Nazik integraz klon sağlamak için CM Margaret Smith Destek finansmanı: Genom Kanada / Genom Prairie NSERC Discovery ve Stratejik Proje hibeleri

1. Kültür Üretimi <ol><li> TY 10 (5 g / l tripton, 3 g / l maya ekstresi, 0.44 g / l kalsiyum klorid dihidrat), ve LB 11 (10 g / l tripton, 5 g / L maya ekstresi, 5 g /: steril sıvı ortam hazırlamak sodyum klorid, pH 7). </li><li> SmUW227: 50 mg / ml spectinomycin ile TY medya (S. meliloti LP-suşu: inşaat kodlanabilir, istek üzerine inşaat detayları zorlanma) () 5 ml içine tek bir koloni inoküle. E.: verilen antibiyotik ile ilave aşağıdaki suşlar LB sıvı ortamları 5 ml içine inoküle coli DH5α 12 10 ug / ml tetrasiklin pJC2, plazmid integraz ifadesi, içeren; E. coli 10 ug / ml Kloramfenikol ile MT616 13, harekete geçirici, ve E. coli DH5α 5 ug / ml gentamisin ile pJH110, plazmid donör kaset, içeren. </li><li> Inkübe E. 37 ve geceleme colideg; sürekli sarsıntı C. Inkübe S. çalkalama ile, iki gün süre ile 30 ° C &#39;de meliloti suşu. Bir S. elde etmek mümkündür Daha büyük bir inokulum (doymuş bir kültür alt kültürü 1:500) ile bir gece kültüre meliloti. </li></ol> 2. Kültür Hazırlama ve Karıştırma <ol><li> 30 saniye süreyle 17,000 x g&#39;de santrifüj ile hücre peleti toplanması ve 1 ml steril% 0.85 NaCl içinde resuspending her bir suş için kültür 1.5 ml yıkanır; tekrarlanan bir kez. </li><li> Steril% 0.85 NaCl 100 ul yılında Pastör pipetiyle pelet. </li><li> Bireysel düz TY Agar yüzeyindeki her bir suş için yıkanmış kültür 10 ul nokta. Bu lekeler kontrol noktalar vardır. </li><li> E. hariç her bir suş 40 ul ekleme coli DH5α 12 steril bir tüp, mix ve düz TY agar Bu karışımı spot 120 ul yılında pJC2 içeren. Bu nokta hiç integraz negatif kontrolüdür. </li><li> Steril bir t, her bir suş 40 ul eklemeUbe, mix ve spot düz TY Agar yüzeyindeki bu karışımın 160 ul. Bu nokta IMCE çiftleşme yerdir. </li><li> Noktalar bir laminer akış kaputu kurumasını bekleyin. </li><li> Contası plakalar ve 30 ° C&#39;da gece boyunca inkübe. </li></ol> 3. Trans-integrants İzolasyonu <ol><li> TY agar plaklarına Streak kontrol noktaları ve IMCE nokta 200 mikrogram / ​​ml streptomisin (SmUW227 streptomisin 14 yüksek düzey direnç sağlayan bir mutasyon taşıdığı Rm1021 dayanmaktadır) ve 30 mg / ml gentamisin ile desteklenmiş. </li><li> IMCE verimlilik hesaplanması için, yaklaşık tekrar süspansiyon ¼ 500 ul steril% 0.85 NaCl IMCE çiftleşme spot. 10 -7 ile 10 kat seri dilüsyonlar olun. Plaka dilüsyonları 10 -2 trans-integrants için seçmek için 200 mikrogram / ​​ml streptomisin ve 30 mg / ml gentamisin, takviye TY Agar plaklarda -5 ile 10. Plaka dilüsyonları 10 -3 through trans-integrants ve potansiyel alıcılar, yani toplam alıcı hem de seçmek için 200 mg / ml streptomisin ile desteklenmiş TY agar 10 -7. </li><li> Markerless trans-integrants izolasyonu için steril% 0.85 NaCl 500 ul yaklaşık IMCE çiftleşme nokta dörtte tekrar süspansiyon. 