親切なインテグラーゼクローンを提供するためのマーガレットCMスミスへ からの資金調達のサポート： ゲノムカナダ/ゲノムプレーリー NSERC発見と戦略プロジェクトの助成金

1。文化の生産<ol><li> TY 10（5 g / lのトリプトン、3 g / lの酵母エキス、0.44 g / lの塩化カルシウムの脱水）、およびLB 11（10 g / lのトリプトン、5g / lの酵母エキス、5gの/：滅菌液体培地を準備します。塩化ナトリウム、pH 7）。 </li><li> 50μg/ mlのスペクチノマイシンとTYメディアの5ミリリットルに（）：SmUW227（建設はリクエストを承り構造の詳細を痛め、別の場所で説明されるS.根粒 LP-ひずみ：）は、単一のコロニーから接種する。 E.：与えられた抗生物質を補充する、LB液体培地5ml中に以下の菌株を接種する大腸菌は DH5α12 10μg/ mlのテトラサイクリンでpJC2、プラスミドインテグラーゼの発現を含む。E.大腸菌 MT616 13、動員、10μg/ mlのクロラムフェニコール、およびE.大腸菌 DH5α5μg/ mlのゲンタマイシンをpJH110、ドナープラスミドカセットを含む。 </li><li>インキュベートE. 37＆一晩大腸菌株度、一定に振とうしながらC。インキュベートS.振盪しながら2日間の30℃における根粒菌株 。 S.を得ることが可能である大規模な接種（飽和文化の1:500継代培養）で一晩根粒の文化。 </li></ol> 2。文化の調製とミキシング<ol><li> 30秒間17000×gで遠心分離によって細胞ペレットを集め、1 mlの滅菌0.85％NaClでの再懸濁することにより、各株の培養液1.5 mlを洗って、一回繰り返します。 </li><li>滅菌した0.85％NaClで100μlのペレットを再懸濁します。 </li><li>個別に平野TY寒天プレート上でそれぞれの株に洗浄した文化の10μlを見つける。これらのスポットは、コントロールスポットです。 </li><li> E.除く各株の40μlを加え大腸菌 DH5α滅菌チューブにpJC2を含む12、ミックス、プレーンTY寒天プレート上で、この混合物の120μlを見つける。このスポットはno-インテグラーゼ陰性コントロールです。 </li><li>無菌tに各株の40μlを加えUBE、ミックス、スポット平野TY寒天プレート上で、この混合物160μlの。このスポットはIMCE交配スポットです。 </li><li>スポットが層流フード内で乾燥させます。 </li><li>シール板と30°Cで一晩インキュベートします。 </li></ol> 3。トランス組込み体の単離<ol><li> 200μg/ mlのストレプトマイシン（SmUW227はストレプトマイシン14に高レベルの耐性を付与する突然変異を運ぶRm1021に基づいています）、30μg/ mlのゲンタマイシンを添加したTY寒天プレート上にストリークコントロールスポットとIMCEスポット。 </li><li> IMCE効率の計算については、約再懸濁し¼500μlの滅菌0.85％NaClでのIMCE交配スポット。 10 -7〜10倍希釈系列を作成します。 TY寒天プレート上10 -5を介してプレート希釈液10 -2は 、トランス組込み体を選択するために、200μg/ mlのストレプトマイシンおよび30μg/ mlのゲンタマイシンを補った。 10 -3 THRプレート希釈液トランス組込み体と潜在的な受信者の両方を選択するために、200μg/ mlのストレプトマイシンを補充したTY寒天プレート上でウワーッ10 -7、合計受信者、すなわち。 </li><li>マーカーレストランス組込み体を単離するための無菌の0.85％NaClで500μlに約IMCE交配スポットの四半期に再懸濁します。 10 -6〜10倍希釈系列を作成します。プレート希釈液200μg/ mlのストレプトマイシンおよび200μg/ mlのX-glucを添加した4 TY寒天プレート上で10 -6〜10 -4。 </li><li> 3日間30℃でプレートをインキュベートします。 </li><li>緑色の光（525 nm）の下に、赤のフィルタ（&gt; 610 nm）で15を介してビューRFPトランス組込み体。 </li><li>トランス組込み体のCFUは、全受信者のCFUと比較してパーセントIMCE効率を計算します。トランス組込み体の約半分は、敏感な彼らは、白とスペクチノマイシン作る真のカセット交換を受けています。 </li><li>マーカーレストランス組込み体（Wを見つけるにはここでドナーベクターは、白いコロニー（拡張潜伏期間、室温で1-2余分な日、X-gluc経由してコロニーの分化を向上させるための画面）pJH110のようにGmのrを含んでいませんが、これはS.必要である根粒 SmUW227）、抗生物質および100μg/ mlのスペクチノマイシンを含むTY寒天プレートなしのTY寒天プレート上でスクリーニングすることによってコロニーのスペクチノマイシン感受性を確認します。 </li></ol> 4。代表的な結果ストレプトマイシンとゲンタマイシン制御筋を添加したTY上でのインキュベーションの3日後には成長しているべきではありません。 IMCEストリークは、図2に見られるように、第二連勝で頭筋上のコンフルエントな成長と多くの植民地を持っている必要があります。合計受信者へのトランス組込み体の割合として表されるトランス統合の効率は、0.5％の範囲でなければなりません。約トランス組込み体の半分はスペクチノマイシンに敏感と白になります彼らは真のカセット交換を受けて表示しています。緑色の光（525 nm）の下、赤フィルター（&gt; 610 nm）で15を介して見たときpJH110からテスターのRFPドナーカセットを含有するトランス組込み体は、識別可能なRFP蛍光を表示する必要があります。 <img src="/files/ftp_upload/3698/3698fig1.jpg" alt="図1"/> 図1コンジュゲートの混合図：pRK600からの転送遺伝子の発現による支援は、プラスミドのすべてが細胞から細胞へランダムに転送されます。 IMCE、インテグラーゼ（int）はヘルパープラスミドpJC2とドナープラスミドpJH110に必要な2つのプラスミドのLP-株の買収により、混合物中のトランス組込み体の作成 ​​にこの転送の結果、。プラスミド非複製ドナー（pJH110）からドナーカセットはLP-マーカー（Spの損失、その結果、染色体上にLP-カセットのマーカーとΦC31インテグラーゼ活性を介して交換されrとuidA）で処理し、得られたトランスインテグラのドナーカセットのメンテナンス（RFPとのGm R）。 <img src="/files/ftp_upload/3698/3698fig2.jpg" alt="図2"/> 図2。 ：寒天表面上の乾燥した細胞混合物を示す非選択TYプレート上に嵌スポットプレートB：上からストレプトマイシン、ゲンタマイシン-X-glucプレート上にストリーク嵌スポットは、Sに、時計回りIMCEを全くインテグ制御を残っていない根粒、E.大腸菌 DH5αを含むpJC2制御、E.大腸菌 DH5aはpJH110コントロールを含む、E.大腸菌 MT616制御、およびS.根粒 UW227コントロールC：。TYストレプトマイシン-X-gluc寒天上で交配スポット再懸濁の10 -2希釈D：TYストレプトマイシン、ゲンタマイシン-X-gluc寒天（青コロニーは単一の組換え体であり、上の交配スポット再懸濁の10 -2希釈白色コロニーは、真の受けたカセット交換）E：。蛍光性の欠如を示すTYストレプトマイシン-X-gluc寒天上で交配スポット再懸濁の10 -2希釈F：2つのレベルを示すTYストレプトマイシン、ゲンタマイシン-X-gluc寒天上で交配スポット再懸濁の10 -2希釈蛍光の（明るいコロニーは青色のコロニーに対応しており、読み取りしかしコロニーが、真のカセット交換なしでlacプロモーターからのリードスルーでのみ、その即時プロモーターとRFPが含まれて受けたベクター配列からプロモーターに起因すると考えられる高いRFPを発現している不在であるベクトル、）。

