現在の研究は、上海科学技術委員会の上海の自治体 (CN) 博士温王に主催の革新資金 (294300 1402 H) によって支えられました。彼の技術支援とこの原稿の科学的助言のため広東省周博士 (組織工学センターの中国国家) に感謝したいと思います。我々 も感謝したい氏 Huitang 夏 (上海第九人民病院) 動物の福祉に貢献しました。

動物を対象とする手順は動物の苦痛を最小限に抑える努力をローカル機関動物のケアと倫理委員会で承認されました。次のプロトコルは、機関動物ケアおよび使用委員会 (IACUC) 上海 9 人によって承認された &#8217; プロトコルと上海交通大学医学部付属病院 s [2017] 番号 063。
 1 です Surgically-Induced ラット膝関節変形性関節症モデルの確立 <li>この手術、250 と 300 g. 間体重の使用 8-12 週齢雄 SD) ラットの</li> <ol>。
	<li>40 mg/kg チレタミンと 40 mg/kg 筋肉内注射を介してチレタミン動物を麻酔します</li>。
	<li>は、左側臥位で加熱ステージに動物を配置します。右膝を徹底的にかみそりで剃る。70% エタノールに続いて 10 %providone ヨード溶液による外科的領域を消毒、2 回以上繰り返すと後 10 %providone ヨード溶液でペイントし、非外科的手術パッド領域をカバーします</li>。
	<li>、Jointcapsules を公開する膝蓋腱に沿って横方向に 2 cm の切開を行う手術のメスを使用しています</li>。
	<li>は、内側側副靱帯のトランセクトを手術用メスを使用します。大腿骨に向かってメニスカスを反映します</li>。
	<li>軟骨が乾くを防ぐために操作中に関節軟骨の表面の滅菌の 0.9% 塩化ナトリウム溶液を滴下します</li>。
	<li>は、ピンセットで内側半月板をつかむし、メス ( 図 1 a) と脛骨の添付ファイルからの狭いポイントで半月板をカットします。避けるために軟骨を負傷します</li>。
	<li>4-0 吸収性縫合糸と関節包を閉じます</li>。
	<li>彼らは意識を取り戻すまで、手術のラットを監視します</li>。
	<li>感染や手術後の痛みを和らげるために筋肉内のルートを通じて 0.05 mg/kg ブプレノルフィン 20 mg/kg のゲンタマイシンを挿入します</li>。
	<li>温度制御動物施設発症コアに 3-8 週の 12 h 明暗サイクルの下でラットを維持します。動物が痛み症状が続く場合、ブプレノルフィン (0.05 mg/kg, i. m.) とゲンタマイシン (20 mg/kg, i. m.) に限定されないなどの鎮痛薬を受け取り続けます</li>。
</ol> 2。ラット コア モデルの組織評価 <ol><li>過剰な CO 2 吸入 (動物の無意識を作るに追加商工会議所の既存の空気に二酸化炭素を 1 分あたり試験室の容積の約 30% のフィル レートとラットを犠牲に2-3 分の最低維持 CO2 流れ呼吸後 1 分以内停止します。)手術後時点 (代表コア誘導 3 から 8 週間までの 8 週間で結果が表示されます) を示した</li> <li>メスと膝継手を切り肌と膝の関節の周りの筋肉を解剖</li>。
	<li>は、室温で 3 日間の 4% パラホルムアルデヒド (PFA) の膝継手を修正します。その後、4 ° C で約 4 週間 20% エチレンジアミン四酢酸 (EDTA) のサンプルをめし、EDTA を 3 日ごとに変更します 。
	注意: パラホルムアルデヒドは有毒である、手袋、眼鏡など適切な個人用保護具 (PPE) を着用します</li>。
	<li>は、自動ティッシュ プロセッサを使用してサンプルを脱水します。フラッシュ サイクルを次のように起動するメニューを設定: 70% のエタノールの 1 h。その後 80% エタノールと 95% のエタノール 1 h のそれぞれ;1 時間の 100% エタノールの 2 サイクルが続くその後、1 h の 2 倍のキシレンのたび、たび 58 ° C 1 時間で 3 回パラフィンします 。
	注意: キシレンは有毒である、ので、適切な PPE は、手袋、眼鏡などを着用します 。
	注: 必要に応じて、Coplin Jar の使用自動ティッシュ プロセッサとして同じセットアップと手動脱水します</li>。
	<li>そのまま接合面の内側面を下に置くし、中央に埋め込まれた組織をパラフィン ブロックに埋め込む 。
	注: 必要に応じて、パラフィン ブロックにサンプルを手動で埋め込む鉗子を使用します</li>。
	<li>回転式ミクロトームで 2 連続切片で 30 μ m 間隔で 5 μ m 矢状セクション内側縁から関節をカットを開始します</li>。
	<li>は、スライド ガラス上のセクションをマウントし、キシレンでスライドを deparaffinize します。2 連続切片の間ヘマトキシリンと 1 つのスライドを染色 &#38; エオシン (H &#38; E) 染色: 0.1% 1%、15 分間ヘマトキシリン 10 s および 0.5% の酢酸水溶液 10 分エオシン。サフラニン O/高速の緑色の染色と他のスライドを染色: 0.1%、0.02% の 15 分間ヘマトキシリン 3 分、1% の高速グリーン酢酸溶液に 10 s および 3 分の 0.1% サフラニン O 
	注意: キシレンは有毒である、眼鏡、手袋などの適切な PPE を着用します</li>。
