著者は、バイオリアクターでの彼らの助けのためにQuinXell技術とCellD、特にロサー・グランマンとドミニーク・ゴーズラン確認したいと思います。私たちは、プルラン/デキストラン多糖類の足場を提供してくれキャサリンルヴィサージュを、感謝します。この作品は、欧州連合（ERC-2014-重心プロジェクトマティス648779）によっておよびAgenceNationaledeラRECHERCHE（ANR）、フランス（MagStemプロジェクトANR-11 SVSE5）によってサポートされていました。

磁気デバイスの1建設注：細胞播種のために使用されるデバイスは、アプリケーションに依存して変化する（ 図1）。磁気チップは（直径750μm）と非常に薄くなければならないので、凝集体を形成するために、細胞数は、2.5×10 5 /集約に限定されます。 1.8センチメートル2月 7日mm厚足場を播種するために、磁石は大きくなければならない（直径3mm）と、足場の孔を通って細胞移動を確実にします。 <ol><li>凝集体形成のための微小磁石を有するデバイスの構築（ 図1A） <ol><li>アルミニウム板を介して0.8 mmのドリルを有するマイクロ孔（直径3センチ、厚さ6mm）を作ります。 </li><li>プレートの各穴に磁気先端（直径750μm）を挿入します。 </li><li>飽和に磁化を確実に永久ネオジム磁石、上でこのディスクを置きます。 </li></ol></li><li>足場の播種用のデバイスの構築（ <strオング&gt;図1B） <ol><li> 2.4センチメートル2つの正方形にハードポリスチレンをカット。 </li><li> 1.6センチメートル2の表面積にわたって等距離9つの小さな磁石（直径3mm、長6ミリメートル）を挿入します。 </li><li>永久ネオジム磁石の上にこのデバイスを置きます。 </li></ol></li></ol> 2.幹細胞標識注：これらは30分（5±0.4 pgの鉄/細胞）で播種するために0.2mMの磁性ナノ粒子で標識しつつ、幹細胞は、30分（2.6±0.2 pgの鉄/細胞）凝集体を形成するために0.1mMの磁性ナノ粒子で標識しました足場。これらのナノ粒子の濃度およびインキュベーション時間は、以前に使用し、MSCおよび他の細胞18、19のために公開し、ナノ粒子がどちらの細胞生存率もMSCの分化能に影響を与えたことが確認されているされています。幹細胞に組み込まれた鉄の塊は、単一のCEを介して測定しました。LL磁気泳動19、20。 <ol><li>近くまで37℃、5％CO 2で、完全な間葉系幹細胞増殖培地（MSCGM）で培養ヒト間葉系幹細胞（MSC）は、（〜90％）をコンフルエンスまで。 </li><li>グルタミンなしロズウェルパーク記念研究所（RMPI）で修飾された無血清マッコイ5A培地および5を含む：0.1又は0.2ミリモルのマグヘマイトクエン酸でコーティングされた鉄酸化物（8 nmの直径コア被着γFe2 O 3）を混合することにより、磁気標識溶液を調製mMクエン酸ナトリウム。 </li><li> 、培地を捨てグルタミンなしの無血清RPMI培地で細胞をすすぎ、150-cm 2の培養フラスコ、すべてのセルをカバーするために必要な最小体積当たりの鉄酸化物ナノ粒子溶液10mLを加えます。 </li><li> 37℃で30分間インキュベートし、5％CO 2、次いでナノ粒子溶液を捨てます。ナンを内部化するためにグルタミンなし無血清RPMI培地で5分間すすぎますまだ形質膜に付着oparticles。 </li><li> RPMI培地を廃棄し、フラスコ当たり完全MSCGM培地25mlを加えます。 37℃で一晩インキュベートし、5％CO 2。 </li></ol> 3.磁気細胞播種<ol><li>新たに50μMのL-アスコルビン酸-2-リン酸、0.1μMデキサメタゾン、1mMピルビン酸ナトリウム、0.35 mMのL-プロリン、1％ユニバーサル培養を添加することにより、L-グルタミンを有するダルベッコ変法イーグル培地（DMEM）、高グルコースを用いて軟骨形成培地を調製インスリン、ヒトトランスフェリン、および亜セレン酸（ITS-プレミックス）、及び10ng / mLの形質転換増殖因子ベータ3（TGF-β3）を含むサプリメント。 </li><li> 150-cm 2の培養フラスコ及び遠心あたり0.05％トリプシン-EDTA 8mLの5分間260×gで解離した細胞を用いた磁気細胞を切り離します。メディアを吸引除去し、再懸濁した細胞を数えます。 </li><li>両方の磁気デバイスの上部にガラス底細胞培養ペトリ皿（34 mm）を置きます。 </li><li> Mにagnetically、凝集体を形成するペトリ皿に軟骨形成培地3mlを加え、穏やかに（16の凝集体まで堆積させることができる）集計当たり2.5×10 5個の標識細胞を含有する可能最小体積（これ以上8以下μL）を堆積させます。それはスフェロイドを形成することができ、それを移動させずに20-30分間ペトリ皿を残し、その後、37℃、5％CO 2インキュベーター中に、16の凝集体を含有するペトリ皿を含む、完全なデバイスを配置します。 </li><li>フォームコントロール凝集同じプロトコルに従い、TGF-β3なし軟骨形成培地を完全培地を交換してください。 <ol><li> 3D集合体構築物を作製するために、8つのダブレットを形成し、融合を開始することを8日目に接触して2骨材を配置します。 11日目に、4つの四つ組を形成する2つのダブレットをマージします。最後に、最終構造を得るために15日目に4つの四つ組を融合。 </li><li>同時に、2.5×10 5を遠心分離することによってフォーム凝集体が260で幹細胞を標識し15; TGF-β3を伴うまたは伴わない軟骨形成培地1.5 mlを15 mLチューブ中で5分間G（サンプルおよびコントロールのための、それぞれ）。 </li></ol></li><li>シード足場を磁気的には、ペトリ皿に各乾燥足場を置きます。プルラン/デキストラン21で作られた多糖類の多孔質足場を使用してください。各足場のために、TGF-β3なし軟骨形成培地350μL中に2×10 6個の標識された幹細胞を希釈し、慎重に足場上に細胞をピペット。 <ol><li>足場内で完全細胞侵入を可能にするために37℃で5分間インキュベートし、次いで穏やかにペトリ皿に（試料又はコントロールのため、それぞれ）TGF-β3の有無にかかわらず軟骨形成培地3mlを加えます。 </li><li>足場の孔および閉じ込めを介して細胞の移動を可能にするために、4日間、37℃、5％CO 2での磁気デバイスで細胞化足場をインキュベートします。 </li></ol></li><li> 2×10 6と同時に、シード足場</s&gt;まで同じ方法に従って幹細胞を標識し、ポジティブコントロールとして均一に播種足場を得るために、磁石なしでインキュベートします。 </li></ol>軟骨細胞への分化4. 注：インキュベーションの4日後、磁石を除去し、ペトリ皿（静的条件）またはバイオリアクター（動的条件）のいずれかで軟骨成熟を続けます。陰性対照サンプルは、TGF-β3なし軟骨形成培地で静的条件下で熟成されます。 <ol><li>静的条件では、同一のシャーレに細胞化スカフォールドまたは凝集体を保ちます。 21日間、1週間に2回軟骨形成培地を変更します。 </li><li>動的条件下では、バイオリアクターを準備します。 <ol><li>製造業者のプロトコルに従って適切な長さでシリコーンチューブを切断します。 </li><li> 500mLの培養チャンバー、チューブ、2ウェイ回転子、及びケージ：すべての材料をオートクレーブ。 </li><li>滅菌microbioにバイオリアクター部品を配置論理的な安全ステーション。製造者の指示に従って2ウェイ回転子に、培養室にチューブを接続します。 </li><li>注意深く滅菌スパチュラを用いて滅菌ケージに細胞化スカフォールドを移します。ケージ当たり2つの足場を置きます。ケージの準備が整ったら、さらに回転中に移動するからそれらを保つために蓋の針に挿入してください。軟骨形成培地で培養室を記入し、ケージを含む蓋を閉じます。 </li></ol></li><li>軟骨形成培地でチューブを埋めるために蠕動ポンプの電源を入れ、気泡を排除します。 </li><li>配置及びバイオリアクターのモータに充填されたチャンバを固定し、アームのチャンバの両方の回転を制御するコンピュータの電源をオン。 </li><li>アームチャンバの両方に毎分5つの回転（RPM）の回転速度を適用します。細胞化スカフォールドの連続供給のために10回転の流量で、蠕動ポンプを調整します。 </li></ol>注：RNA抽出の前に、酵素溶液を用いて足場を消化します。 <ol><li>無血清DMEM培地850 mLにプルラナーゼの100μL（40 U / mL）およびデキストラナーゼの50μLた（60mg / ml）を添加することによって酵素溶液1mLを調製します。 </li><li> 、無血清DMEM培地で2回足場をすすぐメディアを破棄し、足場あたりの酵素液800μLを加えます。穏やかな撹拌下37°Cで15〜30分間インキュベートします。 </li><li>足場が完全に溶解されたときに、1.5 mLのチューブに細胞を含む溶液を転送する、10分間、300×gで遠心分離、注意深く滅菌1×リン酸緩衝生理食塩水（PBS）で2回すすぎ、培地を吸引、遠心分離に300×10分間、G、およびRNA単離溶液で再懸濁細胞。 </li><li> 、凝集体からRNAを抽出し、RNA単離溶液にスフェロイドを配置することと完全RNA抽出を行う前に、ホモジナイザーを使用してそれらをつぶします。 </li><li>製造業者の説明書に従って全RNA抽出用キットを用いてRNAを単離します。 </li><li> 250 ngのランダムプライマー、dNTP混合物1μL（各10mM）、及び40 U / mLのRNase阻害剤を用いて、製造者の指示に従って逆転写酵素を用いて総RNA 400 ngのから相補的DNAを合成します。反応の最終容量は20μLです。反応の終了時に、100μLの最終体積を得るために、蒸留水80μLを加えます。 </li><li>定量的ポリメラーゼ連鎖反応（PCR）のために、10倍希釈のcDNAを用いて、そのようなアグリカン（AGC）及びコラーゲンII（COL II）として目的の遺伝子の相対的発現を定量するための蛍光試薬を含むPCRミックスを使用します。