著者は、彼のタンパク質分析のための彼女の技術的専門知識とアンドリュー·ラーセンのためにブルックイェルムに感謝します。この作品は、代替の研究開発財団（MMHK）グラントとNIH NIAID性感染症と局所殺ウイルス剤共同研究センターIU19 AI062150-01（MMHK）によって一部で賄われていた。我々は感謝して図の再利用のための生殖の生物学を認める。

<ol>
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P., 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=Heterogeneous+breast+tumoroids:+An+in+vitro+assay+for+investigating+cellular+heterogeneity+and+drug+delivery.">Heterogeneous breast tumoroids: An in vitro assay for investigating cellular heterogeneity and drug delivery.</a> J. Biomol Screen. 12, 13-20 (2007).</li><li>Jin, F., 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=Establishment+of+three-dimensional+tissue-engineered+bone+constructs+under+microgravity-simulated+conditions.">Establishment of three-dimensional tissue-engineered bone constructs under microgravity-simulated conditions.</a> Artif Organs. 34, 118-125 (2010).</li><li>Vertrees, R. 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著者らは、開示することは何もありません。

ここに提示RWVバイオリアクター技術の活用は、より生理学的に関連する器官の細胞培養モデルに、現在の細胞培養系を前進させる能力を研究者に提供することがあります。 RWVバイオリアクター細胞培養システムは、タイトジャンクション、ムチン産生、細胞外のプロセス（すなわち微）、および細胞の極性を含む生体のような特性にして3-Dの細胞凝集体を形成する細胞を可能に...

<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>のAlexa Fluor®488 </td><td>インビトロジェン</td><td> A21131 </td><td> 1:500希釈で使用</td></tr><tr><td> FACSDiva </td><td> BD </td><td></td><td>フローサイトメーター</td></tr><tr><td> β-tublin抗体</td><td>カルビオケム</td><td> 654162 </td><td> 1:5000希釈で使用</td></tr><tr><td>バイオプレックス2000 </td><td> BioRad社</td><td> 171-000205 </td><td> V5ソフトウェア</td></tr><tr><td>バイオリアクターとコンポーネント</td><td> Synthecon </td><td> RCCS-4 </td><td></td></tr><tr><td>セルストレーナー</td><td> BDファルコン</td><td> 352340 </td><td> 40μmの細孔径</td></tr><tr><td>コニカルチューブ（50ml）に</td><td>コー​​ニング</td><td> 5-538-60 </td><td></td></tr><tr><td>カバースリップ</td><td> VWR </td><td> 48366067 </td><td></td></tr><tr><td>サイトカインビーズアレイキット</td><td> BioRad社</td><td>カスタム人間キット</td><td></td></tr><tr><td> Cytodexビーズ</td><td>シグマ</td><td> C3275 </td><td></td></tr><tr><td> DPBS </td><td>ギブコ</td><td> 14190 </td><td></td></tr><tr><td> EDTA </td><td>シグマ</td><td> ED-500G </td><td>エチレンジアミン四酢酸</td></tr><tr><td>上皮に特異的な抗体（ESA） </td><td>ケミコン</td><td> CBL251 </td><td> 1:50希釈で使用</td></tr><tr><td>ウシ胎児血清（FBS） </td><td>ギブコ</td><td> 10438 </td><td>不活化熱</td></tr><tr><td> HARV（使い捨て） </td><td> Synthecon </td><td> D-405 </td><td></td></tr><tr><td>塩酸</td><td>シグマ</td><td> 258148 </td><td> 37パーセント</td></tr><tr><td>インボルクリン抗体</td&gt; <td>シグマ</td><td>私は9018 </td><td></td></tr><tr><td>顕微鏡スライド</td><td> VWR </td><td> 16004-368 </td><td></td></tr><tr><td> MTT試薬</td><td> MPバイオメディカル、LLC </td><td> 194592 </td><td> 3 - （4,5 - Dimethylthiazolyl 1-2）-2,5 - ジフェニルテトラゾリウムブロミド</td></tr><tr><td> MUC1抗体（顕微鏡） </td><td>サンタクルス</td><td> SC-7313 </td><td> 1:50希釈で使用</td></tr><tr><td> MUC1抗体（フローサイトメトリー） </td><td> BD Pharmingen社</td><td> 559774 </td><td>また、CD227と呼ばれる、テストあたり20μLを使用し</td></tr><tr><td> パラホルムアルデヒド </td><td>電子顕微鏡学</td><td> 15710 </td><td> DPBSで4％に希釈し</td></tr><tr><td>ペトリ皿（小） </td><td> BDファルコン</td><td> 353002 </td><td></td></tr><tr><td>フィルタ付きポリスチレンチューブ</td><td> BDファルコン</td><td> 352235 </td><td></td></tr><tr><td&gt;ポリスチレンフローチューブ</td><td> BDファルコン</td><td> 352058 </td><td></td></tr><tr><td> PR抗体</td><td> DAKO </td><td> M3569 </td><td> 1:100希釈で使用</td></tr><tr><td>ゴールドを長引かせる</td><td>インビトロジェン</td><td> P36931 </td><td> DAPIと取り付けメディア</td></tr><tr><td> RNeasyミニキット</td><td>キアゲン</td><td> 74903 </td><td></td></tr><tr><td>ドデシル硫酸ナトリウム</td><td>シグマ</td><td> 71725 </td><td></td></tr><tr><td>滅菌袋</td><td> VWR </td><td> 11213-035 </td><td></td></tr><tr><td>コック（片道） </td><td>メデックス</td><td> MX5061L </td><td></td></tr><tr><td>シリンジ（10ml）に</td><td> BD </td><td> 309604 </td><td>ルアーロックチップ</td></tr><tr><td>シリンジ（5mL）を</td><td> BD </td><td> 309603 </td><td>ルアーロックチップ</td></tr><tr><td>トリパンブルー</td><td>インビトロジェン</td><td>T10282 </td><td></td></tr><tr><td> VP5抗体</td><td>サンタクルス</td><td> SC-13525 </td><td> HSV-2抗体クローン6F10、1:5000希釈で使用</td></tr></tbody></table>

culturing and applications of rotating wall vessel bioreactor derived 3d epithelial cell models

