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1。ハンドル：デザインとシェーピング<ol><li>テープで木の板にNR 10炭素手術用メスの刃を固定します。 </li><li>顕微鏡下でリングの中心にタグを付ける。これはメスの刃の木立〜1.2センチ真っ直ぐに配置する必要があります。 </li><li>球形のダイヤモンドビット、2 mmの穴を開け、高速ドリルに取り付けられた。 </li><li>紡錘状または円錐ダイヤモンドビットで穴を広げる。 表1に示すように、最終的な直径は、ラットの体重に応じて変化する。 </li><li>永久マーカーでハンドルの形状を設計する。 図1及び図2は、ステンシルとして使用することができる。 </li><li>ミニベンチグラインダーにドリルを変換し、形状の外刃の部分を削除します。 </li><li>ブラシを回転させ、低速を持つ作品を締めくくる。 </li></ol> 2。近似​​：デザインとシェーピング<ol><li>近似​​は修正ストレート外科クレマーのやコッヘルの鉗子によって得られるS。 </li><li>高速ミニベンチグラインダーを使用して、顕微鏡下で1枝の半分を切り落とした。 </li><li>完全にスライドするハンドルを可能にするために両側に対称の溝を作る。 </li><li>長い腕の縦スリットをカットし、長腕の中心と好位置にハンドルが同じ軸上にあることを確認してください。 </li><li>ハンドルの中央に面した長い腕にノッチを設計します。 </li><li>ブラシを回転させ、低速で送料オフできるだけ薄く丸いブランチを作る。 </li></ol> 3。袖口：デザインとシェーピング<ol><li>カフキャリパーは、ラット体重とひずみに応じて異なります。対照的に、それらの長さが4ミリメートルの周りに保たれるべきである。 </li><li>細かい穴ポリエチレンチューブを取り、NR 10メスで1エッジを丸める。 </li><li>端から木立0.5ミリメートルを設計し、慎重に木立に下る斜面を切り取る。 </li><li>チューブに1つまたは2つの全層ニックを切り取る。これらの意志移植準備と移植時にカフを操作するのに役立ちます。 </li><li>その縁から4ミリメートルでカフをカットします。 </li></ol>

<ol>
	<li>Oldani, G., Lacotte, S., Morel, P., Mentha, G., Toso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+Liver+Transplantation+in+Rats.">Orthotopic Liver Transplantation in Rats.</a> J. Vis. Exp. (65), e4143 (2012).</li><li>Hori, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+and+innovative+techniques+for+liver+transplantation+in+rats:+A+surgical+guide.">Comprehensive and innovative techniques for liver transplantation in rats: A surgical guide.</a> World Journal of Gastroenterology., WJG. 16, 3120 (2010).</li><li>Kashfi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+various+techniques+of+orthotopic+liver+transplantation+in+the+rat.">A review of various techniques of orthotopic liver transplantation in the rat.</a> Transplantation Proceedings. 37 (1), 185-188 (2005).</li><li>Kamada, N., Calne, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+liver+transplantation+in+the+rat.+Technique+using+cuff+for+portal+vein+anastomosis+and+biliary+drainage.">Orthotopic liver transplantation in the rat. Technique using cuff for portal vein anastomosis and biliary drainage.</a> Transplantation. 28, 47 (1979).</li><li>Oldani, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+rat+liver+transplantation+using+Quick+Linker+system:+a+preliminary+result.">A novel technique for rat liver transplantation using Quick Linker system: a preliminary result.</a> Journal of Surgical Research. 149, 303-309 (2008).</li></ol>

著者らは、開示することは何もありません。

現在のレポートでは、より簡単に、ラット肝移植のためのクイック·リンカシステムの設計と構築を説明しています。 ラット肝移植は1990年代初頭（以来毎年発行さ125から180関連論文にも人気のモデルです<a href="http://www.pubmed.com" target="_blank">www.pubmed.com</a> ）。これは、免疫学的およびグラフト保全問題の探査実験を含む広範囲のを可能にする。 &quot;クイックリンカー&quot;キットの使用は、保証内膜ツー内膜に接触して簡単かつ迅速に吻合することができます。ラット肝臓移植の技術的工程の詳細な説明および論議は、以前1に記載されている。 クイックリンカキットは共通の実験室材料からの低価格で構築することができます。遭遇する主な課題は、最終的なブラッシング中に1つのハンドルの分解に関連しており、その問題が狭いパラメーターを設定することによって解決されている0.5ミリメートル（ 図1と図2は、適切な大きさを示す）でハンドルリングのT。ブレードの質に関しては、ステンレス鋼のみが考慮されるべきである。平均炭素鋼の刃に比べてわずかに低い可撓ものの、ステンレススチールブレードは、ハンドルの寿命の使用を可能にする、錆を発症しない。 注目すべきは、現在のハンドルと袖口のデザインが最も効率的な吻合を可能にし、提案された大きさにつながっているプロセスの結果である。カフの直径は、ラットの量（ 表1）に応じて異なる。最小の変動が約4mm（ 図3および図4）の通常の長さは、長さの点で許容することができる。 近似​​については、選択したコッヘルの鉗子は、支点から少なくとも4センチ扱いしている必要があります。これは、動物の腹部外ラックの安全な閉鎖を可能にします。 全体的に、提案されたデバイスは、肝臓とラットの移植片の他のタイプの移植のために簡単に静脈吻合を許可することができます。