参照遺伝子リボソームタンパク質、大、P0（RPLP0）で遺伝子発現のレベルを標準化。 ΔΔCT式- 2で計算を実行、ΔΔCT=ΔCTは、分化状態の場合 - 制御条件のΔCTを意味し、各ΔCTは、目的の遺伝子のCT表す - 参照遺伝子（RPLP0）のCT。 </li><li>平均（SEM）の標準誤差±平均値などの統計的測定値を決定します。 n個の≥2つの独立した実験と解析を行います。遠心分離し、ペレットおよび磁気融合（* P &lt;0.05）との間に統計的差異を分析するためにスチューデントのt検定を使用してください。分化足場の間の制御足場（* P &lt;0.05）と統計的差異を分析するためにクラスカル・ワリス検定（ANOVAは、ノンパラメトリック一方向）で有意性を決定します。 </li></ol> 6.組織学的分析<ol><li>滅菌1×PBSで細胞化スカフォールドまたは凝集体をすすぎ、室温で1時間10％ホルマリン溶液でそれらを修正し、1×PBSでリンス。 </li><li> PBSを削除し、最適なcuttinでサンプルを埋め込みますG温度化合物（OCT）、及び液体窒素中に浸漬イソペンタン浴でそれらを凍結。 -20°Cでサンプルを保管してください。凝集体または細胞化スカフォールド12ミクロン、8ミクロンの凍結切片を得るために、クライオスタットを用いてサンプルを切断。 </li><li> 2分間0.5％トルイジンブルー溶液を用いて凍結切片を染色し、水道水ですすぎ、100％エタノールで脱水し、トルエンを用いて明らかにし、光学顕微鏡用の封入剤でスライドをマウントします。 </li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+toxicity+of+superparamagnetic+iron+oxide+nanoparticles+(SPION).">Potential toxicity of superparamagnetic iron oxide nanoparticles (SPION).</a> Nano Reviews. 1, (2010).</li><li>Lee, J. H., Hur, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffold-free+formation+of+a+millimeter-scale+multicellular+spheroid+with+an+internal+cavity+from+magnetically+levitated+3T3+cells+that+ingested+iron+oxide-containing+microspheres.">Scaffold-free formation of a millimeter-scale multicellular spheroid with an internal cavity from magnetically levitated 3T3 cells that ingested iron oxide-containing microspheres.</a> Biotechnol Bioeng. 111 (5), 1038-1047 (2014).</li><li>Mattix, 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=Biological+magnetic+cellular+spheroids+as+building+blocks+for+tissue+engineering.">Biological magnetic cellular spheroids as building blocks for tissue engineering.</a> Acta Biomaterialia. 10 (2), 623-629 (2014).</li><li>Mazuel, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massive+Intracellular+Biodegradation+of+Iron+Oxide+Nanoparticles+Evidenced+Magnetically+at+Single-Endosome+and+Tissue+Levels.">Massive Intracellular Biodegradation of Iron Oxide Nanoparticles Evidenced Magnetically at Single-Endosome and Tissue Levels.</a> ACS Nano. 10 (8), 7627-7638 (2016).</li><li>Kim, J. 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=High-throughput+generation+of+spheroids+using+magnetic+nanoparticles+for+three-dimensional+cell+culture.">High-throughput generation of spheroids using magnetic nanoparticles for three-dimensional cell culture.