10 -6 10 kat seri dilüsyonlar olun. 200 ug / ml streptomisin ve 200 ug / ml X-gluc ile desteklenmiş dört TY agar plakalar üzerine 10 -6 ila 10 -4 plakası dilüsyonları. </li><li> 3 gün süreyle 30 ° C&#39;de inkübe. </li><li> Yeşil ışık (525 nm) altında ve kırmızı bir filtre (&gt; 610 nm) 15 üzerinden Profil RFP trans-integrants. </li><li> Trans-integrants koloni sayısı toplam alıcı CFU kıyasla yüzde IMCE verimini hesaplayın. Trans-integrants yaklaşık yarısı duyarlı bunları beyaz ve spectinomycin yapım gerçek kaset değişim geçirmiş olacak. </li><li> Markerless trans-integrants (w bulmak içinBurada donör vektör beyaz bir koloni (genişletilmiş bir inkübasyon süresi, oda sıcaklığında 1-2 gün fazladan, X-gluc ile koloniler farklılaşma artıracaktır için ekranda) pJH110 gibi Gm r içermez, bu S. gereklidir meliloti SmUW227), antibiyotikler ve 100 mg / ml spectinomycin içeren bir TY agar olmadan TY agar plaka tarama ile koloninin spectinomycin hassasiyeti onaylayın. </li></ol> 4. Temsilcisi Sonuçlar TY üzerinde inkübasyon üç gün streptomisin ile desteklenmiş ve gentamisin sonra kontrolü çizgiler hiçbir büyüme olmalıdır. IMCE çizgi başı çizgi ve Şekil 2&#39;de görüldüğü ikinci çizgi, birçok koloniler üzerinde birleşik büyüme olmalıdır. Toplam alıcıya trans-integrants bir yüzdesi olarak ifade trans-entegrasyon verimi,% 0.5 aralığında olmalıdır. Yaklaşık trans-integrants yarısı spectinomycin hassas ve beyaz olacakOnlar gerçek kaset döviz uğramıştır gösteren. Yeşil ışık (525 nm) altında ve kırmızı bir filtre (&gt; 610 nm) 15 bakıldığında pJH110 gelen test rfp ​​donör kaset içeren Trans-integrants ayırt RFP floresans göstermesi gerekir. <img src="/files/ftp_upload/3698/3698fig1.jpg" alt="Şekil 1"/> . Şekil 1 Konjugasyon karışımı çizimde: pRK600 gelen transfer genlerinin ifadesi tarafından Destekli, tüm plazmitler hücreden hücreye rastgele aktarılır. IMCE, integraz (int) yardımcı plazmid pJC2 ve donör plazmid pJH110 için gerekli iki plazmit LP-suşu satın alma yoluyla karışımı içinde trans-integrants oluşturulması, bu aktarma sonuçlanır. Plazmid olmayan kopyalayan donör (pJH110) gelen donör kaset (LP-marker kaybına neden kromozom üzerinde LP-kaset işaretleri ile ΦC31 integraz aktivite ile değiştirilir SpR ve uidA) ve sonuçta elde edilen trans-tamamlayıcı olarak donör kaseti (RFP ve GM r) bakım. <img src="/files/ftp_upload/3698/3698fig2.jpg" alt="Şekil 2"/> Şekil 2. A:. Agar yüzeyinde kuru hücre karışımları gösteren bir non-selektif TY plaka üzerinde Çiftleşme nokta plaka B: yukarıdan streptomisin-gentamisin-X-gluc plaka üzerinde çizgili Çiftleşme noktalar, S. içine, saat yönünde IMCE hiçbir integraz kontrolü yapmamışlar meliloti, E. coli DH5α pJC2 kontrolü ihtiva eden, E. coli pJH110 kontrolü, E. içeren DH5a coli MT616 kontrolü, ve S. . meliloti UW227 kontrolü C:. TY streptomisin-X-gluc agar D çiftleşme nokta tabanda 10 -2 dilüsyon: TY streptomisin-gentamisin-X-gluc agar (mavi koloniler tek rekombinantlar vardır üzerinde çiftleşme nokta tabanda 10 -2 dilüsyon, beyaz koloniler gerçek geçirmiş. kaset değişimi) E: floresans eksikliği gösteren TY streptomisin-X-gluc agar çiftleşme nokta tabanda 10 -2 dilüsyon F:. iki seviye gösteren TY streptomisin-gentamisin-X-gluc agar çiftleşme nokta tabanda 10 -2 dilüsyon floresans (parlak kolonileri mavi koloniler karşılık gelir ve salt rağmen koloniler gerçek kaset döviz no lac promotöründen salt üzerinden sadece onun hemen promotor ile RFP içeren geçirmediyse vektör sırası gelen organizatörü nedeniyle muhtemelen daha yüksek rfp ifade var Vektör, hangi yoktur.).

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Çıkar çatışması ilan etti.

