlar işaretler. Bu sinyal VACC TOPO tarafından üretilen ve çift iplikli 5&#39;OH tatili 11,12 içeren DNA ile phagolysosomes için lokalize. Kırmızı floresans (5&#39;PO 4 sonları) etiket çekirdekleri makrofajlar tarafından yuttu apoptotik timositleri. Timositleri Massive aynı anda 5&#39;PO 4 çift iplikli molaları kuşak tarafından eşlik apoptozis tabi ligaz tabanlı etiketleme tarafından görüntülendi. Bu sonları halka şeklinde desenler oluşturan nükleer çevre bulunmaktadır. Tüm hücre çekirdekleri DAPI (mavi floresan) ile counterstaining tarafından görüntülenmiştir.

<ol>
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Çıkar çatışması ilan etti.

Bu videoda, bir çift etiketleme DNA hasarı sensörü montajı ve nasıl kullanılacağını göstermektedir. Sensörü, biyo-moleküler motor, yapay bileşenleri ile bağlantılı bir virüs kodlanan protein VACC TOPO tarafından yönlendirilen bir moleküler makine. Sunulan geliştirme, toksik olmayan moleküler ölçekli cihazların 12,15 tasarımı, biyolojik yapıları, mimarileri ve hücre gerçek parçaları ve bileşenleri adapte savunucuları bir biyo-etkin bir yaklaşım örneğidir. 1: Bu yaklaş?...

<ol><li> Ksilen </li><li> 80 ve% 96 Etanol. </li><li> Distile su (BSA), sığır serum albümin% 2&#39;lik solüsyon. </li><li> Oligonükleotid 1: bir çift-saç tokası, çift floresein ve tetramethylrhodamine ile etiketlenir: </li></ol> GCG AT 5&#39;-AAG GGA SKK GC F GCA GGT CCC TTA ACG CAT R TT-3 &#39;F - FITC-dT; R - Tetramethylrhodamine-dT. BODIPY TR rodamin veya TAMRA gibi diğer kırmızı-kaymıştır fluorophores R olarak yerine tetramethylrhodamine kullanılabilir. Alternatif Oligonükleotid 2 kullanabilirsiniz. Floresein ile etiketlenmiş bir çift saç tokası single (tek bir DNA kırıkları tipi (DNaz II-tipi) tespiti için tek fluorofor taşıyan prob önemli ölçüde daha az pahalı ve rahat bir zaman tek bir DNA break tipi etiketli: 5&#39;-AAG GGA, SKK GC F GCA GGT CCC TTA ACG CAT ATG CGT T-3 &#39;; F - FITC- dT </p&gt; <ol start="5"><li> Oligonükleotid 3. Tetramethylrhodamine-dT - R; 5&#39;-GCG CTA GAC C R G GTC TAG CGC-3: T4 DNA ligaz ile yerinde ligasyon sinyal artırılması için künt uçlu rodamin etiketli saç tokası </li><li> Vaccinia DNA topoizomeraz l (VACC TOPO) - 3000 U / ml (Canlı Teknolojileri). İlk deneylerde reaksiyon karışımı her 25 mcL başına VACC TOPO 215 pmoles (7.1 mg) kullanılır. Ancak, hassasiyet kaybı olmadan topoizomeraz konsantrasyonunu önemli ölçüde azaltılabilir. Daha sonra benzer sonuçlar VACC TOPO (bölüm başına reaksiyon karışımı 25 mcL 1.76 mcg) Bölüm başına dört kat daha az miktarda kullanılır. 880 ng (25 mcL reaksiyon karışımı) enzimin miktarını azaltarak, daha zayıf bir sinyal ve 8.8 ng VACC TOPO saptanabilir sinyal üretti. </li><li> T4 DNA ligaz 5 U / ml (Roche). Bu son derece konsantre ligaz hazırlık deneysel koşullarda en iyi sinyal verir. </li&gt; <li> T4 DNA ligaz için 10 x reaksiyon tamponu: 660 mM Tris-HCl, 50 mM MgCl 2, 10 mM dithioerythritol, 10 mM ATP, pH 7.5 (20 ° C) (Roche). ATP reaksiyon tamponu içinde çözülme-donma tekrarlayan döngüler halinde kolayca yok edilir. Kısım küçük 15-20 mcL bölümlerini ve mağaza tampon - 20 ° C bir kez kullanın. </li><li> Bidistile su PEG-8000 (Sigma)% 30 (w / v) solüsyonu. % 15 PEG-8000 reaksiyon karışımı hacmi dışlama bileşenlerinin etkili konsantrasyonları artan algılama uyarır. </li><li> Distile su proteinaz K (Roche) 20 mg / ml stok solüsyonu. Mağaza - 20 ° C PBS stok hazırlanan reaksiyon kullanımı 50mg/mL çözüm. Kullanmayın. </li><li> DAPI (vektör Laboratuvarlar) ile Vectashield. </li><li> Sodyum bikarbonat tampon: 50m M NaHCO 3, 15m M NaCl, pH 8.2. </li><li> 22x22mm veya 22x40mm cam ya da plastik lamelleri. Plastik lamelleri bir reaksiyon sırasında tercih edilirbölümünden kaldırmak için daha kolay yeniden. </li></ol>

