We thank the laboratory of Dr. Stephen Moss for providing embryonic rat brain tissues. M.D.T. designed the original protocol. This work was funded by National Institutes of Health P41 Tissue Engineering Resource Center Grant EB002520. K.C. was supported with Postdoctoral Fellowship from German Research Foundation (DFG) (CH 1400/2-1).

脳組織の分離プロトコルは、タフツ大学施設内動物管理使用委員会によって承認され、実験動物の管理と使用に関する健康ガイド（施設内動物管理使用委員会B2011-45）の国立研究所に準拠していました。 1.シルク足場の準備<ol><li> カイコの繭からシルク溶液の調製以前に19,20説明したように。 <ol><li>はさみを使用して8等分に各繭をカットします。断片化された繭の5グラム約11繭を使用してください。 （15分） </li><li> 0.02 MのNa 2 CO 3溶液の2リットルを用意し、ホットプレートを用いて沸騰するためにそれを持って来ます。 （15分） </li><li>断片化された繭を5g秤量し、30分間のNa 2 CO 3溶液でそれらを沸騰。スパチュラで沸騰絹を数分おきにかき混ぜます。また、脱ガム引きと呼ばれるこのステップは、親水性タンパク質、セリシンから絹フィブロインを浄化します。 （30分） </li><li&gt;は、手でフィブロインを強く絞りと、残りのセリシンや化学物質を洗い流すために、蒸留水で少なくとも3回、それをすすいでください。 （5分） </li><li>シャーレに湿ったフィブロインを置き、流フードO / Nでフィブロインの抽出物を乾燥させます。 </li><li>次の日は、乾燥フィブロイン量を計量し、ガラスビーカー中フィブロインを配置します。 （15分） </li><li> 9.3 MのLiBr溶液にフィブロインを溶解させるために、4で乾燥したフィブロインの質量を乗じてのLiBr（ml単位）必要な量を計算ゆっくり絹フィブロインを超える臭化リチウム水溶液を注ぎ、スパチュラを使用して、すべてのフィブロイン繊維を浸します。蒸発を防止し、繊維を溶解させるために4時間60°Cでそれを配置するビーカーを覆います。 （15分） </li><li>シリンジを用いて、ビーカーからフィブロイン溶液を収集し、MWCO 3500透析カセットにロードします。 48時間蒸留水に対して透析を行ってください。水に数時間おきに変更します。 </li><li>シリンジを使用して、よりフィブロイン溶液を集めます20分間4℃で9,000 rpmで（〜12,700 XG）の2倍で50ミリリットルコニカルチューブと遠心分離機にカセット。各遠心分離工程の後に新しいチューブに上清を注ぎ、ペレットを捨てます。 （40分） </li><li>乾燥重量を推定することによりフィブロインの濃度を測定します。計量ボートに絹溶液1mlを注ぎます。 2時間60℃のオーブン中でサンプルを乾燥させます。乾燥した絹フィブロインを秤量し、シルク溶液の予想濃度が6-9％（w / v）のある100により得られた重みを乗算して絹フィブロイン溶液の濃度を計算します。 </li><li>蒸留水で希釈することにより（w / v）の6％絹濃度を調整します。 停止ポイント液体絹フィブロインは、密閉容器内で最大1ヶ月間、4℃で保存することができます。 </li></ol></li><li>シルク溶液から多孔質足場の調製。 <ol><li>顆粒を分離するために、粒状のNaClをふるいは、後のステップで使用される500-600μmで、大きさ。顆粒を破棄Sは500ミクロン以下および600ミクロンを超えるサイズに。 （15分） </li><li>ポリテトラフルオロエチレン（PTFE）型（ 図1）への6％絹溶液30mlを注ぎます。静かに絹の上にふるいにかけられ、NaClの60グラムを散乱。塩の均一な層を得るために、コンテナをタップします。絹の重合に室温で48時間インキュベートします。 </li><li>重合を完了し、残りの液体を蒸発させるために1時間60℃のオーブンで足場を有するPTFE製の金型を置きます。 </li><li>塩を浸出するために48時間蒸留水2リットルを含むビーカー中のPTFE金型のコンテンツを配置します。水1日あたり2〜3回に変更します。塩が完全に溶出されたときに、金型からのスポンジ足場を削除ポイントの停止 ：スポンジ脱水から足場を防ぐために密閉容器中で4℃の水に浸漬し保存することができます。 </li><li>準備ができたら、直径5mmの生検パンチで足場を切り出します。高さが約2mmに到達するために足場をプレカット。 Puの直径2mmの生検パンチ（ 図2A）で足場の中心をNCH。 （1時間） </li><li> 。 ポイントの停止 （湿潤サイクル、121℃、20分）して滅菌水に浸漬した足場をオートクレーブ：スポンジ脱水から足場を防ぐために密閉容器中で4℃の水に浸漬し保存することができます。 </li><li>計画された細胞播種の前に、滅菌した0.1 mg / mlのポリ-D-リジン（PDL）溶液中で足場を浸します。 37℃で1時間インキュベートします。 </li><li>足場は非結合PDLを除去するためにリン酸緩衝生理食塩水（PBS）で3回洗浄します。 （30分） </li></ol></li></ol>ラット皮質ニューロンの2の単離<ol><li>承認された動物プロトコルが得られた後、以前にPacificiとペルッツィ27で説明したように日胚から18（E18）Sprague-Dawleyラットを皮質を解剖。 （2時間） </li><li> 3で20分間（ウシ膵臓から）、0.3％のDNアーゼIを有する0.25％トリプシン5mlの10皮質をインキュベート7°C。 </li><li> 1mg / mlの大豆タンパク質の等容積を添加することによってトリプシンを不活性化します。 </li><li>単一細胞懸濁液が生成されるまでピペットで10 mlのパスツールピペットを用いて皮質ダウン20回粉砕します。穏やかであると気泡の形成を避けます。 （10分） </li><li> 5分間127×gで細胞懸濁液を遠心。 </li><li>再懸濁培地10ml中の細胞ペレット（Neurobasal培地、1×B27サプリメント、1×グルタマックス、1％ペニシリン/ストレプトマイシン）。細胞を数えます。予想される細胞濃度が約2×10 7 / mlです。 （20分） </li></ol> 3.アセンブリと文化を構築<ol><li>細胞と足場播種。 <ol><li>無菌の足場と細胞培養フード内のすべての必要な調理器具を移動します。滅菌ピンセットを使用して、ウェルごとに足場を割り当てる、96ウェル細胞培養プレート中で足場を置きます。 （10分） </li><li>前の細胞種にそれらを平衡化するために、細胞培養培地中での足場を浸しる。 37℃で1時間インキュベートします。 （10分） </li><li>足場からのメディアの過剰を吸引します。 </li><li>細胞懸濁液/足場の100μLを適用します。 （10分） </li><li>足場への細胞接着を可能にするために37°のCO / Nで細胞をインキュベートします。 </li><li>翌朝は、非付着細胞を吸引し、200μl/ウェルの新鮮な培養培地を適用します。 （10分） </li></ol></li><li>コラーゲンマトリックスに埋め込む足場。 （2時間） <ol><li>私は氷の上にコラーゲン10倍PBS、水、1NのNaOHおよびラットの尾を置きます。製造者の指示に従って、コラーゲンのワーキング溶液を調製します。細胞を播種した構築物は、（1時間まで）準備ができるまで、氷上に維持します。 </li><li>インキュベーターから細胞を播種した絹の構造を削除し、メディアの過剰を吸引。 </li><li>滅菌ピンセットを使用すると、ウェルプレートの空のウェルに足場を転送し、3 mg / mlのコラーゲン溶液の100μlの各足場を浸します。バック組織培養プレートを置きコラーゲンの重合を可能にするために30分間インキュベーターインチ</li><li>ウェル/予熱した細胞培養培地100μlを適用します。文化一週間構築物は、培地の半分だけボリュームを交換することにより、毎日培地を交換すること。 </li></ol></li></ol> 4.顕微鏡分析<ol><li>生/死染色（1時間） <ol><li>ヨウ化プロピジウム（PI）を蒸留水に1mg / mlの（1.5ミリモル）のストック溶液を調製します。 </li><li>アセトン中のフルオレセインジアセテート（FDA）の2mg / mlのストック溶液を調製します。 </li><li> PBSで希釈した10μMのPIおよび0.15μM、FDAを含むワーキング溶液を調製します。水浴中で37℃に溶液を予め温めます。 </li><li>血清エステラーゼを除去するために、予め温めておいたPBSで細胞を洗浄。 PBSを吸引除去します。 </li><li> 2分間細胞に予め温めておいた作業溶液を適用し、PBSで洗ってください。 </li><li> FDAの元λ= 490 nは、画像への落射蛍光顕微鏡を使用する細胞（PIの元λ= 490 nmのは、EMλは570nmでの=mは、全角λ）は514nmのを=。染色は、40分かけて安定しています。長時間の染色は、細胞と細胞内でのPI / FDAの共局在によってPI取り込みに起因する非特異的な染色をもたらす可能性があります。 </li></ol></li><li>免疫染色<ol><li>培養物の所望の時点で、RTで30分間、4％パラホルムアルデヒド（PFA）で細胞を固定します。 PFAの毒性に定着工程は、化学ヒュームフード内で行われるべきであるヒューム。 </li><li> 。 ポイントの停止 3倍PBSで足場を洗う：PFA-固定構築物は、さらなるステップに進む前に数日間4℃でPBS中に保存することができます。 </li><li> 4℃で一次抗体のO / Nを含有するPBS中の0.2％トリトン、0.25％ウシ血清アルブミン（BSA）中で足場をインキュベートします。 </li><li>次の日は、染色溶液を廃棄し、結合していない一次抗体を除去するためにPBSで足場を3×3​​0分を洗います。 </li><li> 0.2％トリトン中に希釈した二次抗体とサンプル、PBS中の0.25％BSAで2インキュベートRTで時間。 </li><li>染色溶液を除去し、未結合の二次抗体を除去し、PBSで足場を3×3​​0分を洗います。 </li><li>画像1μmのステップとサンプルの100μmのZ-セクションを取ることによって20Xの目的とした共焦点顕微鏡を用いて足場。細い神経突起を可視化するために画像の解像度は、最小1,024×1024ピクセルでなければなりません。 </li></ol></li><li> RNA、DNAおよびタンパク質の単離 注：RNA、DNAおよびタンパク質の分離は、製造業者の説明書に従って商業AllPrep DNA / RNA /タンパク質ミニキットを用いて行われます。 <ol><li>滅菌ピンセットを使用すると、2ミリリットル滅菌チューブに培養プレートの外に足場を転送します。チューブあたり1足場を置きます。 </li><li>各チューブにRLT緩衝液200μlを追加します。 </li><li>滅菌microscissorsでそれを断片化することによって構造物を破砕。 </li><li>破壊された組織を均質化するために、スピンカラムにチューブの内容を転送します。微量でフルスピードで2分間スピン。これは、スピンカラムを通過する際に溶解物を均質化します。 </li><li>フロースルーを収集し、製造業者のプロトコルに従ってRNA、DNAおよびタンパク質を単離するために、すぐにそれを使用しています。 </li><li>核酸の収量互換性のある他の方法（例えば、光度計）を定量化します。 </li><li>製造業者の指示に従ってBCAタンパク質アッセイを使用して、タンパク質収量を定量化。波長562nmでマイクロプレートリーダーを用いたアッセイの結果を測定します。 注：細胞密度に関連して構築あたりの核酸およびタンパク質の予想収量は、 図4に示されています。 </li></ol></li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unified+framework+for+clustering+and+quantitative+analysis+of+white+matter+fiber+tracts.">A unified framework for clustering and quantitative analysis of white matter fiber tracts.</a> Med Image Anal. 12 (2), 191-202 (2008).</li><li>Vepari, C., Kaplan, 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=Silk+as+a+biomaterial.">Silk as a biomaterial.</a> Prog Polym Sci. 32 (8-9), 8-9 (2007).</li><li>Yao, D., 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=Salt-leached+silk+scaffolds+with+tunable+mechanical+properties.">Salt-leached silk scaffolds with tunable mechanical properties.</a> Biomacromolecules. 13 (11), 3723-3729 (2012).</li><li>Kim, U. J., Park, J., Kim, H. J., Wada, M., Kaplan, 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=Three-dimensional+aqueous-derived+biomaterial+scaffolds+from+silk+fibroin.">Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin.</a> Biomaterials. 26 (15), 2775-2785 (2005).</li><li>Gil, E. S., Mandal, B. B., Park, S. H., Marchant, J. K., Omentetto, F. G., Kaplan, 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=Helicoidal+multi-lamellar+features+of+RGD-functionalized+silk+biomaterials+for+corneal+tissue+engineering.">Helicoidal multi-lamellar features of RGD-functionalized silk biomaterials for corneal tissue engineering.</a> Biomaterials. 31 (34), 8953-8963 (2010).</li><li>Hopkins, A. 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=Silk+hydrogels+as+soft+substrates+for+neural+tissue+engineering.">Silk hydrogels as soft substrates for neural tissue engineering.</a> Adv Funct Mater. 23 (41), 5140-5149 (2013).</li><li>Pacifici, M., Peruzzi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+embryonic+neural+cells:+A+quick+protocol.">Isolation and culture of rat embryonic neural cells: A quick protocol.</a> Vis. Exp. (63), e3965 (2012).</li></ol>

このビデオ記事の出版は、コーニングが主催しています。