IMCE tekniği önceden tasarlanmış suşu LP-lokusunda tek bir attB çevrili DNA kaset verimli entegrasyon sağlar. Istenen yapı donör kaset oluşturmak için RFP yerine klonlanmış sonra, tekniğin son derece güçlü hale, müteakip DNA arıtma ve dönüşüm gerektirmez. Bu antibiyotik direnci trans-integrants ve diğer değil faktörlerin oluşturulması nedeniyle belirli olabilmesi için, uygun büyüme kontrolleri dahil olduğunu önemlidir. IMCE yaklaşık% 0.5 v...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Streptomisin </td><td> Bioshop Kanada A.Ş. </td><td> STP101 </td><td></td></tr><tr><td> Spectinomycin </td><td> Bioshop Kanada A.Ş. </td><td> <a href="https://ssl34.alentus.com/bioshopcanada/detail.asp?Pin=SPE201">SPE201</a> </td><td></td></tr><tr><td> Gentamisin </td><td> Bioshop Kanada A.Ş. </td><td> GTA202 </td><td></td></tr><tr><td> Choramphenicol </td><td> Bioshop Kanada A.Ş. </td><td> CLR201 </td><td></td></tr><tr><td> Tetrasiklin </td><td> Bioshop Kanada A.Ş. </td><td> TET701 </td><td></td></tr><tr><td> Kanamisin </td><td> Bioshop Kanada A.Ş. </td><td> KAN201 </td><td></td></tr><tr><td> Bakteriyolojik notu agar </td><td&gt; Bioshop Kanada A.Ş. </td><td> AGR001 </td><td></td></tr><tr><td> Tripton </td><td> Bioshop Kanada A.Ş. </td><td> TRP402 </td><td></td></tr><tr><td> Maya Ekstraktı </td><td> Bioshop Kanada A.Ş. </td><td> YEX401 </td><td></td></tr><tr><td> Sodyum klorür </td><td> Bioshop Kanada A.Ş. </td><td> SOD001 </td><td></td></tr><tr><td> Kalsiyum Klorür </td><td> Bioshop Kanada A.Ş. </td><td> CCL444 </td><td></td></tr><tr><td> X-gluc </td><td> Altın Biyoteknoloji A.Ş. </td><td> G1281C1 </td><td></td></tr><tr><td> E. coli suşu MT616 </td><td> Istek üzerine </td><td></td><td> Ayrıca laboratuar dışında kullanılan </td></tr><tr><td> E. coli suşu pJC2 </td><td> Isteği ile evde, mevcut </td><td></td><td></td></tr><tr><td> E. coli suşu pJH110 </td><td> Reques tarafından evde, mevcutt </td><td></td><td></td></tr><tr><td> SmUW227 gerginlik </td><td> Isteği ile evde, mevcut </td><td></td><td></td></tr></tbody></table>

site-specific bacterial chromosome engineering: &#934;c31 integrase mediated cassette exchange (imce)

Bakteriyel kromozom stably Mega baz aralığında 1 yabancı DNA korumak için de kullanılabilir. Kromozom entegrasyon gibi plazmid çoğaltma, plazmid istikrar, plazmid uyumsuzluk ve plazmid kopya sayısı varyans gibi konular circumvents. Bu yöntem, Streptomyces faj (Φ) C31 2,3 den site-specific integraz kullanır. AttB ve attP (sırasıyla 34 ve 39 bp) 4: ΦC31 integraz iki spesifik DNA bölgeleri arasında doğrudan bir rekombinasyon katalizler. Bu rekombinasyon istikrarlı ve 5 geri almaz. Bir spectinomycin-direnç geninden, Aada (SPR), ve E. oluşan A &quot;tampon iniş&quot; (LP) sekansı attP siteleri çevrili coli ß-glukuronidaz geni (uidA) Sinorhizobium meliloti, Ochrobactrum anthropi ve bir intergenik bölgede Agrobacterium tumefaciens kromozomlarını entegre edilmiştir, duyuyorumsırasıyla pC odağı ve teta odağı,. S. meliloti Bu protokolde kullanılır. Bir stuffer kırmızı floresan proteini (rfp) geni ve antibiyotik direnç geni kuşatan attB siteleri içeren Mobilizable donör vektörler aynı zamanda inşa edilmiştir. Bu örnekte gentamisin dirençli plazmid pJH110 kullanılır. RFP geni 6 SPH I ve Pst I ile istenen yapı ile ikame edilebilir Alternatif attB siteleri tarafından çevrili sentetik bir yapı gibi pK19mob 7 gibi bir mobilizable vektörüne alt-klonlandı olabilir. ΦC31 integraz geni (pHS62 8 den klonlanmış) ile ifade pRK7813 9 plazmid bir mobilizable geniş bir aralığı üzerinde ana, lac promotör tarafından tahrik edilir. Bir eşleşme tetraparental protokolü LP suşu böylece donör kaset ile LP sırayla değiştirme belirteçler içine donör kaset transfer etmek için kullanılır. Bu hücreler vardır trans-integrants. Trans-integrants% 0.5 arasında, tipik bir verim ile oluşturulanlardır. Trans-integrants tipik olarak 5-bromo-4-kloro-3-indolil-beta-D-glukuronik asit (X-gluc) kullanılarak antibiyotik duyarlılığı veya mavi-beyaz tarama ile taranmıştır olanlar 500-1,000 koloniler içinde bulunurlar. Bu protokol, trans-integrants oluşturma ve izole etmek için çiftleşme ve seçme işlemleri içerir.