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

Doğal olarak ortaya çıkan bio-moleküler makineler her canlı hücrenin çalışması ve çeşitli tasarımlar 1-6 gösterilecek. Ancak yönlü hareket, yani moleküler motorlar uyumlu cihazlar ve ölçekli aşağı mekanik parçalar (jantlar, aks, kolye vb) 7-9 yapay moleküler makineler merkezlerinin geliştirilmesi. Bu nanoworld ortamlarda 10, atalet ve momentum korunumu gibi, bu cihazlar için gerekli olan fiziksel özellikleri, kullanışlı olmasa bile, makro makineleri taklit eder . Nanoworld belirli koşulları, mekanik macromachines şemaları izleyin ve biyolojik moleküllerin evrimi geliştirilen mekanizmalar kullanmayın alternatif tasarımlar, yararlanabilirsiniz. Bunun yanı sıra, nano-tasarım amaçları için gerçek biyolojik moleküllerin adapte nanoteknoloji ürünleri oluşturabilecek potansiyel tehlikeleri azaltır. Burada, böyle bir biyo-uyumlu inşa montaj ve uygulamasını göstermekyapay bileşenleri ile doğal olarak ortaya çıkan moleküler makine birleştirir emi-yapay moleküler cihaz. Enzimoloji noktadan itibaren, bir yapı, bir tasarımcı floresan enzim-substrat kompleksi yararlı belirli bir işlevi gerçekleştirmek için bir araya koymak. Bu meclis, bir 12 içerdiğinden, tanımı gereği bir moleküler makine . Ancak, mühendislik bölümü ile entegrasyon - floresan çift saç tokası - DNA hasarını 12 etiketleme yeni bir görev için yeniden yönlendirir. Bizim yapı 32-mer DNA ve enzim vaccinia topoizomeraz I (VACC TOPO) toplanır. Makine daha sonra iki floresan etiketli detektör birimleri (Şekil 1) imal kendi malzeme kullanır. Birimlerden biri (yeşil floresan) VACC TOPO kovalent 3&#39;end ve başka bir birim (kırmızı floresan) bağlı bir terminal 3&#39;OH ile ücretsiz saç tokası taşır. Birimleri, kısa ömürlü ve hızlı bir şekilde, orijinal bir yapı, sonradan recleaves içine geri yenilemek.DNA yokluğunda bu iki ünite sürekli döngüsel bir şekilde ayrı ve religate kırar. DNA hasarı, topoizomeraz taşıyan dedektör ünitesi seçici floresan etiketleme 5&#39;OH (DNaz II tipi sonları): 11,12 künt uçlu DNA kırıkları verdiği doku kesitlerinde . T4 DNA ligaz 13,14 çözüm varsa oligonükleotid bölünme sonra oluşan ikinci, enzim ücretsiz saç tokası, 5&#39;PO 4 künt uçlu sonu (DNaz I-tipi sonları) 11,12 Arter olacak. T4 DNA ligaz bir doku bölüm veya 5&#39;PO 4 künt uçlu sonları ile DNA içeren bir çözüm için eklendiğinde, ligaz, yarı kararlı bir enzim-DNA kompleksleri oluşturarak, 5&#39;PO 4 DNA biter tepki verir . Künt sona erdi toka, böylece 5&#39;PO 4 künt uçlu DNA kırıkları etiketleme, DNA ligaz ve kovalent bağlantı toka serbest bu kompleksleri ile etkileşim olacaktır . Bu gelişme th için yeni bir pratik yaklaşım örneğidire moleküler makineler tasarım ve sabit hücre ve dokularında apoptozis ve DNA hasarı tespit için yararlı bir sensör sağlar.

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

Biz topoizomeraz bir protein tarafından desteklenmektedir moleküler ölçekli cihaz montaj ve uygulama göstermektedir. Inşa uçları iki farklı fluorophores ekleyerek doku kesitlerinde DNA kırıkları başlıca iki tipi etiketler moleküler biyo-sensör.

Yarı yapay Moleküler Makine ve Kullanımı DNA Hasar Tespiti için in vitro Meclisi

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 Görüntüleme.  Baylor College of Medicine. Biz topoizomeraz bir protein tarafından desteklenmektedir moleküler ölçekli cihaz montaj ve uygulama göstermektedir. Inşa uçları iki farklı fluorophores ekleyerek doku kesitlerinde DNA kırıkları başlıca iki tipi etiketler moleküler biyo-sensör.

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.

Nükleik Asitler, Proteinler ve Küçük Moleküller Reagentless Algılama Elektrokimyasal DNA Biyosensörler Fabrikasyon

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. ...

Biyomühendislik

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: İnsan Microarrayed hücrelerde DNA hasarına ölçülmesi için bir Yüksek verim 96 Oyuklu Platformu

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

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).

Yeniden İnşa İnsan Kornea gibi Epitelyal (RHCE) Doku Modeli kullanılarak Göz tahriş Kimyasalların Tehlike Tanımlaması Göz tahrişi Testi (EIT)

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 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 ...

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.

Robot sıvı işleme derleme modüler DNA cihazların otomatik

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.

Hücrelerin ultra hızlı Mıknatıslanma için katyonize Magnetoferritin sentezi

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.

İnşaat ve in Vitro mezenkimal kök/Stromal hücrelerdeki Osteojenik farklılaşma güçlendirmeye yönelik bir elektriksel stimülasyon odası kullanımı

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.

İnsan Nötrofil Swarming in vitro Kemotaktik ve Moleküler Analizi için Biyopartikül Mikrodizileri

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.

Uzun Süreli Nanomalzeme Maruziyetini Takiben İn vitro Genotoksisite Testi için Gelişmiş 3D Karaciğer Modelleri

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

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.