</a> Biomaterials. 34 (34), 8555-8563 (2013).</li><li>Shimizu, K., Ito, A., Honda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+cell-seeding+into+3D+porous+scaffolds+by+use+of+magnetite+nanoparticles.">Enhanced cell-seeding into 3D porous scaffolds by use of magnetite nanoparticles.</a> J Biomed Mater Res B Appl Biomater. 77 (2), 265-272 (2006).</li><li>Sensenig, R., Sapir, Y., MacDonald, C., Cohen, S., Polyak, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+nanoparticle-based+approaches+to+locally+target+therapy+and+enhance+tissue+regeneration+in+vivo.">Magnetic nanoparticle-based approaches to locally target therapy and enhance tissue regeneration in vivo.</a> Nanomedicine (Lond). 7 (9), 1425-1442 (2012).</li><li>Waldman, S. D., Couto, D. C., Grynpas, M. D., Pilliar, R. M., Kandel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-axial+mechanical+stimulation+of+tissue+engineered+cartilage:+review.">Multi-axial mechanical stimulation of tissue engineered cartilage: review.</a> Eur Cell Mater. 13, 66-74 (2007).</li><li>Takahashi, 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=Compressive+force+promotes+sox9,+type+II+collagen+and+aggrecan+and+inhibits+IL-1beta+expression+resulting+in+chondrogenesis+in+mouse+embryonic+limb+bud+mesenchymal+cells.">Compressive force promotes sox9, type II collagen and aggrecan and inhibits IL-1beta expression resulting in chondrogenesis in mouse embryonic limb bud mesenchymal cells.</a> J Cell Sci. 111 (14), 2067-2076 (1998).</li><li>Campbell, J. J., Lee, D. A., Bader, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+compressive+strain+influences+chondrogenic+gene+expression+in+human+mesenchymal+stem+cells.">Dynamic compressive strain influences chondrogenic gene expression in human mesenchymal stem cells.</a> Biorheology. 43, 455-470 (2006).</li><li>Vunjak-Novakovic, 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=Bioreactor+cultivation+conditions+modulate+the+composition+and+mechanical+properties+of+tissue-engineered+cartilage.">Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage.</a> J Orthop Res. 17 (1), 130-138 (1999).</li></ol>