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利害の衝突が宣言されません。

IMCE技術は、以前に設計された株のLP-遺伝子座への単一attB挟まれたDNAカセットの効率的な統合が可能になります。希望するコンストラクトがドナーカセットを作成するRFPの代わりにクローン化されると、技術は非常に堅牢な作り、その後のDNA精製と変換を必要としません。それは抗生物質耐性は、トランス組込み体の作成とない他の要因によるものであることを確実にするため?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>ストレプトマイシン</td><td> Bioshopカナダ社</td><td> STP101 </td><td></td></tr><tr><td>スペクチノマイシン</td><td> Bioshopカナダ社</td><td> <a href="https://ssl34.alentus.com/bioshopcanada/detail.asp?Pin=SPE201">SPE201</a> </td><td></td></tr><tr><td>ゲンタマイシン</td><td> Bioshopカナダ社</td><td> GTA202 </td><td></td></tr><tr><td> Choramphenicol </td><td> Bioshopカナダ社</td><td> CLR201 </td><td></td></tr><tr><td>テトラサイクリン</td><td> Bioshopカナダ社</td><td> TET701 </td><td></td></tr><tr><td>カナマイシン</td><td> Bioshopカナダ社</td><td> KAN201 </td><td></td></tr><tr><td>細菌学的等級寒天</td><td&gt; Bioshopカナダ社</td><td> AGR001 </td><td></td></tr><tr><td>トリプトン</td><td> Bioshopカナダ社</td><td> TRP402 </td><td></td></tr><tr><td>酵母エキス</td><td> Bioshopカナダ社</td><td> YEX401 </td><td></td></tr><tr><td>塩化ナトリウム</td><td> Bioshopカナダ社</td><td> SOD001 </td><td></td></tr><tr><td>塩化カルシウム</td><td> Bioshopカナダ社</td><td> CCL444 </td><td></td></tr><tr><td> X-gluc </td><td>ゴールドバイオ株式会社</td><td> G1281C1 </td><td></td></tr><tr><td> 大腸菌 MT616株</td><td>リクエストも承ります</td><td></td><td>また、我々の研究室の外で使用</td></tr><tr><td> 大腸菌 pJC2株</td><td>リクエストにより、家の中で、利用可能な</td><td></td><td></td></tr><tr><td> 大腸菌 pJH110株</td><td> requesによって家の中で、利用可能なトン</td><td></td><td></td></tr><tr><td> SmUW227ひずみ</td><td>リクエストにより、家の中で、利用可能な</td><td></td><td></td></tr></tbody></table>