	<li>ラット 6 で変形性関節症の研究の社会インターナショナル (OARSI) 病理組織評価システムを使用して染色スライドをスコアします</li>。
</ol> 3。蛍光色素は、ヒト間葉系幹細胞をラベリング <ol><li>1 mM の濃度で 100% エタノールの細胞ラベリング ソリューションの準備をしました。光から保護して細胞ラベリング ソリューションと 6 ヶ月間常温保存します</li>。
	<li>成長ダルベッコの標準的な細胞培養プロシージャと haMSCs &#39; s 変更イーグル &#39; s 培 (DMEM) 1% ペニシリン、ストレプトマイシンと 10% 牛胎児血清 (FCS) 10 cm シャーレで</li>。
	<li>1 mM EDTA 合流前の約 80% 点と 2 mL 0.25% トリプシン haMSCs をデタッチします</li>。
	<li>培地 4 mL のトリプシンを中和し、室温で 3 分間 800 x g でセルをスピンします</li>。
	<li>5 mL 無血清培地の 1 x 10 6 セル/ml の密度でセルを中断します</li>。
	<li>50 を追加することによって、セルのラベル μ L は 5 mL 50 分の 37 ° C で培養の無血清細胞ラベリング ソリューション</li>
	<li>800 x g で 3 分間でラベル付きセルをスピン</li>
	<li>5 mL リン酸バッファー生理食塩水 (PBS) 3 分間 800 x g で 2 セルを洗浄して回以上</li>。
	<li>Cy5 のフィルター セットと蛍光顕微鏡を用いたラベル付きの haMSCs を確認してください</li>。
	<li>トリパン ブルー染色でセルをカウントし、2.5 倍の注射用 100 μ L の PBS で 10 の 6 の実行可能なセルを取得します</li>。
</ol> 4。生体内で蛍光イメージング トラック haMSCs <ol><li>(ステップ 1)、手術後 8 週間は 25 mg/kg チレタミンとチレタミン 25 mg/kg の筋肉内投与によりコア ラットを麻酔します</li>。
	<li>右膝を徹底的に関節部を公開するかみそりで剃る。70% エタノールで関節部を消毒します</li>。
	<li>2.5 x 10 26 G 注射針を使用して注入 6 標識三角形領域の中央に 100 μ L の PBS 中に浮遊細胞は内側膝蓋靭帯、内側の大腿骨顆と脛骨の内側顆 (によって形成されます。 図 1 b).</li>
	<li>イメージング ソフトウェアを開き、体内 の発光イメージング システムを初期化します</li>。
	<li>イメージング システムの中央ステージでの位置や部屋のドアを閉じる位置ラット臥</li>。
	<li>チェック、&#34; 蛍光 &#34; 640 の設定チェック ボックスと蛍光フィルターの選択励起、nm および 680 nm の発光 ( 図 2 a) ため</li>。
	D. 設定中 (8) にピクセルビニングに <li>選択フィールドのビュー 2 と露出に F/ストップの自動時間します。最適な維持します。全体の研究 ( 図 2 a) で同等の結果を取得する一貫性のある露出時間</li>。
	<li>はステージと麻酔からの回復のためのモニターから動物を削除します 。
	注: 加熱パッド上の場所の動物のタオル地の 2-3 層で覆われている</li>。
	<li>は、放射効率の蛍光 ( 図 2 b) 測定のための単位を選択します。利益 (率 ROI) 分析のための領域を選択し、それぞれのイメージ ( 図 2) から蛍光信号を定量化します</li>。
	<li>は 生体内 イメージング研究 haMSCs 縦保持関節の順に各ラットのための手順を繰り返します 。
	注: 動物のイメージングを提案 &#39; 蛍光信号を用いたイメージング システム注入後最初の週 2 日に一度と一度毎週まで信号が検出できません</li>。
</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=Coculture+of+human+nucleus+pulposus+cells+with+multipotent+mesenchymal+stromal+cells+from+human+bone+marrow+reveals+formation+of+tunnelling+nanotubes.">Coculture of human nucleus pulposus cells with multipotent mesenchymal stromal cells from human bone marrow reveals formation of tunnelling nanotubes.</a> Mol Med Rep. 9 (2), 574-582 (2014).</li><li>Wang, W., 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=Human+adipose-derived+mesenchymal+progenitor+cells+engraft+into+rabbit+articular+cartilage.">Human adipose-derived mesenchymal progenitor cells engraft into rabbit articular cartilage.</a> Int J Mol Sci. 16 (6), 12076-12091 (2015).</li></ol>