著者は、開示することは何もありません。

ここで紹介するテクニックは、磁性ナノ粒子の内在化に依存しているため、彼らは、細胞内局在したらまず、一つの重要な問題は、ナノ粒子の結果です。鉄ナノ粒子は、それらのサイズ、コーティング、及び露光19の時間に応じて潜在的な毒性または障害分化能力、22をトリガすることができることは事実です。カプセル化された鉄ナノ粒子は、マ?...

磁性ナノ粒子は、既に磁気共鳴画像（MRI）用造影剤としてクリニックで使用されており、それらの治療への応用が拡大し続けます。例えば、最近、標識された細胞は、移植1、2、3の定義された部位に外部磁界を用いてin vivoで操作することができ、向けることができ、および/または維持することが示されています。再生医療において、それらは血管組織5、6、7、8、骨、および軟骨9を含むインビトロ 4 で編成組織を操作するために使用することができます。 関節軟骨は、損害が発生したときに、非常に限られた細胞外マトリックス成分の修理を行う、無血管環境に浸漬されます。このため、researc用時間は、現在、欠損部位に移植することができ硝子軟骨置換のエンジニアリングに焦点を当てています。他のものは軟骨12,13に分化する間葉系幹細胞（MSC）の能力を強調しながら、自己置換を生成するために、いくつかの研究グループは、セルソース10、11のような自家軟骨細胞の使用を模索しています。彼らの骨髄採取はかなりシンプルで、自分の表現型14を失う危険健全な軟骨細胞の犠牲を必要としないので、ここで再現した以前の研究では、我々は、MSCを選択しました。 幹細胞の軟骨形成分化を開始するために不可欠早い段階で彼らの結露です。細胞凝集体は、一般に遠心分離またはマイクロマス培養15のいずれかを用いて形成されています。しかし、これらの縮合方法neitheR厚い足場内の細胞クラスタを作成する可能性も凝集体の融合を制御する可能性を提示します。本稿では、MSC磁気標識及び磁気吸引力を利用した幹細胞を凝縮する革新的なアプローチについて説明します。この技術は、ミリメートルスケール軟骨組織9を得るために互いに凝集体の融合を介して足場フリー3D構築物を形成することが証明されています。厚いと大きな足場の磁気播種はまた、改変された組織のサイズを大きくする移植のためより容易に有用な形状を設計、および軟骨修復における臨床応用の可能性を多様化する可能性を可能にしました。ここでは、天然多糖類、プルラン、およびデキストランからなる多孔質足場にMSCの磁気シーディングのために、私たちの詳細プロトコルは、足場は以前幹細胞16、17を閉じ込めるために使用されます。軟骨形成分化がfinallましたyは細胞の高い密度で播種した足場のマトリックスコアに連続的な栄養素とガス拡散を確実にするために、バイオリアクターで行います。細胞に栄養、軟骨形成成長因子、およびガスを提供するだけでなく、バ​​イオリアクターは、機械的刺激を提供しました。全体として、バイオリアクター内で動的成熟と組み合わされた幹細胞を閉じ込めるために使用される磁気技術は、著しく軟骨形成分化を向上させることができます。

