Name: establishment of a clinic-based biorepository
Uploaded: 2017-05-29T14:00:00
Description: Watch this Scientific Journal Video about Establishment of a Clinic-based Biorepository at JoVE.com

この研究は、中西部大学健康科学研究助成助成金、EEHに授与された資金、およびKJLに授与された中西部大学研究およびスポンサープログラムの教員補助プログラムの助成を受けて行われました。 Affiliated LaboratoriesおよびAffiliated Dermatologyが追加サポートを提供しました。彼らの技術的支援のためにSarah Potekhen、Jamie Barto、Stefani Fawks、Cody Jording、Stacie Schimke、Heather Kissel、Ali Zaidiに感謝します。

ヒト組織サンプルに関する全ての実験は、Western IRB（WIRB Protocol＃20142461）によって承認された。これらの処置のための組織収集の単一の基準は、生検で証明されたSCCの診断である。除外基準は元のIRBプロトコルで概説されていませんが、血液媒介性伝染性疾患の患者のサンプルは使用されませんでした。皮膚組織、血液、および唾液の採取に先立ち、インフォームドコンセントが得られた。 M...

<ol>
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W., Gao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transition+in+oral+squamous+cell+carcinoma+triggered+by+transforming+growth+factor-beta1+is+Snail+family-dependent+and+correlates+with+matrix+metalloproteinase-2+and+-9+expressions.">Epithelial-mesenchymal transition in oral squamous cell carcinoma triggered by transforming growth factor-beta1 is Snail family-dependent and correlates with matrix metalloproteinase-2 and -9 expressions.</a> Int J Oncol. 37, 663-668 (2010).</li><li>Vaught, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+in+biospecimen+research.">Developments in biospecimen research.</a> Br Med Bull. 114, 29-38 (2015).</li></ol>

著者らの知る限り、このプロトコールは、費用対効果と迅速なアプローチの両方で皮膚組織サンプルの臨床調達に焦点を当てた最初のものです。 Mohsマイクロサージャリーを受けている患者は、通常、特定の時間帯にスケジュールされ、収集はこれらの期間に限定されます。サンプル収集には、患者のケアに携わる医療補助員、腫瘍サンプルを処理するモース組織学者、サンプルの採取およ?...

