Name: in vivo tracking of human adipose-derived mesenchymal stem cells in a rat knee osteoarthritis model with fluorescent lipophilic membrane dye
Uploaded: 2017-10-08T14:00:00.000+00:00
Duration: 7 min 31 s
Description: 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

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>

<ol>
	<li>Loeser, R. F., Goldring, S. R., Scanzello, C. R., Goldring, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritis:+a+disease+of+the+joint+as+an+organ.">Osteoarthritis: a disease of the joint as an organ.</a> Arthritis Rheum. 64 (6), 1697-1707 (2012).</li><li>Lane, N. E., Shidara, K., Wise, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritis+year+in+review+2016:+clinical.">Osteoarthritis year in review 2016: clinical.</a> Osteoarthritis Cartilage. 25 (2), 209-215 (2017).</li><li>Wang, W., Cao, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+osteoarthritis+with+mesenchymal+stem+cells.">Treatment of osteoarthritis with mesenchymal stem cells.</a> Sci China Life Sci. 57 (6), 586-595 (2014).</li><li>Burke, J., 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=Therapeutic+potential+of+mesenchymal+stem+cell+based+therapy+for+osteoarthritis.">Therapeutic potential of mesenchymal stem cell based therapy for osteoarthritis.</a> Clin Transl Med. 5 (1), 27 (2016).</li><li>Bendele, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+osteoarthritis.">Animal models of osteoarthritis.</a> J Musculoskelet Neuronal Interact. 1 (4), 363-376 (2001).</li><li>Gerwin, N., Bendele, A. M., Glasson, S., Carlson, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+OARSI+histopathology+initiative+-+recommendations+for+histological+assessments+of+osteoarthritis+in+the+rat.">The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the rat.</a> Osteoarthritis Cartilage. 18, S24-S34 (2010).</li><li>Janusz, M. J., 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=Induction+of+osteoarthritis+in+the+rat+by+surgical+tear+of+the+meniscus:+Inhibition+of+joint+damage+by+a+matrix+metalloproteinase+inhibitor.">Induction of osteoarthritis in the rat by surgical tear of the meniscus: Inhibition of joint damage by a matrix metalloproteinase inhibitor.</a> Osteoarthritis Cartilage. 10 (10), 785-791 (2002).</li><li>Li, M., 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=In+vivo+human+adipose-derived+mesenchymal+stem+cell+tracking+after+intra-articular+delivery+in+a+rat+osteoarthritis+model.">In vivo human adipose-derived mesenchymal stem cell tracking after intra-articular delivery in a rat osteoarthritis model.</a> Stem Cell Res Ther. 7 (1), 160 (2016).</li><li>Zhou, B., 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=Administering+human+adipose-derived+mesenchymal+stem+cells+to+prevent+and+treat+experimental+arthritis.">Administering human adipose-derived mesenchymal stem cells to prevent and treat experimental arthritis.</a> Clin Immunol. 141 (3), 328-337 (2011).</li><li>Desando, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-articular+delivery+of+adipose+derived+stromal+cells+attenuates+osteoarthritis+progression+in+an+experimental+rabbit+model.">Intra-articular delivery of adipose derived stromal cells attenuates osteoarthritis progression in an experimental rabbit model.</a> Arthritis Res Ther. 15 (1), 22 (2013).</li><li>Riester, S. M., 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=Safety+Studies+for+Use+of+Adipose+Tissue-Derived+Mesenchymal+Stromal/Stem+Cells+in+a+Rabbit+Model+for+Osteoarthritis+to+Support+a+Phase+I+Clinical+Trial.">Safety Studies for Use of Adipose Tissue-Derived Mesenchymal Stromal/Stem Cells in a Rabbit Model for Osteoarthritis to Support a Phase I Clinical Trial.</a> Stem Cells Transl Med. 6 (3), 910-922 (2017).</li><li>Bai, X., 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=Tracking+long-term+survival+of+intramyocardially+delivered+human+adipose+tissue-derived+stem+cells+using+bioluminescence+imaging.">Tracking long-term survival of intramyocardially delivered human adipose tissue-derived stem cells using bioluminescence imaging.</a> Mol Imaging Biol. 13 (4), 633-645 (2011).</li><li>Wolbank, S., 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=Labelling+of+human+adipose-derived+stem+cells+for+non-invasive+in+vivo+cell+tracking.">Labelling of human adipose-derived stem cells for non-invasive in vivo cell tracking.</a> Cell Tissue Bank. 8 (3), 163-177 (2007).</li><li>Heymer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+oxide+labelling+of+human+mesenchymal+stem+cells+in+collagen+hydrogels+for+articular+cartilage+repair.">Iron oxide labelling of human mesenchymal stem cells in collagen hydrogels for articular cartilage repair.</a> Biomaterials. 29 (10), 1473-1483 (2008).</li><li>Hemmrich, K., Meersch, M., von Heimburg, D., Pallua, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applicability+of+the+dyes+CFSE,+CM-DiI+and+PKH26+for+tracking+of+human+preadipocytes+to+evaluate+adipose+tissue+engineering.">Applicability of the dyes CFSE, CM-DiI and PKH26 for tracking of human preadipocytes to evaluate adipose tissue engineering.