<table><tbody><tr><td>Iron oxide (maghemite) nanoparticules (&amp;gamma;-Fe&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;)</td><td>PHENIX - University Paris 6</td><td>Made and given by C. M&amp;eacute;nager</td><td>Mean diameter of 8.1 nm and negative surface charge</td></tr><tr><td>Polysaccharide Pullulan/Dextran scaffolds</td><td>LIOAD - University Nantes</td><td>Made and given by C. Le Visage</td><td>Prepared from a 75:25 mixture of pullulan/dextran in alkaline conditions (10&amp;nbsp;M NaOH). Porosity: 185-205&amp;nbsp;&amp;micro;m; Thickness: 7&amp;nbsp;mm; Surface area: 1.8&amp;nbsp;cm&lt;sup&gt;2&lt;/sup&gt;.</td></tr><tr><td>TisXell Regeneration System</td><td>QuinXell Technologies</td><td>QX900-002</td><td>Biaxial bioreactor with 500 mL culture chamber&amp;nbsp;</td></tr><tr><td>Cage for scaffolds: Histosette II M492&amp;nbsp;</td><td>VWR</td><td>720-0909</td><td></td></tr><tr><td>Mesenchymal Stem Cell (MSC)</td><td>Lonza</td><td>PT-2501</td><td>Three independant batches have been used</td></tr><tr><td>MSCGM BulletKit medium</td><td>Lonza</td><td>PT-3001</td><td>For the complete medium, add the provided BulletKit (containing serum, glutamine and antibiotics) to the MSCGM medium</td></tr><tr><td>DMEM with Glutamax I</td><td>Life Technologies</td><td>31966-021</td><td>No sodium pyruvate, no HEPES</td></tr><tr><td>RPMI medium 1640, no Glutamine</td><td>Life Technologies</td><td>31870-025</td><td>No sodium pyruvate, no HEPES</td></tr><tr><td>PBS w/o CaCl&lt;sub&gt;2&lt;/sub&gt; w/o MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Life Technologies</td><td>14190-094</td><td></td></tr><tr><td>0.05% Trypsin-EDTA (1x)</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>Penicillin (10,000&amp;nbsp;U/mL) / Streptomycin (10,000&amp;nbsp;&amp;micro;g/mL)</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>ITS Premix Universal Culture Supplement (20x)</td><td>Corning</td><td>354352</td><td></td></tr><tr><td>Sodium pyruvate solution 100 mM</td><td>Sigma</td><td>S8636</td><td></td></tr><tr><td>L-Ascorbic Acid 2-phosphate</td><td>Sigma</td><td>A8960</td><td>Prepare the concentrated solution (25 mM) in distilled water extemporaneously</td></tr><tr><td>L-Proline</td><td>Sigma</td><td>P5607</td><td>Prepare the 175 mM stock solution diluted in distilled water and store at 4&amp;nbsp;&amp;deg;C</td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D4902</td><td>Prepare the 1 mM stock solution diluted in Ethanol 100% and store at -20&amp;nbsp;&amp;deg;C</td></tr><tr><td>TGF-beta 3 protein 10&amp;nbsp;&amp;micro;g</td><td>Interchim</td><td>30R-AT028</td><td></td></tr><tr><td>Tri-sodium citrate</td><td>VWR</td><td>33615.268</td><td>Prepare the 1&amp;nbsp;M stock solution diluted in distilled water and store at 4&amp;nbsp;&amp;deg;C</td></tr><tr><td>Pullulanase from &lt;em&gt;Bacillus acidopullulyticus&lt;/em&gt;</td><td>Sigma</td><td>P2986</td><td></td></tr><tr><td>Dextranase from &lt;em&gt;Chaetomium erraticum&lt;/em&gt;</td><td>Sigma</td><td>D0443</td><td></td></tr><tr><td>NucleoSpin RNA Extraction Kit</td><td>Macherey-Nagel</td><td>740955.5</td><td></td></tr><tr><td>SuperScript II Reverse Transcriptase</td><td>Life Technologies</td><td>18064-014</td><td></td></tr><tr><td>Random Primer - Hexamer&amp;nbsp;</td><td>Promega</td><td>C1181</td><td>500 &amp;micro;g/mL: Use diluted 1/2 and put 1 &amp;micro;L per sample</td></tr><tr><td>Recombinant RNAs in ribonuclease inhibitor</td><td>Promega</td><td>N2511</td><td>40 U/&amp;micro;L: put 1 &amp;micro;L per sample</td></tr><tr><td>PCR nucleotide dNTP mix (10&amp;nbsp;mM each)</td><td>Roche</td><td>10842321</td><td></td></tr><tr><td>SyBr Green PCR Master Mix</td><td>Life Technologies</td><td>4368708</td><td></td></tr><tr><td>Step One Plus Real-Time PCR System</td><td>Life Technologies</td><td>4381792</td><td></td></tr><tr><td>Formalin solution 10% neutral buffered</td><td>Sigma</td><td>HT5012</td><td></td></tr><tr><td>OCT solution</td><td>VWR</td><td>361603E</td><td></td></tr><tr><td>Isopentane</td><td>Sigma</td><td>M32631</td><td></td></tr><tr><td>Toluidine blue O</td><td>VWR</td><td>1.15930.0025</td><td></td></tr><tr><td>Ethanol absolute</td><td>VWR</td><td>20821.310</td><td></td></tr><tr><td>Toluene</td><td>VWR</td><td>1.08323.1000</td><td></td></tr><tr><td>Mounting medium Pertex</td><td>Histolab</td><td>840</td><td></td></tr><tr><td>RPLP0 Primer for qPCR</td><td>Eurogentec</td><td></td><td>5&#039;-TGCATCAGTAC&lt;br/&gt; CCCATTCTATCAT-3&#039;;&lt;br/&gt; 5&#039;-AAGGTGTAATC&lt;br/&gt; CGTCTCCACAGA-3&#039;</td></tr><tr><td>Aggrecan Primer for qPCR</td><td>Eurogentec</td><td></td><td>5&#039;-TCTACCGCTGCGAGGTGAT-3&#039;; 3&#039;-TGTAATGGAACACGATGCCTTT-5&#039;</td></tr><tr><td>Collagen II Primer for qPCR</td><td>Eurogentec</td><td></td><td>5&#039;-ACTGGATTGACCCCAACCAA-3&#039;; 3&#039;-TCCATGTTGCAGAAAACCTTCA-5&#039;</td></tr></tbody></table>

3d magnetic stem cell aggregation and bioreactor maturation for cartilage regeneration

