この研究は国立衛生研究所からの米国退役軍人局（ワシントンDC）、助成金0852048、0731201、および0854430国立科学財団（アーリントン、バージニア州）から、および助成金RO1 DK080897（ベセスダ、MD）によってサポートされていました。氏カンナは、ビル＆メリンダ·ゲイツ財団（ワシントン州シアトル）から氏エドワード·ロス博士モニカ·モヤで寛大な寄付から支援を受けた。

ここでプロトコルは多層アルギン酸マイクロビーズ（ 図1）を生成するための3つのステップの手順について説明します。まず、アルギン酸マイクロビーズ（ 図2A）が形成されている。この手順は、以下のセクション1に記載されている。細胞やタンパク質は、配信システムとして動作するために、このステップのマイクロビーズに追加することができます。次のステップでは、マイクロビーズ上に選択透過層の形成を含み、第2章で説明されています。最後のステップは、追加のアルギン酸塩層の形成を伴い、第3節で説明されています。ビーズの表面（ 図2B）の外側にこの層の形態は、移植後のシステムへの細胞応答を誘導する治療用分子（ 図2C）をカプセル化し、提供するために使用することができます。 1。アルギン酸マイクロビーズの調製<ol><li> 1mL中にLVMアルギン酸の15グラムを溶解することによって1.5％（w / v）のLVMアルギン酸溶液を調製内層アルギン酸塩溶液（25 mM HEPES緩衝DI水にある118軒のNaCl、5.6 mMの塩化カリウム、2.5 mMのMgCl 2、pH7.4に調整）。アルギン酸塩の電源が完全に透明で粘性のある溶液を形成するために溶解するまでボルテックスで混和します。注：このプロトコルは、アルギン酸マイクロカプセル内の膵島のカプセル化のための最適な条件を記述する1アルギン酸ミクロスフェアの濃度と組成は、他のアプリケーション（例えば、薬物送達、組織工学など）に注意するためのプロパティを調整するために変更することができます。膵島のカプセル化膵島microencapsulatorにロードする前にこの段階でアルギン酸塩溶液に添加することができます。 </li><li> DI水に100 mMのCaCl 2及び10mMのHEPES緩衝液に溶解し、pH7.4に調整することにより、架橋溶液（純水で22 mM CaCl 2を ）準備します。注意：このようなBr 2のような他の二価陽イオン+、Srの2 +などがアルギン酸ゲルdの性質に応じて、Ca 2 +の代わりに使用することができますesired。 </li><li>シリンジに25ゲージの針を追加することにより、2チャンネルエアジャケットアルギン酸microencapsulatorを設定し、針がエアジャケットの中央にあることを確認するために、各側にバルブを調整します。異なるゲージの針がターゲットアルギン酸マイクロビーズのサイズに応じて、このステップに使用することができます。 このステップはまた、注射器で行うことができ、microencapsulatorが使用可能であるべきではありません。シリンジにアルギン酸塩溶液を追加し、希望するマイクロビーズのサイズに基づいて、ニードルゲージを選択します。 </li><li>直接針の下に架橋溶液10 mLを入れたフラスコを置きます。溶液中で攪拌棒を配置します。 </li><li>マイクロビーズを形成するために、アルギン酸を架橋するためには、CaCl 2溶液に直接滴を注入します。連続的に攪拌しながら、少なくとも15分間硬化させるために架橋溶液中でビーズをインキュベートします。 </li><li> 15 mLの遠心チューブにビーズを転送します。 、残液を除去すると2分ごとに生理食塩水で0.2％CaCl 2で3回洗浄を行います。 </li></ol> 2。ポリ-L-オルニチンでコーティングMicroBeadsを<ol><li>生理食塩水のポリ-L-オルニチン（PLO）の0.1％（w / v）溶液3 mLを準備します。それは完全に透明な溶液を形成し、溶解するまでボルテックスでソリューションを配置します。ポリ-L-リジン（PLL）は、選択透過性と同様のレベルで、その結果このステップのためにPLOの代わりに使用できます。 </li><li> PLO溶液にアルギン酸ビーズを転送します。ポリカチオン、ポリアニオン複合体を形成し、アルギン酸塩と相互作用するPLOに十分な時間を可能にするために30分間ボルテックス上に置きます。 30分の終わりには、アルギン酸マイクロビーズ周りにはっきりと見える白いコーティングがあるはずです。 </li><li> PLOのソリューションを削除し、2分ごとに生理食塩水0.2％CaCl 2で3回洗浄を行います。 </li></ol> 3。外アルギン酸を作成するには、レイえー<ol><li>外側の層を作成するために使用される所望の濃度のアルギン酸塩溶液を準備します。 （1.1）上記のような溶液を調製する。外層のサイズは、（ 図3）を使用アルギン酸の組成および濃度の影響を受けています。 </li><li>セルストレーナーにアルギン酸ビーズを転送します。可能な限りマイクロビーズを乾燥するために、余分な溶液を吸収するためにキムワイプを使用しています。 </li><li>パラフィルムの表面にマイクロビーズを転送します。必要に応じて、例えば、ガラスあるいはプラスチック製のペトリ皿のような他の滑らかな表面は、パラフィルムの代わりに使用できます。 </li><li>アルギン酸マイクロビーズ上（3.1）で調製したアルギン酸塩溶液を転送します。アルギン酸溶液の体積は、完全にアルギン酸ビーズをカバーする必要がありますすることができます。マイクロビーズは、45分間アルギン酸塩溶液に残ってみましょう。 </li><li>マイクロビーズに結合していない過剰なアルギン酸塩溶液を削除するには、ピペットを使用しています。 </li><li>のマイクロビーズを転送22mmのCaCl 2の溶液へ。これは、別個の外側のアルギン酸塩層を形成し、その結果、マイクロビーズの周りにアルギン酸塩溶液を架橋。 </li><li> 2分ごとに生理食塩水に22 mMのCaCl 2で3回洗浄を行います。顕微鏡下でマイクロビーズを表示すると、コアのマイクロビーズの周りに形成され見られるように別個の外側のアルギン酸塩層のために許可する必要があります。 </li></ol> 4。代表的な結果<img src="/files/ftp_upload/3388/3388fig1.jpg" alt="図1" /> 図1多層アルギン酸ビーズを作成する手順の概略。 Khanna らから許可を得て複製。 J BIOMEDマーテル解像度A. 11月95（2）、632から40（2010）。 <img src="/files/ftp_upload/3388/3388fig2.jpg" alt="図2" /> 図2（A）と（B）アルギン酸マイクロビーズの位相コントラストイメージです。 （）マイクロビーズを示しています。後の合成工程（1.7）、（B）ステップ（3.7）の完了後に存在する別個の外側のアルギン酸塩層を表示した。 （C）外側の層にカプセル化された蛍光標識したBSA蛋白質のFITCイメージです。 Khanna らから許可を得て複製。 J BIOMEDマーテル解像度A. 11月95（2）、632から40（2010）。 <img src="/files/ftp_upload/3388/3388fig3.jpg" alt="図3" /> 図は3つ。外側のアルギン酸塩層のサイズが使用アルギン酸塩の組成及び濃度に基づいて変化させることができる。我々の結果は、増加するアルギン酸塩濃度を有する外層のサイズが増加すると、LVMおよびLVGアルギン酸の両方の、ことを示し、そのLVGアルギン酸塩は、同じ濃度でLVMアルギン酸よりも厚い外層が得られます。 Khanna らから許可を得て複製。 J BIOMEDマーテル解像度A. 11月95（2）、632から40（2010）。 <img src="/files/ftp_upload/3388/3388fig4.jpg" alt="図4"/&gt; FGF-1の図4。リリース、外側のアルギン酸塩層からの血管新生増殖因子タンパク質は、使用するLVMとLVGアルギン酸塩の濃度に基づいて変化した。 （A）と（B）％の放出を示し、（C）と（D）別の外層製剤の時間に対するFGF-1の対応する質量放出を示す。すべての初期5時間（AとC）内の条件、30日（BおよびD）までの低線量連続リリースのために展示バースト放出があります。 Khanna らから許可を得て複製。 J BIOMEDマーテル解像度A. 11月; 95（2）、632から40（2010） <a href="http://www.jove.com/files/ftp_upload/3388/3388fig4large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。