Here we described the guidelines to assemble a compartmentalized 3D brain-like tissue model. The model is characterized by dense polarized culture of neurons resulting in the development of two morphologically different compartments: one containing densely packed cell bodies, second containing pure axonal networks. Overall, the scaffold architecture and growth pattern mimic brain cortical tissue including the six laminar layers and white matter tracts21.
The donut-shaped silk protein-based tissue model allows for modifications and tuning of mechanical properties, versatile structural forms, hydrogel fillers and cellular components. Thus, this tissue model establishes a base platform for a wide range of studies. Silk is a favorable candidate for biomaterial platforms due to its biocompatibility, aqueous-based processing, and robust mechanical and degradation properties22. The silk matrix also serves as a stable anchor to reduce collagen gel contraction over time in culture. The properties such as silk concentration and porosity of the scaffold used in this model have been previously adjusted to achieve optimal cell growth and mechanical properties resulting in brain-like tissue elasticity18,23,24. We suggest to keep these parameters constant to ensure the successful outcome and reproducibility of the experiments.
The 3D neuronal network formation was achieved by combining two types of biomaterials with different mechanical properties: a stiff scaffold to provide neuronal anchoring and a softer gel matrix to permit axon penetration and connectivity in 3D. Selective material preferences of the silk scaffold and soft hydrogel provide the underlying principle for compartmentalizing the neurons from the axonal connections. Due to the inert nature of the silk scaffold, functionalization with poly-D-lysine is required for neuronal attachment. However, other cell adhesion promoting factors can be applied such as RGD25 or fibronectin26. To achieve the 3D network formation the silk scaffold needs to be filled with hydrogel in a timely manner as soon as neurons are attached to the scaffold. Likewise, the collagen matrix filler can be replaced by other hydrogels, thus allowing the study of the influence of 3D extracellular environments on axonal network formation to serve as a platform to evaluate novel hydrogels in terms of neuronal compatibility. Additionally, apart from rat cortical neurons other neuronal sources such as hippocampal neurons or induced pluripotent cell (iPSc)-derived neurons can be utilized. Moreover, the tissue model can be used to study heterocellular interactions by including glial cells along with neurons and to build more complex brain-like tissue.