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Site-specific Bacterial Chromosome Engineering: &amp;#934;C31 Integrase Mediated Cassette Exchange (IMCE)

Önceden hazırlanmış alıcı suşlar olarak adlandırılan iniş takımı suşları, içine faiz yabancı DNA entegre hızlı ve verimli bir yöntem tarif edilmiştir. Metodu ΦC31 integraz konjugasyonu ve ifadesi ile, belirli bir lamine suşun iniş pedi odağına içine bir DNA kasetinin site-specific entegrasyon sağlar.

Siteye özgü Bakteriyel Kromozom Mühendislik: ΦC31 integraz Aracılı Kaset Borsası (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

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Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

15.5K Görüntüleme.  University of Waterloo. Önceden hazırlanmış alıcı suşlar olarak adlandırılan iniş takımı suşları, içine faiz yabancı DNA entegre hızlı ve verimli bir yöntem tarif edilmiştir. Metodu ΦC31 integraz konjugasyonu ve ifadesi ile, belirli bir lamine suşun iniş pedi odağına içine bir DNA kasetinin site-specific entegrasyon sağlar.

Video: Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Akciğer Mühendisliği Prosedürü

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Biyomühendislik

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

İkincil Elektrosprey İyonizasyon Kütle Spektrometresi (Sesi-MS) ile karakterize Bakteriyel uçucu

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

Mikroelektrod Diziler Kullanarak siRNA Bağlanan Hücre Yüksek verim, Siteye özgü Transfeksiyon (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Kardiyak Doku Mühendisliği kılcal Kuvvetleri Litografi

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

Fibroblast aracılı Fibrin Jel Sıkıştırmayı Gözlemleme ve Ölçme

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

Binder Bakteriyel Selüloz kullanmak Sağlam Doğal Elyaf preform üretim

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea (ureolysis) in naturally occurring environments through secretion of an enzyme urease. This process of ureolysis, through a chain of chemical reactions, leads to the formation of calcium carbonate precipitates. This is known as Microbiologically Induced Calcite Precipitation (MICP). The proper culture protocols for MICP are detailed here. Finally, visualization experiments under different modes of microscopy were performed to understand various aspects of the precipitation process. Techniques like optical microscopy, Scanning Electron Microscopy (SEM) and X-Ray Photo-electron Spectroscopy (XPS)&#160;were employed to chemically characterize the end-product. Further, the ability of these precipitates to clog pores inside a natural porous medium was demonstrated through a qualitative experiment where sponge bars were used to mimic a pore-network with a range of length scales. A sponge bar dipped in the culture medium containing the bacterial cells hardens due to the clogging of its pores resulting from the continuous process of chemical precipitation. This hardened sponge bar exhibits superior strength when compared to a control sponge bar which becomes compressed and squeezed under the action of an applied external load, while the hardened bar is able to support the same weight with little deformation.

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

Mikrobiyolojik Kalsit Yağış Bağlı tarafından Aracılı<em&gt; Sporosarcina pasteurii</em

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Bir ıntrinsic Vaskülarizasyon tarafından Doku Mühendisliği<em&gt; İn Vivo</em&gt; Doku Mühendisliği Odası

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Parazit Mikro / Nanoparçacık Hücre Mühendisliği mikroakışkan Tampon Değişim

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Hücre Kültürü Uygulamaları için Hidrojeller Işık aracılı Oluşumu ve Desenlendirme

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...