<ol>
	<li>Darrabie, M. D., Kendall, W. F., Opara, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+Poly-L-+Ornithine-coated+alginate+microcapsules.">Characteristics of Poly-L- Ornithine-coated alginate microcapsules.</a> Biomaterials. 26, 6846-6852 (2005).</li><li>Yamagiwa, K., Kozawa, T., Ohkawa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+alginate+composition+and+gelling+conditions+on+diffusional+and+mechanical+properties+of+calcium-alginate+gel+beads.">Effects of alginate composition and gelling conditions on diffusional and mechanical properties of calcium-alginate gel beads.</a> J. Chem. Eng. Jpn. 28, 462-467 (1995).</li><li>Amsden, B., Turner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+characteristics+of+calcium+alginate+gels.">Diffusion characteristics of calcium alginate gels.</a> Biotechnol. Bioeng. 65, 605-610 (1999).</li><li>Uriel, S., Brey, E. M., Greisler, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+low+levels+of+fibroblast+growth+factor-1+promote+persistent+microvascular+network+formation.">Sustained low levels of fibroblast growth factor-1 promote persistent microvascular network formation.</a> Am. J. Surg. 192, 604-609 (2006).</li><li>Moya, M. L., Lucas, S., Francis-Sedlak, M., Liu, X., Garfinkel, M. R., Huang, J. J., Cheng, M. H., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+delivery+of+FGF-1+increases+vascular+density+in+comparison+to+bolus+administration.">Sustained delivery of FGF-1 increases vascular density in comparison to bolus administration.</a> Microvasc. Res. 78, 142-147 (2009).</li><li>Moya, M. L., Cheng, M. H., Huang, J. J., Francis-Sedlak, M. E., Kao, S. W., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+FGF-1+loaded+alginate+microbeads+on+neovascularization+and+adipogenesis+in+a+vascular+pedicle+model+of+adipose+tissue+engineering.">The effect of FGF-1 loaded alginate microbeads on neovascularization and adipogenesis in a vascular pedicle model of adipose tissue engineering.</a> Biomaterials. 31, 2816-2826 (2010).</li><li>Khanna, O., Moya, M. L., Greisler, H. P., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayered+Microcapsules+for+the+Sustained-Release+of+Angiogenic+Proteins+From+Encapsulated+Cells.">Multilayered Microcapsules for the Sustained-Release of Angiogenic Proteins From Encapsulated Cells.</a> Am. J. Surg. 200, 655-658 (2010).</li><li>Khanna, O., Moya, M. L., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+multilayered+alginate+microcapsules+for+the+sustained-release+of+fibroblast+growth+factor-1.">Synthesis of multilayered alginate microcapsules for the sustained-release of fibroblast growth factor-1.</a> J. Biomed. Mater. Res. A. 95, 632-640 (2010).</li></ol>