As shown previously, our model can be utilized to evaluate neuronal functionality in 3D microenvironment with a variety of assays such as cell viability, gene expression, LC-MS/MS and electrophysiology, thus demonstrating physiological relevance of the model18. Other methods, which are frequently utilized to evaluate 3D tissue-engineered constructs, such as immunostaining and microscopy8,9,11 can also be used to assess cell distribution and extent of axonal network formation. However, it has to be noted, that due to the high density of the collagen matrix, the penetration of antibodies and depth of the imaging is limited to few hundred micrometers. Moreover, the signal to noise ratio may be affected by nonspecific background fluorescence. This can be overcome by using lipophilic cell tracers and genetically expressed fluorescent proteins which diminish the need for immunostaining11. Alternatively, the imaging can be performed with 2-photon microscopy instead of usual one-photon technique, which may reduce the signal loss, photobleaching, and can be extended to several hundred micrometers of depth.
Summarizing, the silk and collagen-based brain-like tissue offers a robust platform to study neuronal networks in 3D and is a starting point for the development of more advanced models of neurological disorders in the future. Independent from the mode of evaluation, the relative simplicity of this tissue model supports its utility, success and reproducibility.

中枢神経系（CNS）は、血管構造、機能性、感染性または変性を伴う種々の障害の影響を受けることができます。推定680万の人々が世界中で成長している社会経済的負担1を表し、神経学的障害の結果として毎年死にます。しかし、障害のほんの数は、利用可能な治療法があります。したがって、神経障害に罹患している患者のための革新的な治療戦略のための重要な必要性が存在します。残念ながら、多くのCNS標的治療による生理学的に関連する機能の読み出しと急性および慢性影響の評価を可能にしない不十分な前臨床研究モデルの利用、一部には、臨床試験中に失敗。 過去数十年にわたる重要な研究努力にもかかわらず、CNSの構造と機能についての未知の膨大な量があります。より多くの知識を得るために、動物モデルは、頻繁に私達でありますEDは、特に前臨床試験において、例えば、外傷性脳損傷（TBI）、または認知症のような病理学的状態をモデル化します。しかし、動物は、遺伝子発現および代謝2-4、CNSの解剖学だけでなく、機能の両方で人間とは大きく異なります。一方、2D インビトロ培養は、細胞生物学を研究するための一般的な方法であり、日常的に薬物発見のために使用されます。しかし、二次元細胞培養物は、人間の脳5-7と比較して複雑さおよび生理学的関連性を欠いています。動物モデルにより提供される2次元細胞培養研究や複雑さの低コストと単純に代わるはありませんが、3D組織工学は 、in vitroおよびin vivoのアプローチで 、2Dの間に存在するギャップを埋めるために改良された研究のモデルを生成することができます。 3D組織工学は、生体材料足場が提供する3D細胞 - 細胞相互作用および細胞外の合図によって達成より生理学的に関連する実験条件を提供します。 Desp3D培養物の値の背後にある重要な証拠をITEは、このような幹細胞由来オルガノイド文化8-10としてわずか数3D CNS組織モデルは、現在ありますが、11を neurospheroidsヒドロゲル培養12,13を分散させました。多層リソグラフィー14、および3D印刷15を含む高度な技術の方法は、肺、肝臓、及び腎臓の組織を研究するために利用されています。しかし、そのような皮質アーキテクチャと生物学を模倣するように区画ニューロンの成長を可能にする3D CNSモデルの欠如があります。神経細胞体から神経突起の分離された成長は、以前に軸索道のトレース、カルシウム流入、ネットワークアーキテクチャや活動の研究を可能にする微細加工16,17を使用することにより、2D培養で実証されています。この考えは、細胞体と軸索路は、脳18の階層化アーキテクチャを模倣異なる区画に配置されている3D偏神経組織を開発するために私たちに影響を与えました</sup&gt;。我々のアプローチは、限られた容量で高密度の細胞を収容し、コラーゲンゲルに密な軸索ネットワークの伸長を可能にするユニークなシルク足場の設計の使用に基づいています。ここでは、足場の製造および神経文化などの脳のような組織の完全な組み立て手順を示しています。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Na2CO3</td>
			<td>Sigma-Aldrich</td>
			<td>223530</td>
			<td>-</td>
		</tr>
		<tr>
			<td>LiBr</td>
			<td>Sigma-Aldrich</td>
			<td>213225</td>
			<td>-</td>
		</tr>
		<tr>
			<td>MWCO 3500 dialysis cassettes&#160;</td>
			<td>Thermo Fisher</td>
			<td>66110</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Heating plate</td>
			<td>Fisher Scientific</td>
			<td>Isotemp</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Centrifuge 5804 R</td>
			<td>Eppendorf</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sieve</td>
			<td>Fisher Scientific</td>