軟骨工学は、ネイティブの組織に似たin vitro機能インプラントを作成するのが困難に起因する課題です。最近、自家代替品の開発のための探求のアプローチは、軟骨細胞への幹細胞の分化に関与します。この軟骨形成を開始するには、幹細胞の圧縮度が要求されます。したがって、我々は、細胞アトラクタとして小型化磁界源を使用して、厚い足場と足場フリー内の両方、磁気凝縮細胞の実現可能性を実証しました。この磁気アプローチはまた、凝集融合を誘導する足場を含まない構築するために使用される組織化、三次元（3D）のサイズの数ミリメートルを組織しました。強化されたサイズを有することに加えて、磁気駆動の融合によって形成された組織は、コラーゲンIIの発現の有意な増加を提示し、同様の傾向がアグリカン発現のために観察されました。ネイティブの軟骨が力トンに供した通り帽子は、その立体構造に影響を与えた、ダイナミックな成熟も行いました。機械的な刺激を提供するバイオリアクターは、21日間にわたって培養物に磁気播種足場を使用しました。バイオリアクターの成熟は、主として細胞化足場に軟骨形成を向上させます。これらの条件下で得られた細胞外マトリックスは、コラーゲンIIおよびアグリカンに富んでいました。この作業は、改善された軟骨形成分化、足場フリーの両方に多糖類の骨格内のバイオリアクター中で標識された幹細胞の磁気凝集及び動的成熟の革新的な可能性を概説します。

まず、凝集体は、個々の幹細胞（ 図2A）を標識した2.5×10 5を堆積することにより微小磁石を用いて形成することができます。これらの単一集合体（サイズ〜0.8ミリメートル）は、より大きな構造にシーケンシャル、磁気的に誘導し融合のおかげで融合させることができます。例えば、軟骨成熟の8日目に、凝集体がダブレットを形成するように対?...

Watch this Scientific Journal Video about 3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration at JoVE.com

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

幹細胞から軟骨形成は、培養条件の微調整が必​​要です。ここで、我々は、細胞を集光するための軟骨形成を開始するために不可欠なステップを磁気アプローチを提示します。また、当社は、バイオリアクター内の動的成熟は、細胞の構造に機械的刺激を適用し、軟骨細胞外マトリックスの産生を増強することを示しています。

軟骨再生のための3次元磁気幹細胞凝集およびバイオリアクター成熟

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

3d-magnetic-stem-cell-aggregation-bioreactor-maturation-for-cartilage

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.7K ビュー.  UMR 7057 CNRS and University Paris Diderot. 幹細胞から軟骨形成は、培養条件の微調整が必​​要です。ここで、我々は、細胞を集光するための軟骨形成を開始するために不可欠なステップを磁気アプローチを提示します。また、当社は、バイオリアクター内の動的成熟は、細胞の構造に機械的刺激を適用し、軟骨細胞外マトリックスの産生を増強することを示しています。 

ビデオ: 3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

組織工学のための二軸メカニカルローディングバイオリアクターの設計

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

生物工学

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

臍帯血由来誘導多能性幹細胞からの軟骨形成ペレット形成

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

発生生物学

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

エンジニアリング3D細胞化血管組織再生のためのコラーゲンゲル

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

関節軟骨細胞培養軟骨の生成のための3Dハイドロゲルの足場

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

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

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

軟骨と皮膚組織類縁ミキシング ユニット Bioink Precellularization の小説のパッシブ バイオプリンティング

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

三次元ペプチド足場で哺乳動物の細胞を培養

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Construction, Characterization, and Regenerative Application of Self-Assembled Human Mesenchymal Stem Cell Aggregates

Mesenchymal stem cells (MSCs), characterized by their self-renewal ability and multilineage differentiation potential, can be derived from various sources and are emerging as promising candidates for regenerative medicine, especially for regeneration of the tooth, bone, cartilage, and skin. The self-assembled approach of MSC aggregation, which notably constructs cell clusters mimicking the developing mesenchymal condensation, allows high-density stem cell delivery along with preserved cell-cell interactions and extracellular matrix (ECM) as the microenvironment niche. This method has been shown to enable efficient cell engraftment and survival, thus promoting the optimized application of exogenous MSCs in tissue engineering and safeguarding clinical organ regeneration. This paper provides a detailed protocol for the construction and characterization of self-assembled aggregates based on umbilical cord mesenchymal stem cells (UCMSCs), as well as an example of the cranial bone regenerative application. The implementation of this procedure will help guide the establishment of an efficient MSC transplantation strategy for tissue engineering and regenerative medicine.

This study describes a method to construct aggregates based on the self-assembly of human mesenchymal stem cells and identifies the morphological and histological characteristics for the regenerative treatment of cranial bone defects.

Mesenchymal stem cells (MSCs), characterized by their self-renewal ability and multilineage differentiation potential, can be derived from various sources ...

Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited self-repairing capacity and, most importantly, no definitive treatments, tendinopathy still influences patients&#8217; life quality negatively. Tendon-derived stem cells (TDSCs), as primary precursor cells of tendon cells, play an essential role in both the development of tendinopathy, and functional and structural restoration after tendinopathy. Thus, a method that can in vitro mimic the in vivo differentiation of TDSCs into tendon cells would be useful. Here, the present protocol describes a method based on a three-dimensional (3D) uniaxial stretching system to stimulate the TDSCs to differentiate into tendon-like tissues. There are seven stages of the present protocol: isolation of mice TDSCs, culture and expansion of mice TDSCs, preparation of stimulation culture medium for cell sheet formation, cell sheet formation by culturing in stimulation medium, preparation of 3D tendon stem cell construct, assembly of the uniaxial-stretching mechanical stimulation complex, and evaluation of the mechanical stimulated in vitro tendon-like tissue. The effectiveness was demonstrated by histology. The entire procedure takes less than 3 weeks. To promote extracellular matrix deposition, 4.4 mg/mL ascorbic acid was used in the stimulation culture medium. A separated chamber with a linear motor provides accurate mechanical loading and is portable and easily adjusted, which is applied for the bioreactor. The loading regime in the present protocol was 6% strain, 0.25 Hz, 8 h, followed by 16 h rest for 6 days. This protocol could mimic cell differentiation in the tendon, which is helpful for the investigation of the pathological process of tendinopathy. Moreover, the tendon-like tissue is potentially used to promote tendon healing in tendon injury as an engineered autologous graft. To sum up, the present protocol is simple, economic, reproducible and valid.

A three-dimensional uniaxial mechanical stimulation bioreactor system is an ideal bioreactor for tenogenic-specific differentiation of tendon-derived stem cells and neo-tendon formation.

腱由来幹細胞の発熱分化を誘導する三次元単軸機械刺激バイオリアクターシステムの適用

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited ...

医学

Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

This study describes a method for producing chondrocytic spheroids by aggregating cells into spheroids under low-adhesion conditions using gravity, followed by culturing the resulting spheroids in mini-bioreactors.

重力下での低接着条件と自家製ミニバイオリアクターを用いた種々の軟骨細胞からのスフェロイド作製

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a ...

軟骨再生のための3次元磁気幹細胞凝集およびバイオリアクター成熟

要約

さらに動画を探す

この動画の章

組織工学のための二軸メカニカルローディングバイオリアクターの設計

臍帯血由来誘導多能性幹細胞からの軟骨形成ペレット形成

エンジニアリング3D細胞化血管組織再生のためのコラーゲンゲル

関節軟骨細胞培養軟骨の生成のための3Dハイドロゲルの足場

軟骨欠損のヒト間葉系幹細胞を標識Ferumoxidesの注入

軟骨と皮膚組織類縁ミキシングユニット Bioink Precellularization の小説のパッシブバイオプリンティング

三次元ペプチド足場で哺乳動物の細胞を培養

Construction, Characterization, and Regenerative Application of Self-Assembled Human Mesenchymal Stem Cell Aggregates

腱由来幹細胞の発熱分化を誘導する三次元単軸機械刺激バイオリアクターシステムの適用

重力下での低接着条件と自家製ミニバイオリアクターを用いた種々の軟骨細胞からのスフェロイド作製

軟骨再生のための3次元磁気幹細胞凝集およびバイオリアクター成熟

要約

さらに動画を探す

この動画の章

組織工学のための二軸メカニカルローディングバイオリアクターの設計

臍帯血由来誘導多能性幹細胞からの軟骨形成ペレット形成

エンジニアリング3D細胞化血管組織再生のためのコラーゲンゲル

関節軟骨細胞培養軟骨の生成のための3Dハイドロゲルの足場

軟骨欠損のヒト間葉系幹細胞を標識Ferumoxidesの注入

軟骨と皮膚組織類縁ミキシング ユニット Bioink Precellularization の小説のパッシブ バイオプリンティング

三次元ペプチド足場で哺乳動物の細胞を培養

Construction, Characterization, and Regenerative Application of Self-Assembled Human Mesenchymal Stem Cell Aggregates

腱由来幹細胞の発熱分化を誘導する三次元単軸機械刺激バイオリアクターシステムの適用

重力下での低接着条件と自家製ミニバイオリアクターを用いた種々の軟骨細胞からのスフェロイド作製

軟骨と皮膚組織類縁ミキシングユニット Bioink Precellularization の小説のパッシブバイオプリンティング