利害の衝突が宣言されません。

アルギン酸塩は藻類から抽出された、1,4 &#39;-β-D-マンヌロン酸（M）とα-L-グルロン酸（G）2,3の単位で構成されている天然の酸性多糖類である。シンプルなゲル化が発生したときに、例えばCa 2のような二価陽イオン、+、のSr 2 +、またはBa 2 +、隣接するアルギン酸塩の鎖間のイオン架橋を形成するG-モノマーと対話します。アルギン酸マイクロビーズは?...

<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（オプション） </td></tr><tr><td> Pronova純LVGアルギン酸</td><td>ノヴァ·マトリックス</td><td> 4200006 </td><td>アルギン酸製剤の様々な利用可能です。アルギン酸塩の選択は、サイズを含むマイクロビーズの終わりの特性、機械的特性、および輸送に影響を与えます。使用される組成物は、特定のアプリケーション用に最適化する必要があります。 </td></tr><tr><td> Pronova純LVMアルギン酸</td><td>ノヴァ·マトリックス</td><td> 4200206 </td><td>アルギン酸製剤の様々な利用可能です。アルギン酸塩の選択は、サイズを含むマイクロビーズの終わりの特性、機械的特性、および輸送に影響を与えます。使用される組成物は、特定のアプリケーション用に最適化する必要があります。 </td></tr><tr><td>ポリ-L-オルニチン塩酸塩</td><td> Sigma-Aldrich社</td><td> P2533 </td><td></td></tr></tbody></table>