同所性ラット肝移植の技術は、可視化実験紙1の最近の刊行物に示されている。門脈および外肝静脈吻合を迅速リンカーアシストカフ技法を用いて行った。この技術は、それが簡単かつ迅速に吻合、良好な血行動態を生じる可能にするように、元のカマダの手法2-4の他のリビジョンのうちから選択され、より短い学習曲線1,5で実現することができる。 クイックリンカー技術は、ハンドルの使用を必要とし、近似と袖口は、その全てが本報告書に記載の技術に係る一般的な実験室用の材料で設計することができる。

<table border="1">
<tbody>
 <tr>
 <td>Tool / Reagent</td>
 <td>Company</td>
 <td>Catalogue n.</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>Porta cuff</td>
 <td>altecweb or 
 smiths-medical</td>
 <td>Coil 01-96-1729 
 800/100/420</td>
 <td>Bore : variable 
 Wall : 0.30 mm 
 Length: 3.8-4.2 mm 
 <a href="http://altecweb.com" target="_blank">altecweb.com</a> or 
 <a href="http://smiths-medical.com" target="_blank">smiths-medical.com</a></td>
 </tr>
 <tr>
 <td>IVC cuff</td>
 <td>altecweb or 
 smiths-medical </td>
 <td>Coil 01-96-1733 
 800/100/540</td>
 <td>Bore : variable 
 Wall : 0.30 mm 
 Length: 3.8-4.2 mm 
 <a href="http://altecweb.com" target="_blank">altecweb.com</a> or 
 <a href="http://smiths-medical.com" target="_blank">smiths-medical.com</a></td>
 </tr>
 <tr>
 <td>Mini-drill </td>
 <td>Dremel</td>
 <td>100 series</td>
 <td>35,000 rpm 
 <a href="http://dremel.com" target="_blank">dremel.com</a></td>
 </tr>
 <tr>
 <td>Diamond wheel point</td>
 <td>Dremel</td>
 <td>7103</td>
 <td>2 mm 
 <a href="http://dremel.com" target="_blank">dremel.com</a></td>
 </tr>
 <tr>
 <td>Diamond wheel point</td>
 <td>Dremel</td>
 <td>7105</td>
 <td>4.4 mm 
 <a href="http://dremel.com" target="_blank">dremel.com</a></td>
 </tr>
 <tr>
 <td>Diamond wheel point</td>
 <td>Dremel</td>
 <td>7144</td>
 <td>2.4 mm 
 <a href="http://dremel.com" target="_blank">dremel.com</a></td>
 </tr>
 <tr>
 <td>Cut-off wheels</td>
 <td>Dremel</td>
 <td>420</td>
 <td>dia: 23.8 mm 
 thickness: 0.5 mm 
 <a href="http://dremel.com" target="_blank">dremel.com</a></td>
 </tr>
 <tr>
 <td>Quick-linker handles</td>
 <td>dltchiropody (DLT)</td>
 <td>PAR 10</td>
 <td>modified scalpel blades 
 <a href="http://dltchiropody.co.uk" target="_blank">dltchriopdy</a></td>
 </tr>
 <tr>
 <td>Quick-linker approximator</td>
 <td>pharmap</td>
 <td>UD-04-275</td>
 <td>modified Kocher's forceps 
 <a href="http://pharmap.ch" target="_blank">pharmap.ch</a></td>
 </tr>
 </tbody>
</table>

manufacturing devices and instruments for easier rat liver transplantation

同所性ラットの肝臓移植は、 &quot;クイックリンカー&quot;デバイスの使用と最近のJoveの論文で示されてきた人気のモデルです。この手法は、合理的な学習曲線の後に簡単に静脈カフanatomosesが可能になります。デバイスは、メスの刃から刻まれている2つのハンドル、、細かい穴ポリエチレンチューブから設計された1つのコッヘル鉗子を変更することによって得られた近似と、袖口から構成されている。全体のプロセスは、低コストで使用した一般的な実験室用材料で行うことができる。本報告は、必要な部品の設計のためのステップ·バイ·ステップ·プロトコルを提供し、ステンシルを含む。