孟李明浩、温王が現在の従業員と細胞生物医学グループのストック オプションの保有者 (Nasdaq: CBMG)。他の著者は利益相反を宣言しません。

我々 はベンチから枕元にコアの再生幹細胞治療をもたらすことができる前に、安全基準と体内の幹細胞療法の研究は必要緊急です。ただし、疾患の病理組織学的環境は、永続性と移植 haMSCs10の体内で重要な役割を果たしています。最近、私たちのグループを示した haMSCs の関節内注入の永続化をもはや病理コア環境よりも通常の条件8下注射をしました。炎...

膝変形性関節症 (コア) は、関節軟骨の損失や世界の1のまわりの高齢者の主要な慢性疾患になった進歩的な炎症から生じる退行性疾患です。ただし、抗炎症薬、物理的なサプリメントや手術の手順を使用して現在の治療症状痛み2の一時的な救済を提供することがあります。
膝変形性関節症、軟骨の再生と免疫調節特性3、潜在的な多能性分化のための有望な再生治療薬となっているひと脂肪由来間葉系幹細胞 (haMSCs) 4。生体内でアクションのメカニズムを調査する薬理学的ルートと比べると、小さなコア動物モデルでライブの haMSCs を追跡する現在の理論的根拠と臨床応用の前に haMSC 療法の有効性を確立するために有益です。前臨床試験の内側半月 (MM) は、一貫性のある再現性5比較的実現可能なモデルを提供するラットでは, コアを誘導するために関節の機械的負荷を不安定化します。MM によるコアの発症は、膝前十字靭帯断裂単独あるいは部分内側半月6より前です。したがって、コアの病理学的微小環境と注入された haMSCs の長期的な相互作用は多くの場合8MM7,投与ラットにおける評価されます。
HaMSCs の薬効は広く報道されると、関連する知識をされているが (IA) の関節内注射による注入 haMSCs の生体内で持続性は乏しい9,10です。したがって、様々 な細胞ラベリング方法が開発されて、immunohistology11、ルシフェラーゼ12、緑の蛍光蛋白質13トランスフェクション、鉄酸化物の磁気共鳴画像 (MRI)14 のラベリングなど、および多数蛍光セル染料8,15,16。手間のかかる組織解析と比較して、生体内の非侵襲的イメージングはリアルタイム配信や蛍光信号10,17の付いた細胞の動態を検出する光学デバイスを採用しています。機能の生きているセルイメージ投射のため cytocompatible の蛍光標識、幹細胞移植18後細胞活動を明らかにする洗練された放射性無料トラッキング手法です。また、多色蛍光脂溶性色素は、アミノ反応親水性染料や蛍光蛋白質が改良されたセル透磁率および増強蛍光量子収量19を含む上の利点を所有しています。
こうして、ここで含まれるプロトコル利用赤い脂溶性 carbocyanines ラベルの付いたセルを励起するレーザーでした (DilC18(5))、遠赤色蛍光 Dil (dialkylcarbocyanines) アナログ20であります。赤いシフト励起と発光スペクトルされる干渉を回避したし、深い組織の生きている動物の8時間の長期間画像をことができます。この方法で追跡細胞の生体内での付いたでしたを理解し、現在の幹細胞再生治療の向上に不可欠な動物モデルでの haMSCs などの移植の幹細胞を監視するためです。

<table><tbody><tr><td>Matrx VMR animal anesthesia system</td><td>Midmark</td><td>VIP3000</td><td></td></tr><tr><td>4-0 suture</td><td>Shanghai Jinhuan</td><td>KC439</td><td></td></tr><tr><td>Razor</td><td>Pritech</td><td>LD-9987</td><td></td></tr><tr><td>Gentamicin</td><td>Zhejiang Jindakang Animal Health Product Co., Ltd.</td><td>None</td><td></td></tr><tr><td>0.9% Sodium chloride solution</td><td>Hunan Kelun Pharmaceutical Co., Ltd.</td><td>H43020455</td><td></td></tr><tr><td>Penicillin</td><td>Shanghai Kangfu chemical pharmaceutical Co., Ltd.</td><td>None</td><td></td></tr><tr><td>Buprenorphine</td><td>Tianjin Pharmaceutical Research Institute Pharmaceutical Co., Ltd.