がんやバイオマーカーの研究は質の高いヒトの組織サンプルの供給に依存しており、限られた供給は研究1,2を妨げている。多くの皮膚科学的研究は、供給不足、品質の変化、およびヒト組織の使用に関連するコストによって制限される。大規模な専用バイオバンクを構築するコストは約200万ドルと推定されており、これらのコストは人体組織の使用を多くの研究者の手の届かないところに置きます。さらに、研究サンプルを生成し保存するプロセスは、注意深く実行されなければ、臨床操作に影響を与え、患者のケアを遅らせるリスクをもたらす。推奨されるベストプラクティスとサンプルのバリデーション4,5,6に従って、皮膚癌サンプルに焦点を当てた費用効果の高いクリニックベースのバイオリポジトリが確立されました。 <pこのプロトコルは、扁平上皮癌（SCC）、基底細胞癌（BCC）およびメラノーマ皮膚癌を除去するために多量のモース顕微鏡手術を行う皮膚科診療所で開発されました。ボランティアドナーは、この患者集団から募集することができます。収集場所にバイオリポジトリを設置して、治療を遅らせることなく、同意された患者の組織や血液を迅速に採取することが重要です。同じ診療所から採取した検体を集めることは、採取技術のばらつきを最小限に抑え、検体の品質のばらつきを最小限に抑えます。これは、下流のアプリケーション7,8で問題になります。 Mohs顕微鏡手術技術の目標は、可能な限り多くの健康な組織を保存しながら、すべての癌組織を確実に除去することです。この手順は、腫瘍組織の薄層の漸進的除去を含む。それぞれの連続する層は彼のものです皮膚科医によって（腫瘍組織を凍結切除し、H＆E染色を行った後に）論理的に検査して、すべての癌組織が除去されたかどうかを決定する。患者がオフィスに留まる間、組織の次の層の切除および検査が実行される。この手法は、SCC 9の最良の治療選択肢と考えられています。この時点で、創傷は閉鎖され、治癒および美容的外観を改善するために、隣接する正常組織（ANT）が頻繁に摘出される。したがって、腫瘍を除去するためのこの外科的処置は、将来の研究のために組織学的に特徴付けられた組織を収集するのに理想的に適している。 腫瘍組織、隣接する正常組織、唾液、および血液サンプルを取得するための調達手順は、通常の職務に最小限の影響を及ぼすように設計されています（ 図1 ）。医療アシスタントは、手順のために患者を準備しながら血液採取を行う。 Mohs手順の完了後、M<html>オハイオ組織学者は標本の追加の組織スライドを準備し、その組織をバイオリポジトリに移す。バイオリポジトリの確立に関連するコストには、凍結保存フリーザの購入、適度な臨床検査スペースの作成、および在庫追跡プログラムの開発が含まれる。 <img alt="図1" src="/files/ftp_upload/55583/55583fig1.jpg" /> 図1：サンプル収集と責任スタッフのシーケンス。患者のチェックインと患者の同意が得られると、医療アシスタントは頬の綿棒を採取して採血を行う。皮膚科医および医療補助員は、腫瘍を切除し、創傷を閉鎖し、その間SCCおよびANT標本をそれぞれ収集する。専用の実験技術者が血液を処理し、組織培養、保存、およびバイオリポジトリへの入室についてSCCおよびANT標本を区分する。ftp_upload / 55583 / 55583fig1large.jpg &quot;target =&quot; _ blank &quot;&gt;この図の拡大版を表示するには、ここをクリックしてください。 収集されたサンプルの多様性は、さまざまな実験的アプローチを可能にします（ 図2 ）。患者から採取した試料は、頬拭き（唾液も必要に応じて採取することができる）、全血、および切除組織である。口腔スワブおよび全血由来のサンプルは、遺伝子タイピングおよび組織適合のために、処理することなく保存される。将来の分析のために、全血を白血球（WBC）と血漿画分に分離する。モース処理後、凍結した腫瘍を液体窒素に直接入れ、-80℃の冷凍庫に移す。新鮮な生存可能な腫瘍組織およびANT試料を、以前の技術10,11の改変を用いて培養し、次いで凍結保存する。収集中、各サンプルタイプの数は、スプレッドシートに記録されてから<html>正確な処理を容易にする在庫追跡プログラム（ 表1 ）。 <img alt="図2" src="/files/ftp_upload/55583/55583fig2.jpg" /> 図2：臨床医ベースのバイオリポジトリのサンプル収集と処理の概要。頬側スワブおよび血液サンプルを患者から採取し、下流の遺伝子型決定および組織適合のために保存する。全血は、白血球（WBC）単離およびCTC分析ならびに血漿採取および液体生検分析のためにさらに処理される。 Mohs手順中に切除された組織は、診断目的のために組織学的に処理され、その後、組織学的スライドはさらなる免疫組織化学的分析のために実験的に使用され得る。切除された組織試料が十分に大きい場合、新鮮な組織の一部を取り出し、タンパク質およびRNAの単離および培養細胞系の確立のために切片化する。55583 / 55583fig1large.jpg &quot;target =&quot; _ blank &quot;&gt;この図の拡大版を見るには、ここをクリックしてください。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>コレクション日： </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>患者1 </td><td>患者2 </td><td>患者3 </td><td>患者4 </td><td>患者5 </td></tr><tr><td>頭文字と誕生日</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>目の色</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>サンプルタイプ</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>サンプルの場所</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>唾液</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> W穴の血</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>プラズマ</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>腫瘍の生存率</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>組織正常Viable </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>腫瘍モース液体窒素</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>組織正常液体窒素</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>スライド</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> 表1：サンプルコレクションを記録するチェックリスト収集された各サンプルと共に追跡され、記録されたデータには、患者のイニシャル、生年月日、および肌の色のための目の色、試験片除去のオン。唾液サンプル数、採血量、および採取した生存および保存された組織標本の数も、後の使用のための参考文献として記録される。 <a href="http://ecsource.jove.com/files/ftp_upload/55583/Table1.xlsx">このファイルをダウンロードするにはここをクリックしてください。</a> サンプル収集手順を検証するために、各サンプルタイプはダウンストリームアプリケーションでテストされています。以前の技術12の改変を用いて、腫瘍およびANTはタンパク質およびRNA単離に首尾よく用いられ、DNA単離のために潜在的に使用され得る。組織切片から確立された生存外植片を顕微鏡検査し、保存された組織スライドを免疫組織化学および免疫蛍光のために使用した。 本明細書に記載されたプロトコールに従って、このモデルを他の皮膚科診療所、他の腫瘍型（メラノーマなど）にまで拡張することが可能であり、、およびヒト癌への多面的研究のためのヒト組織サンプルを提供するための他の外科的専門性および実践を含む。このプロトコルのわずかな変更は、他のプラクティスにとって必要である可能性が高いが、原則として、このプロトコルは、患者治療の過程で収集された患者サンプルを日常的に廃棄する任意の外科手術に適用可能である。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BD Vacutainer Plastic Blood Collection Tubes with K3EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>02-685-2B</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscopy Sciences Double Edge Blades</td>
			<td>ThermoFisher Scientific</td>
			<td>50-949-411</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Medium Point General Purpose Forceps</td>
			<td>ThermoFisher Scientific</td>
			<td>16-100-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium Microcentrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>05-408-138</td>
			<td></td>
		</tr>
		<tr>
			<td>Lonza Walkersville KGM Keratinocyte Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>NC9791321</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscopy Sciences Tissue TEK OCT Compound</td>
			<td>ThermoFisher Scientific</td>
			<td>50-363-579</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica CM1950 Cryostat</td>
			<td>Leica Biosystems</td>
			<td>14047743909</td>
			<td></td>
		</tr>
		<tr>
			<td>Frosted Microscope Slides</td>
			<td>ThermoFisher Scientific</td>
			<td>12-550-343</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Rapid-Chrome H&#38;E Frozen Section Staining Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>99-900-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Long-Term Storage Cryogenic Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>03-337-7X</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 mL Conical Centrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>Boca Scientific BuccalT-Swab&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>NC9679349</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Signaling Technology 10x RIPA Buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>50-195-822</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease and Phosphatase Inhibitor Cocktail</td>
			<td>ThermoFisher Scientific</td>
			<td>PI78443</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5424R Microcentrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>05-401-203</td>
			<td></td>
		</tr>
		<tr>
			<td>Pellet Morter Cordless Homogenizer</td>
			<td>ThermoFisher Scientific</td>
			<td>12-141-3</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse Free Pellet Pestle</td>
			<td>ThermoFisher Scientific</td>
			<td>121-141-364</td>
			<td></td>
		</tr>
		<tr>
			<td>Forma SteriCycle CO2 Incubator</td>
			<td>ThermoFisher Scientific</td>
			<td>13-998-089</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>SH30071.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Advanced DMEM/F-12</td>
			<td>ThermoFisher Scientific</td>
			<td>12-634-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Penicillin-Streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco 1M Hepes</td>
			<td>ThermoFisher Scientific</td>
			<td>15-630-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Cell Culture 35 mm with Vent</td>
			<td>ThermoFisher Scientific</td>
			<td>1256591</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CFH monoclonal antibody clone OX-24</td>
			<td>Abnova</td>
			<td>MAB12583</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-p40 (p63 delta) antibody</td>
			<td>Abnova</td>
			<td>ABX-144A</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti MUC-1 antibody</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-7313</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Snail + Slug (phospho S246)</td>
			<td>Abcam</td>
			<td>Ab63568</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Phallodin</td>
			<td>Molecular Probes&#160;</td>
			<td>A12380</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI</td>
			<td>Molecular Probes&#160;</td>
			<td>P36941</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axio Imager Z1 Microscope with Axiocam camera</td>
			<td>Zeiss</td>
			<td>4300009901</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX51 phase contrast with DP72 camera</td>
			<td>Olympus</td>
			<td>IX511F3</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of a clinic-based biorepository