generation of alginate microspheres for biomedical applications

アルギン酸をベースとする材料がよいため、親水性、生体適合性、および物理アーキテクチャの生物医学アプリケーションのためにかなりの注目を集めている。アプリケーションは、細胞のカプセル化、薬物送達、幹細胞培養、組織工学の足場が含まれています。実際に、臨床試験が現在の島が、I型糖尿病の治療薬としてPLOコーティングされたアルギン酸ビーズ中にカプセル化されている実行されている。しかし、島の多数は移植後の生存率が低いことに起因する効果のために必要とされています。局所的にカプセル化されたセルの周囲に微小血管ネットワーク形成を刺激する能力は、酸素、ブドウ糖やその他の重要な栄養素の改善されたトランスポートを介して自分の可能性を高める可能性があります。線維芽細胞成長因子1（FGF-1）は、血管形成を刺激し、虚血組織中の酸素濃度を向上させることができる天然の成長因子である。それはSUSTに配信されたときにFGF-1の有効性が強化されていますainedファッションではなく、単一の大規模ボーラス投与。膵島カプセル化システムからの成長因子のローカルの長期のリリースでは、潜在的に機能的な移植の転帰を改善する、移植細胞に向かって直接血管の成長を刺激する可能性があります。この記事では、我々は、生物医学アプリケーションで使用するためのアルギン酸ミクロスフェアを調製するための手順を概説します。さらに、我々は多層アルギン酸ビーズを生成するために開発する方法を説明します。細胞は、外側のアルギン酸塩層の内側のアルギン酸コア、新生タンパク質にカプセル化することができます。この外側の層からのタンパク質の放出は、移植膵島に向かって直接ローカル微小血管ネットワークの形成を刺激するだろう。

Watch this Scientific Journal Video about Generation of Alginate Microspheres for Biomedical Applications at JoVE.com

Generation of Alginate Microspheres for Biomedical Applications

以下のセクションでは、我々は、生物医学アプリケーションで使用するためのアルギン酸ミクロスフェアを調製するための手順を概説します。我々は、特に1型糖尿病の潜在的な治療法として細胞やタンパク質のカプセル化という二重の目的のために多層アルギン酸ミクロスフェアを作成するためのテクニックを示しています。

バイオメディカル応用のためのアルギン酸ミクロスフェアの生成

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

generation-of-alginate-microspheres-for-biomedical-applications

Research

JoVE Journal

Medicine

20.6K ビュー.  Illinois Institute of Technology. 以下のセクションでは、我々は、生物医学アプリケーションで使用するためのアルギン酸ミクロスフェアを調製するための手順を概説します。我々は、特に1型糖尿病の潜在的な治療法として細胞やタンパク質のカプセル化という二重の目的のために多層アルギン酸ミクロスフェアを作成するためのテクニックを示しています。

ビデオ: Generation of Alginate Microspheres for Biomedical Applications

Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer specimens and clinical data to be available to researchers within the Thoracic Oncology Research Program. This database also captures a large volume of genomic and proteomic data obtained from various tumor tissue studies. A team of clinical and basic science researchers, a biostatistician, and a bioinformatics expert was convened to design the database. Variables of interest were clearly defined and their descriptions were written within a standard operating manual to ensure consistency of data annotation. Using a protocol for prospective tissue banking and another protocol for retrospective banking, tumor and normal tissue samples from patients consented to these protocols were collected. Clinical information such as demographics, cancer characterization, and treatment plans for these patients were abstracted and entered into an Access database. Proteomic and genomic data have been included in the database and have been linked to clinical information for patients described within the database. The data from each table were linked using the relationships function in Microsoft Access to allow the database manager to connect clinical and laboratory information during a query. The queried data can then be exported for statistical analysis and hypothesis generation.

A thoracic oncology database was developed to serve as a comprehensive repository for clinical and laboratory data for the purposes of translational research. The database will serve translational cancer researchers within the Thoracic Oncology Research Program. This database is adaptable to other cancer models, as well as other human diseases.

包括的な胸部腫瘍学データベースの世代 - トランスレーショナルリサーチのためのツール

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer ...

医学

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

臨床および前臨床研究用途のための半自動循環腫瘍細胞（CTC）アッセイの適応

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

次世代シーケンシングのための収集と唾液DNAの抽出

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

臨床応用のためのヒト羊膜上皮細胞の単離、凍結保存と文化

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

磁気マイクロスフェアの眼注射によって誘発実験緑内障

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

ヒト下垂体腺腫細胞の三次元アルギン酸ビーズ文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Kinomicプロファイリングと薬物反応検査のための3D人間バイオゲル培養システムと患者由来の神経膠芽腫細胞を用いた微小腫瘍の発生

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. This model consists of generating conditioned media from organs (lymph node, bone, lung, and brain) isolated from normal athymic nude mice. The model system has been validated by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and evaluate the role of specific organ-derived soluble factors in driving the process of cancer metastasis.

This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells.

乳がん転移挙動に関する器官特異的影響を研究するためのオルガン付きメディアおよびアプリケーションの生成

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused ...

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

Drug Repurposing Hypothesis Generation Using the &quot;RE:fine Drugs&quot; System

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

薬物転用仮説生成の使用」RE：罰金薬「システム

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

サンプリング戦略とブタの生物医学的モデルからバイオバンク組織サンプルの処理

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...