</td><td>None</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>16005</td><td>Dilute to final concentration of 10% in PBS</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884</td><td>Dilute to final concentration of 20% in PBS</td></tr><tr><td>0.1% Hematoxylin Solution, Mayer&amp;rsquo;s</td><td>Sigma-Aldrich</td><td>MHS16</td><td></td></tr><tr><td>0.5% Eosin Y solution, alcoholic</td><td>Sigma-Aldrich</td><td>HT110116</td><td></td></tr><tr><td>Safranin O</td><td>Sigma-Aldrich</td><td>S8884</td><td></td></tr><tr><td>Fast Green</td><td>Sigma-Aldrich</td><td>F7258</td><td></td></tr><tr><td>Shandon Excelsior ESTM Tissue Processor</td><td>Thermo Fisher</td><td>A78400006</td><td></td></tr><tr><td>Shandon Histocentre&amp;trade; 3 Tissue Embedding Center</td><td>Thermo Fisher</td><td>B64100010</td><td></td></tr><tr><td>Fully Automated Rotary Microtome</td><td>Leica</td><td>RM2255</td><td></td></tr><tr><td>DiD</td><td>Molecular Probes, Life&lt;br/&gt; Technologies</td><td>V-22887</td><td></td></tr><tr><td>D-MEM High Glucose</td><td>Sigma-Aldrich</td><td>D5648</td><td></td></tr><tr><td>PBS</td><td>GIBCO, Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Invitrogen</td><td>25200-114</td><td></td></tr><tr><td>10 cm Petri Dish</td><td>Corning</td><td>V118877</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman</td><td>Optima MAX-TL</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Olympus</td><td>BX53</td><td></td></tr><tr><td>0.4% Trypan Blue solution</td><td>Sigma-Aldrich</td><td>93595</td><td></td></tr><tr><td>Titetamme</td><td>Virbac (Zoletil 50)</td><td>1000000188</td><td></td></tr><tr><td>Zolazepam</td><td>Virbac (Zoletil 50)</td><td>1000000188</td><td></td></tr><tr><td>Sterile hyposermic syringe for single use 26G</td><td>Shanghai Misawa Medical Industry</td><td>None</td><td></td></tr><tr><td>IVIS Spectrum In Vivo Imaging System</td><td>PerkinElmer</td><td>124262</td><td></td></tr><tr><td>Living Imaging 4.0 software</td><td>PerkinElmer</td><td>None</td><td></td></tr></tbody></table>