皮膚癌（ 例えば、扁平上皮細胞癌、基底細胞癌および黒色腫）の発生率は、過去数年間にわたって増加している。生物医学研究研究のための皮膚腫瘍サンプルが並行して要求されることが期待される。しかしながら、組織の入手可能性は、バイオリポジトリを確立するコストと、臨床操作を妨害しない臨床サンプルを得るために利用可能なプロトコルがないために限定されている。モース外科手術の日にルーチンの臨床手順に最小限の影響を与える皮膚腫瘍および関連する血液および唾液サンプルを収集および処理するためのプロトコルが確立された。腫瘍試料は、モース組織工学者による生検で証明された皮膚悪性腫瘍のための第1層のモース手術を受けている患者から収集され、処理される。隣接する正常組織は、手術終了時に収集される。採取することができる追加のサンプルは、全血および口腔スワブ。通常は廃棄される組織サンプルを利用することにより、診療所と臨床検査室の設定に基づいていくつかの重要な利点を提供するバイオリポジトリが生成された。これらには、幅広い収集サンプルが含まれています。病理学的報告を含む、識別されていない患者記録へのアクセス;典型的なドナーの場合、フォローアップ訪問中の追加サンプルへのアクセス。

腫瘍およびANTは、タンパク質単離において首尾よく使用され、ウェスタンブロッティングによって試験された。 図3に示すように、皮膚扁平上皮癌腫マーカー（Muc1 14 、FHL-1 15 、およびp40 16,17 ）は、本発明者らの診療所ベースのバイオリポジトリに収集および保存された患者サンプルで検出することが...