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

細菌の染色体を安定的にメガ塩基の範囲1に外来DNAを維持するために使用されることがあります。染色体への統合は、このようなプラスミド複製、プラスミドの安定性、プラスミド不和合性、プラスミドコピー数の変動などの問題を回避します。このメソッドは、 放線菌ファージ（Φ）C31 2,3から、サイト固有のインテグラーゼを使用しています。 attBとattP（それぞれ34と39 bpの、）4：ΦC31インテグラーゼは、2つの特定のDNA部位の間の直接的な組換えを触媒する。この組換えは、安定していると5を戻すことはできません。スペクチノマイシン耐性遺伝子、aadA（SPR）、およびE.から成る&quot;ランディングパッド&quot;（LP）シーケンスattP部位によって挟まれた大腸菌 β-グルクロニダーゼ遺伝子（uidA）Sinorhizobium根粒、Ochrobactrum anthropiと、遺伝子間領域におけるAgrobacterium tumefaciensの染色体に組み込まれており、 午前それぞれpCの軌跡、そしてTETA軌跡、S.根粒は、このプロトコルで使用されています。スタッファー赤色蛍光タンパク質（RFP）遺伝子と抗生物質耐性遺伝子に隣接するattB部位を含む可動性ドナーベクターも構築されている。この例では、ゲンタマイシン耐性プラスミドpJH110が使用されます。 RFP遺伝子の 6は SPH I と Pst Iを用いて所望の構築物で置き換えることができますまたattB部位に隣接した合成構造などpK19mob 7のような可動性ベクターにサブクローン化されたかもしれません。 ΦC31インテグラーゼ遺伝子の発現（pHS62 8からクローニングされた）pRK7813 9可動性プラスミド広宿主範囲に、lacプロモーターによって駆動されます。 tetraparental交配プロトコルは、それによってドナーカセットでLPシーケンスにマーカーを置き換えるLP株にドナーカセットを転送するために使用されます。これらの細胞はトランジスタです。S-組込み体。トランス組込み体は、0.5％の典型的な効率で形成される。トランス組込み体は、通常、抗生物質感受性または5 - ブロモ-4 - クロロ-3 - インドリル-β-D-グルクロン酸（X-gluc）を使用して青白色スクリーニングでスクリーニング第一500〜1,000コロニー内にあります。このプロトコルは、トランス組込み体を作成し、単離するための交配と選択プロシージャが含まれています。

Watch this Scientific Journal Video about Site-specific Bacterial Chromosome Engineering: Î¦C31 Integrase Mediated Cassette Exchange IMCE at JoVE.com

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

既製のアクセプター株、と呼ばれる着陸パッド株に興味のある外来DNAを統合するための迅速かつ効率的な方法が記載されている。メソッドは、ΦC31インテグラーゼの結合と表現を通して、与えられた菌株の人工着陸パッドの座にDNAカセットの部位特異的に統合することができます。

サイト固有の細菌染色体工学：ΦC31インテグラーゼを介したカセット取引所（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 ...

site-specific-bacterial-chromosome-engineering-c31-integrase-mediated

Research

JoVE Journal

Bioengineering

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

15.5K ビュー.  University of Waterloo. 既製のアクセプター株、と呼ばれる着陸パッド株に興味のある外来DNAを統合するための迅速かつ効率的な方法が記載されている。メソッドは、ΦC31インテグラーゼの結合と表現を通して、与えられた菌株の人工着陸パッドの座にDNAカセットの部位特異的に統合することができます。

ビデオ: 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.

肺のエンジニアリングのための手順

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

生物工学

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.

二次エレクトロスプレーイオン化質量分析法（SESI - MS）を用いて細菌揮発性物質の特性評価

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).

微小電極アレイを用いたsiRNAの付着細胞への高効率、サイト固有のトランス体（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.

心臓組織工学のためのキャピラリーフォースリソグラフィ

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.

線維芽細胞媒介フィブリンゲル圧縮の観察と定量

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.

バインダーとしてのバクテリアセルロースを利用堅牢な天然繊維プリフォームの製造

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.

微生物学的に誘導された方解石沈殿がによって媒介されます<em&gt;スポロサルシナパステウリイ</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.

中に組み込み血管新生による組織工学<em&gt;インビボ</em&gt;ティッシュ・エンジニアリング商工会議所

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.

干渉のないマイクロ/ナノ粒子の細胞工学のためのマイクロ流体緩衝液交換

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.

細胞培養アプリケーションのためのヒドロゲルの光媒介形成とパターニング

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