in vivo tracking of human adipose-derived mesenchymal stem cells in a rat knee osteoarthritis model with fluorescent lipophilic membrane dye

膝変形性関節症 (コア)、ひと脂肪由来間葉系幹細胞 (haMSC) 療法の臨床応用を支援するため、セルの永続性の有効性と動物モデルにおける haMSCs の体内を調べた。脂溶性色素と haMSCs の細胞膜にラベルを付ける方法を示しました。その後、心術のコアを持つラットの標識細胞の関節内注入は、生体内イメージング システム動的に監視されました。我々 は、親油性を採用 carbocyanines でした (DilC18 (5))、周囲の組織から自然の緑色蛍光の励起を避けるために赤色レーザーを活用した遠赤色蛍光 Dil (dialkylcarbocyanines) アナログ。またの赤いシフトの発光スペクトルは、生きている動物の深い組織イメージングとラベリングのプロシージャは細胞毒性または haMSCs に機能障害生じなかった許可でした。このアプローチは、KOA ラットモデルにおける haMSCs の効率的な追跡方法であると示されています。最適な投与経路と前臨床試験で他のソースからの MSCs の投与量を決定するこのメソッドのアプリケーションが使えます。

コア モデルを誘導するために MM は SD ラット (図 3) の右ひざ関節で行われました。手術後 8 週間ラットを犠牲に、膝継手の連続切片を両方 H で行った &#38; E、サフラニン O/高速グリーン染色 (図 4)。H の &#38; E 染色、関節軟骨の表面の展示手術膝手術をせず通常の関節よりも粗いボーダー。サフラニン-O/高速緑染色 (赤染色) ...

Watch this Scientific Journal Video about In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye at JoVE.com

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

生体内でひと脂肪由来間葉系幹細胞が脂溶性膜の蛍光色素をラット膝関節変形性関節症モデルでの追跡

このプロトコルでは、効率的に遠赤色蛍光 (IA) の関節内注入によるラット膝変形性関節症 (KOA) モデルで、セルの永続性とひと脂肪由来間葉系幹細胞 (haMSCs) の体内を監視する方法について説明します。

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

in-vivo-tracking-human-adipose-derived-mesenchymal-stem-cells-rat

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

8.9K ビュー.  Cellular Biomedicine Group, China. このプロトコルでは、効率的に遠赤色蛍光 (IA) の関節内注入によるラット膝変形性関節症 (KOA) モデルで、セルの永続性とひと脂肪由来間葉系幹細胞 (haMSCs) の体内を監視する方法について説明します。