Watch this Scientific Journal Video about Establishment of a Clinic-based Biorepository at JoVE.com

Establishment of a Clinic-based Biorepository

皮膚腫瘍は、しばしば、モース顕微鏡手術後に捨てられる。臨床支援スタッフが、臨床操作を妨害することなく、下流の実験室用途のための皮膚腫瘍（ 例えば、扁平上皮細胞癌、基底細胞癌、および黒色腫）試料を効果的に処理および保存することを可能にするプロトコールがここに記載される。

クリニックベースのバイオリポジトリの設立

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is ...

The overall goal of this protocol is to enable clinical support staff in a dermatology practice to effectively process and store cutaneous tumor samples for downstream laboratory applications, without interfering with routine clinical operations. This protocol was designed to enable medical assistants and Mohs histotechnicians to collect and store cutaneous tumor samples for future biomedical research projects, as part of their daily clinical operations. The main advantage of this protocol is that excised tissues that are normally discarded and destroyed are instead collected and preserved for research, using procedures, which minimally impact routine clinical operations.The variety of samples collected enables correlations between protein and RNA biomarkers in the tissue and plasma samples with patient outcome data. In principle, this protocol, once slightly modified is applicable to any surgical practice, that routinely discards patient samples, gathered in the course of patient treatment. Demonstrating the procedure will be Stefani Fawks, a Mohs histotechnician, and Dr.Mitchell Manway, a research fellow in our practice.To begin, use the tissue received from the Mohs surgeon and with a scalpel, excise a 10 to 50 milligram sample from the center of the apical side of the tissue. With tweezers, transfer the viable tissue to a labeled 1.5 milliliter tube, prefilled with culture medium. Make sure the tissue is entirely immersed in the medium.Place the sample in a refrigerator and store it at four degrees Celsius, for no longer than 24 hours before further processing. Harvest at least a 10 cubic millimeter tissue fragment from the apical side of the excised tissue, when the tumor sample is large enough, and such removal will not interfere with routine Mohs processing. Next, place the remaining unprocessed tumor sample on a cryostat tissue holder.Embed the tissue in an optimal cutting temperature compound, and freeze it within the holder to prepare histological slides. Using a cryostat, cut the tissue into seven micron thick sections, and layer up to three sections over a slide. After sectioning, fix the tissue by immersing the slides in acetone for one to two seconds.Then, allow the slides to air dry. Keeping the remaining sample in a cryostat chamber, pry the tissue from the cryo embedding medium, and transfer it to a labeled cryogenic tube. Immediately transfer the labeled vial into liquid nitrogen.Transfer of the tissue to a cryo vial after Mohs processing must be performed in the negative 20 degrees Celsius cryostat chamber. If performed properly and the tissue is kept frozen, this step will enable the successful extraction of RNA from post-Mohs tissue samples. Next, obtain adjacent normal tissue from the Mohs surgeon.Excise a sample from the apical side of the adjacent normal tissue. Then, using tweezers, transfer the excised sample to a labeled 1.5 milliliter tube filled with culture medium. Immerse the tissue in the same culture medium.Place it into an appropriate sample bag, and store at four degrees Celsius, until subsequent processing. Collect approximately 10 milliliters of venous blood into EDTA-coated collection tubes. Combine the blood samples in a sterile 15 milliliter centrifuge tube.Then, transfer approximately one milliliter of the blood sample into a 1.5 milliliter cryogenic tube, avoiding bubbles. Store this whole blood sample at minus 80 degrees Celsius, until subsequent genotyping and tissue matching. Next, centrifuge the remaining blood sample at 1000 times G for 30 minutes to obtain blood plasma.After spinning, collect and divide the plasma until one milliliter aliquots in 1.5 milliliter cryogenic tubes. And store the samples at minus 80 degrees Celsius for use during future liquid biopsy analyses. Enter the data from the checklist of patient samples, along with patient information and diagnosis, subtracted from the patient chart to the biorepository database.Finally, record the localization of the de-identified samples in the biorepository database, and move the samples to the research lab. Immediately after, transfer the tissue samples into 1.5 milliliter tubes, prefilled with appropriate extraction solutions, making sure the tissue is completely immersed. Store the samples at minus 80 degrees Celsius until further processing.Presented here are the images of representative Western blots of known squamous cell carcinoma markers, mucin one, FHL-one, and p40, obtained for adjacent normal tissue and a tumor sample. The quality of the RNA isolated from Mohs samples was first assessed using gel electrophoresis, followed by RNA integrity analysis. RNA integrity number values achieved for normal and tumor samples indicate that high quality and intact RNA was obtained.Cytometry profiles of the bands were plotted as fluorescence units versus nucleotide number for representative RNA samples from tumor and normal patient tissue. A squamous cell carcinoma tissue explant was imaged using phase contrast microscopy and shown to be a mixed keratinocyte and fibroblast culture, that might be subcultured or used for future cell line development. Finally, in the squamous cell carcinoma tissue sections, positive staining for the epithelial to mesenchymal transition marker was shown.Once mastered, this protocol will extend the standard work flow of the clinic staff to less than 30 minutes, if it is performed properly. The majority of the steps are performed by the existing clinical staff after patient care is completed. While attempting this protocol, it is important to maintain accurate record keeping, linking the samples with the patient's history and diagnosis.As this information is often needed for downstream applications. This technique will help pave the way for researchers in the field of dermatology for the development of liquid biopsies and biomarkers in squamous cell carcinomas and melanoma. After watching this video, you should have a good understanding of how to implement a clinic-based biorepository for cutaneous cancer samples, generated as part of daily practice.