</a> Cells Tissues Organs. 184 (3-4), 117-127 (2006).</li><li>Shim, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+and+in+vivo+fate+of+intra-articularly+transplanted+human+bone+marrow-derived+clonal+mesenchymal+stem+cells.">Pharmacokinetics and in vivo fate of intra-articularly transplanted human bone marrow-derived clonal mesenchymal stem cells.</a> Stem Cells Dev. 24 (9), 1124-1132 (2015).</li><li>Chen, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+safety+study+on+intrathecal+delivery+of+autologous+mesenchymal+stromal+cells+in+rabbits+directly+supporting+Phase+I+human+trials.">A safety study on intrathecal delivery of autologous mesenchymal stromal cells in rabbits directly supporting Phase I human trials.</a> Transfusion. 55 (5), 1013-1020 (2015).</li><li>Chan, M. M., Gray, B. D., Pak, K. Y., Fong, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+in+vivo+imaging+of+arthritis+in+a+collagen-induced+murine+model+with+phosphatidylserine-binding+near-infrared+(NIR)+dye.">Non-invasive in vivo imaging of arthritis in a collagen-induced murine model with phosphatidylserine-binding near-infrared (NIR) dye.</a> Arthritis Res Ther. 17, 50 (2015).</li><li>Texier, I., 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=Cyanine-loaded+lipid+nanoparticles+for+improved+in+vivo+fluorescence+imaging.">Cyanine-loaded lipid nanoparticles for improved in vivo fluorescence imaging.</a> J Biomed Opt. 14 (5), 054005 (2009).</li><li>Honig, M. G., Hume, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+carbocyanine+dyes+allow+living+neurons+of+identified+origin+to+be+studied+in+long-term+cultures.">Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures.</a> J Cell Biol. 103 (1), 171-187 (1986).</li><li>Rahmati, M., Mobasheri, A., Mozafari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+mediators+in+osteoarthritis:+A+critical+review+of+the+state-of-the-art,+current+prospects,+and+future+challenges.">Inflammatory mediators in osteoarthritis: A critical review of the state-of-the-art, current prospects, and future challenges.</a> Bone. 85, 81-90 (2016).</li><li>Detante, O., 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=Intravenous+administration+of+99mTc-HMPAO-labeled+human+mesenchymal+stem+cells+after+stroke:+in+vivo+imaging+and+biodistribution.">Intravenous administration of 99mTc-HMPAO-labeled human mesenchymal stem cells after stroke: in vivo imaging and biodistribution.</a> Cell Transplant. 18 (12), 1369-1379 (2009).</li><li>Hu, S. L., 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=In+vivo+magnetic+resonance+imaging+tracking+of+SPIO-labeled+human+umbilical+cord+mesenchymal+stem+cells.">In vivo magnetic resonance imaging tracking of SPIO-labeled human umbilical cord mesenchymal stem cells.</a> J Cell Biochem. 113 (3), 1005-1012 (2012).</li><li>Xia, Q., 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=Intra-articular+transplantation+of+atsttrin-transduced+mesenchymal+stem+cells+ameliorate+osteoarthritis+development.">Intra-articular transplantation of atsttrin-transduced mesenchymal stem cells ameliorate osteoarthritis development.</a> Stem Cells Transl Med. 4 (5), 523-531 (2015).</li><li>Jasmin, , 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=Optimized+labeling+of+bone+marrow+mesenchymal+cells+with+superparamagnetic+iron+oxide+nanoparticles+and+in+vivo+visualization+by+magnetic+resonance+imaging.">Optimized labeling of bone marrow mesenchymal cells with superparamagnetic iron oxide nanoparticles and in vivo visualization by magnetic resonance imaging.</a> J Nanobiotechnology. 9, 4 (2011).</li><li>Lehmann, T. 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

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

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

強化された骨形成のためのヒト脂肪由来間質細胞の単離および濃縮

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

発生生物学

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

フィブリン塊における同種間葉系幹細胞の注入によってウサギの膝関節における骨軟骨欠損の治療

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

医学

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

軟骨欠損のヒト間葉系幹細胞を標識Ferumoxidesの注入

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

生物学

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, ...

免疫・感染学

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 ...

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

ラットの両側膝蓋腱損傷モデルの幹細胞療法の評価

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.

Infrapatellar fat pad mesenchymal&#160;stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.

ヤギ窒息関節の膝蓋下脂肪パッドからの間葉系幹細胞の単離、増殖、および分化

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded ...

生物工学

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

潜在的な軟骨再生のための脂肪組織由来間質血管分数の生産の臨床プロトコル

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...

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.

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.

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