ビデオ: In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

光イメージングによる非侵襲的検出のための蛍光色素で標識幹細胞

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

生物学

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

で小動物の筋肉の生着のダイナミクスの追跡インビボでの蛍光イメージング

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

蛍光SAPC-DOPSナノベシクルを用いて脳腫瘍、関節炎のin vivo光イメージング中

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

医学

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

マウス骨髄単球の追跡<em&gt;インビボ</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

免疫・感染学

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

以下のための大腿骨のウィンドウチャンバーモデル<em&gt;インビボ</emマウス骨髄内&gt;細胞トラッキング

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

馬腱でテクネチウム-99m標識骨髄間葉系幹細胞のin vivoイメージングと追跡中

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

実験的炎症性腸病気のマウスモデルで使用するため、腸管へのホーミングを増加するヒト間葉系幹細胞の超音波ガイド下心臓内注射

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD). In premature infants, BPD is a major complication despite early use of surfactant therapy, optimal ventilation strategies, and noninvasive positive pressure ventilation. Because pulmonary inflammation plays a crucial role in the pathogenesis of BPD, corticosteroid use is one potential treatment to prevent it. Nevertheless, systemic corticosteroid treatment is not usually recommended for preterm infants due to long-term adverse effects. Preclinical studies and human phase I clinical trials demonstrated that use of mesenchymal stromal cells (MSCs) in hyperoxia-induced lung injuries and in preterm infants is safe and feasible. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and glucocorticoid treatment has emerged as a new strategy to treat newborns with respiratory disorders. The developmental stage of rat lungs at birth is equivalent to that in human lungs at 26&#8722;28 week of gestation. Hence, newborn rats are appropriate for studying intratracheal administration to preterm infants with respiratory distress to evaluate its efficacy. This intratracheal instillation technique is a clinically viable option for delivery of stem cells and drugs into the lungs.

Described is a protocol for performing intratracheal transplantation of mesenchymal stromal cells (MSCs) through intratracheal injection in term neonatal rats. This technique is a clinically viable option for delivery of stem cells and drugs into neonatal rat lungs to evaluate their efficacy.

期産期新生児ラットにおける幹細胞の気管内植在

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

可視化と系統特異的マーカーと間葉系細胞脂肪細胞の微分の潜在性の定量化

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Intraspinal Cavity Injection of Human Mesenchymal Stem Cells and Tracking their Migration into the Rat Brain

Mesenchymal stem cells (MSCs) have been studied for the treatment of various diseases. In neurodegenerative diseases involving defects in both the brain and the spinal cord, the route of administration is very important, because MSCs must migrate to both the brain and the spinal cord. This paper describes a method for administering MSCs into the spinal canal (intraspinal cavity injection) that can target the brain and spinal cord in a rat model. One million MSCs were injected into the spinal canals of rats at the level of lumbar vertebrae 2-3. After administration, the rats were euthanized at 0, 6, and 12 h post-injection. Optical imaging and quantitative real-time polymerase chain reaction (qPCR) were used to track the injected MSCs. The results of the present study demonstrated that MSCs administered via the spinal cavity could be detected subsequently in both the brain and spinal cord at 12 h. Intraspinal cavity injection has the advantage of not requiring general anesthesia and has few side effects. However, the drawback of the low migration rate of MSCs to the brain must be overcome.

<meta charset="utf8"></head><body>ヒト間葉系幹細胞の脊髄腔内注射とラット脳への移動の追跡

Several routes of administration can be used to deliver mesenchymal stem cells (MSCs) to the brain. In the present study, MSCs were delivered throughout the neuraxis and brain via intra-spinal cavity injection. MSCs were injected into the spinal cavities of rats, and stem cell migration was tracked and quantified.

ヒト間葉系幹細胞の脊髄腔内注射とラット脳への移動の追跡

Mesenchymal stem cells (MSCs) have been studied for the treatment of various diseases. In neurodegenerative diseases involving defects in both the brain ...