身体の経験、そのアーキテクチャに影響を与える環境条件、細胞間の通信、および全体的な関数の細胞や組織。 in vitroでの細胞培養モデルで正確に目的の組織を模倣するためには、文化の発展環境が考慮すべき重要な側面です。一般的に使用される従来の細胞培養システムのフラット2次元（2-D）不浸透性の表面に上皮細胞を伝播します。多くの従来の細胞培養系から学んだされていますが、多くの知見は、潜在的に生理学的に関連する微小環境の欠如の結果として、人間の臨床試験または組織外植片で再現可能ではありません。 ここでは、革新的な回転壁容器（RWV）バイオリアクター技術を用いて、2次元細胞培養の培養条件の境界の多くを克服する培養系を説明します。我々と他の器官RWVから派生したモデルはstructurを再現することが示されている同様に人間の外植組織1-6外部からの刺激への電子、機能、本物の人間の反応。 RWVバイオリアクターは、低生理流体剪断条件下で上皮細胞の増殖を可能にする懸濁培養システムです。バイオリアクターは、2つの異なる形式で、高アスペクト回転容器（HARV）または彼らは曝気ソースによって異なっている低速回転側容器（STLV）に来る。上皮細胞は、多孔質、コラーゲンコートしたマイクロビーズ（図1A）との組み合わせで、選択したバイオリアクターに追加されます。細胞がバイオリアクターの定数自由落下（図1B）の間に成長の足場としてビーズを利用しています。バイオリアクターによって提供された微小環境は細胞は、しばしば標準的な2-D培養条件（図1D）の下で観察されなかっ生体に似た特性で表示（3-D）は、3次元凝集体を形成することができます。これらの特性は、タイトジャンクショ​​ン、MUCが含まれていin vivoでのタンパク質の局在化、およびその他の上皮細胞型固有のプロパティで私たちの生産、基底/心尖部の向き、。 完全に分化した3-D集計に上皮細胞の単層からの進行は、細胞の1型、7月13日に基づいて変わります。バイオリアクターからの定期的なサンプリングでは、上皮細胞凝集体形成、細胞分化マーカーと生存率（図1D）のモニタリングが可能になります。一度細胞分化および凝集体形成が確立され、細胞がバイオリアクターから採取され、2-D細胞について行った同様のアッセイは、いくつかの考慮事項（図1E-G）で3-D集約に適用することができます。本研究では、我々はどのようにRWVバイオリアクターシステムと潜在的なアッセイおよび3-D集計を実行することができます分析の様々な文化の3-D上皮細胞凝集体への詳細な手順について説明します。これらの分析は含まれていますが、M /構造が、これらに限定されないorphological分析（共焦点、走査型および透過型電子顕微鏡）、サイトカイン/ケモカインの分泌と細胞内シグナル伝達（サイトメトリービーズアレイおよびウェスタンブロット解析）、遺伝子発現解析（リアルタイムPCR）、薬物/毒物分析と宿主 - 病原体相互作用。これらのアッセイの利用は、メタボロミクス、トランスクリプトミクス、プロテオミクス、他のアレイ·ベースのアプリケーションなど、より詳細なと広大な研究のための基盤を設定します。私たちの目標は、多様な科学的興味を持つ研究者によって使用されるように容易な、堅牢なシステムでは、 生体組織の人間を再現器官の3次元モデルを生成するために、培養ヒト上皮細胞の非従来型の手段を提示することである。

Watch this Scientific Journal Video about Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models at JoVE.com

Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

上皮細胞が3-D細胞凝集体の形成をもたらす生理的条件下で成長することができ、回転細胞培養システムが記載されています。凝集体は、ディスプレイを生成<emin vivoでの&gt;</em&gt;のような特性は、従来の培養モデルで観察され、科学的な調査の多数のためのより正確な器官のモデル系としては機能しません。

回転壁容器バイオリアクター由来3D上皮細胞モデルの培養とその応用

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. ...

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Research

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Biology

26.1K ビュー.  University of Arizona College of Medicine - Phoenix. 上皮細胞が3-D細胞凝集体の形成をもたらす生理的条件下で成長することができ、回転細胞培養システムが記載されています。凝集体は、ディスプレイを生成のような特性は、従来の培養モデルで観察され、科学的な調査の多数のためのより正確な器官のモデル系としては機能しません。

ビデオ: Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

粉末試料の絶対量子収率測定

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

生物学

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

滑液における培養ヒト関節軟骨細胞用の3Dシステム

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

I ·クライオキットを使用してマウスの精子の凍結保存と回復

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

マイクロアレイを用いた植物細胞壁ポリマーの糖鎖プロファイリング

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

エア·液界面での人間の鼻上皮細胞の培養

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

ヒト骨格筋からの血管由来の多能前駆体の単離

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

の組織化学的染色<em&gt;シロイヌナズナ</em&gt;二次細胞壁の要素

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

地図状委縮の目の大きいモデルにヒト胚性幹細胞由来網膜色素上皮細胞の網膜下移植

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

糸状菌の生細胞イメージングのための膜および細胞壁選択的蛍光色素の応用

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

腸内ビリ上皮によるオルガノイドの3D培養

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...