establishment-of-a-clinic-based-biorepository

Research

JoVE Journal

Medicine

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

マウス同所性膀胱異種移植片の最小侵襲性の確立

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

医学

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

全組織生検からヒト上皮EnteroidsとColonoidsの確立

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

マウスモデルにおけるUTIとCAUTIの樹立とその性状

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

プレート骨接合によって安定化マウスにおけるセグメント別大腿臨界サイズ欠陥モデルの確立

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

ウサギにおける完全なダクリオアデネクトー切除術を用いた重症ドライアイモデルの確立

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.

ブタ静脈移植病モデルの確立と評価

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the ...

Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is similar in color to the thyroid gland. The lack of effective animal models in preclinical research is a severe limitation for the development of PG identification techniques. This protocol allows for the establishment of a simple and effective rat model for PG identification. In this model, black iron oxide nanoparticles (IONPs) are injected locally in the thyroid gland and rapidly diffuse within the thyroid gland but not the PG. A negatively stained PG and a positively stained thyroid gland can be easily identified by the naked eye without requiring external microscopes. The position of the PG can be identified by increasing the color contrast between the thyroid gland and the PG, based on the color of the black IONPs. This rat model is low-cost and convenient for PG identification, and the IONPs are a novel PG contrast agent.

To date, the development of parathyroid gland (PG) identification methods is limited by the lack of animal models in preclinical research. Here, we establish a simple and effective rat model for intraoperative PG imaging and evaluate its effectiveness by using iron oxide nanoparticles as a novel PG contrast agent.

術中副甲状腺イメージングのための簡便かつ効果的なラットモデルの確立

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is ...

RVボリューム過負荷マウスモデルを確立するための修正動静脈瘻手術

Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model

Right ventricular (RV) volume overload (VO) is common in children with congenital heart disease. In view of distinct developmental stages,the RV myocardium may respond differently to VO in children compared to adults. The present study aims to establish a postnatal RV VO model in mice using a modified abdominal arteriovenous fistula. To confirm the creation of VO and the following morphological and hemodynamic changes of the RV, abdominal ultrasound, echocardiography, and histochemical staining were performed for 3 months. As a result, the procedure in postnatal mice showed an acceptable survival and fistula success rate. In VO mice, the RV cavity was enlarged with a thickened free wall, and the stroke volume was increased by about 30%-40% within 2 months after surgery. Thereafter, the RV systolic pressure increased, corresponding pulmonary valve regurgitation was observed, and small pulmonary artery remodeling appeared. In conclusion, modified arteriovenous fistula (AVF) surgery is feasible to establish the RV VO model in postnatal mice. Considering the probability of fistula closure and elevated pulmonary artery resistance, abdominal ultrasound and echocardiography must be performed to confirm the model status before application.

著者スポットライト:出生後右室容積過負荷マウスモデルの確立と確認  

This protocol presents the establishment and confirmation of a postnatal right ventricular volume overload (VO) model in mice with abdominal arteriovenous fistula (AVF), which can be applied to investigate how VO contributes to postnatal heart development.