5 approximatorsと11ハンドルを含む5つのキットが、製造されている。ワンハンドルはブラッシングの最終時に壊した。この問題は、ハンドルのリングが狭すぎていたという事実によるものであり、それさ0.5mmのハンドルリングの狭い部分を設定することによって解決されている。いいえ問題はapproximatorsの整形時に発生していません。 <table border="1"><tbody><tr><td> ラット重量（g） </td><td> ポルタハンドル（内径）（mm）の </td><td> ポルタカフ（外径）（mm）の </td><td> IVCハンドル（内径）（mm）の </td><td> IVCカフ（外径）（mm）の </td></tr><tr><td> 120-180 </td><td> 2.2 </td><td> 1.7 </td><td> 2.6 </td><td> 2.08 </td></tr><tr><td> 180から270 </td><td> 2.6 </td><td> 2.08 </td><td> 3.4から3.7 </td><td> 3.0から3.25 </td></tr><tr><td> &gt; 270 </td><td> 2.9<html></td><td> 2.42 </td><td> 4.2 </td><td> 3.7 </td></tr></tbody></table> 表1。 <img src="/files/ftp_upload/50380/50380fig1.jpg" alt="図1" fo:content-width="6in" fo:src="/files/ftp_upload/50380/50380fig1highres.jpg"/> 図1。ポルタは、ステンシルを扱う。1:1成型ハンドルを導くために使用することができますスケールで印刷された場合。 <a href="https://www.jove.com/files/ftp_upload/50380/50380fig1large.jpg" target="_blank">より大きい数字を表示するには、ここをクリックしてください</a> 。 <img src="/files/ftp_upload/50380/50380fig2.jpg" alt="図2" fo:content-width="6in" fo:src="/files/ftp_upload/50380/50380fig2highres.jpg"/> 図2。 IVCは、ステンシルを扱う。1:1成型ハンドルを導くために使用することができますスケールで印刷された場合。 <a href="https://www.jove.com/files/ftp_upload/50380/50380fig2large.jpg" target="_blank">より大きい数字を表示するには、ここをクリックしてください</a> 。 コンテンツ &quot;&gt; <img src="/files/ftp_upload/50380/50380fig3.jpg" alt="図3" fo:content-width="4.6in" fo:src="/files/ftp_upload/50380/50380fig3highres.jpg" /> 図3。ポルタとIVCカフ側面図。グラフトのv.porta、横いずれかに受信者の容器ながら、中央の溝に固定する必要があります。この方便は、最適内膜ツー内膜に接触することができます。 <img src="/files/ftp_upload/50380/50380fig4.jpg" alt="図4" fo:content-width="4.1in" fo:src="/files/ftp_upload/50380/50380fig4highres.jpg"/> 図4。 180〜270グラムの重量を量るラットに適しポルタとIVCの袖口正面。キャリパーが表されます。

Watch this Scientific Journal Video about Manufacturing Devices and Instruments for Easier Rat Liver Transplantation at JoVE.com

Manufacturing Devices and Instruments for Easier Rat Liver Transplantation

我々は簡単に同所ラット肝移植のための &quot;クイックリンカ&quot;デバイスの設計について説明します。

簡単ラット肝移植のための製造装置や計測器

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This ...

manufacturing-devices-instruments-for-easier-rat-liver

Research

JoVE Journal

Bioengineering

8.6K ビュー.  University of Geneva Hospitals. 我々は簡単に同所ラット肝移植のための "クイックリンカ"デバイスの設計について説明します。

ビデオ: Manufacturing Devices and Instruments for Easier Rat Liver Transplantation

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

ミササイトメトリー：CyTOFマスメーターで毎日のチューニングと実行した細胞サンプルのためのプロトコル

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

生物工学

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 &#956;l. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease.

This protocol details a method to isolate extracellular vesicles (EVs), small membranous particles released from cells, from as little as 10 &#956;l serum samples. This approach circumvents the need for ultracentrifugation, requires only a few minutes of assay time, and enables the isolation of EVs from samples of limited volumes.

細胞外小胞の単​​離および特徴のための紙ベースのデバイス

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

振動圧力潅流装置内のラット肝臓の脱細胞化のための手順

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

Fabricating Cotton Analytical Devices

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.

To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.

コットン分析デバイスを製造します

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

最適化されたセットアップおよびヒステリシス測定場の磁区イメージングのためのプロトコル

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

ラットでの肝部分工学生体内での基盤として新しい手術手技

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

Here we present a protocol to design and fabricate custom microfluidic devices with minimal financial and time investment. The aim is to facilitate the adoption of microfluidic technologies in biomedical research laboratories and educational settings.

研究・教育応用用カスタムマイクロ流体デバイスの迅速な製造

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

生体組織の刺激のための電界・磁場装置

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

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

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

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

マイクロ流体混合プラットフォームを用いた遺伝子導入のための脂質ナノ粒子の作製と特性化

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

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

2次元ヒト幹細胞由来心筋細胞における心収縮性調節療法の評価

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...