			<td>-</td>
			<td>No. 270, No. 35, No. 30</td>
		</tr>
		<tr>
			<td>PTFE mold</td>
			<td>-</td>
			<td>-</td>
			<td>made in house (Figure 1) 10 cm diameter, 2 cm height</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71382</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biopsy Punch 5mm</td>
			<td>World Precision Instrument</td>
			<td>501909</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biopsy Punch 2mm</td>
			<td>World Precision Instrument</td>
			<td>501908</td>
			<td>-</td>
		</tr>
		<tr>
			<td>poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407-5MG</td>
			<td>final concentration 10 ug/ml, dissolved in water</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D1283-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T4049-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td>final concentration 0.3%</td>
		</tr>
		<tr>
			<td>Soybean protein</td>
			<td>Sigma-Aldrich</td>
			<td>T6522-100MG</td>
			<td>final concentration 1 mg/ml</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>warm up to 37&#176;C before use</td>
		</tr>
		<tr>
			<td>B27 supplement 50x</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Penicilin Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Collagen I, rat tail, 100 mg</td>
			<td>Corning</td>
			<td>354236</td>
			<td>final concentration 3 mg/ml</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td>corrosive</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Sigma-Aldrich</td>
			<td>P4170-10MG</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>Fluorescein Diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378-5G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Olympus MVX10</td>
			<td>Olympus</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>toxic, final concentration 4%</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-10G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>anti-&#946;IIITubulin antibody&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T2200</td>
			<td>rabbit 1:500</td>
		</tr>
		<tr>
			<td>anti-rabbit Alexa-546</td>
			<td>Molecular Probes</td>
			<td>A11010</td>
			<td>goat 1:250</td>
		</tr>
		<tr>
			<td>goat serum</td>
			<td>Sigma-Aldrich</td>
			<td>G9023-5ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Leica SP2 confocal microscope</td>
			<td>Leica</td>
			<td>-</td>
			<td>objective 20x</td>
		</tr>
		<tr>
			<td>QIAshredder</td>
			<td>Qiagen</td>
			<td>79654</td>
			<td>-</td>
		</tr>
		<tr>
			<td>AllPrep DNA/RNA/Protein Mini Kit</td>
			<td>Qiagen</td>
			<td>80004</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ethyl alcohol, Pure</td>
			<td>Sigma-Aldrich</td>
			<td>E7023&#160;</td>
			<td>-</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>63689</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000c/2000 UV-Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BCA protein assay</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Plate reader Spectramax M2</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td>absorbance 562 nm</td>
		</tr>
		<tr>
			<td>Micro Scissors, Economy, Vannas-type</td>
			<td>TedPella</td>
			<td>1346</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

engineered 3d silk-collagen-based model of polarized neural tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

ドーナツ状にソリッドシルクスポンジのプレカットは、神経組織（ 図2A）に似た区画化アーキテクチャを実現するために、ユニークでシンプルなアイデアを表しています。小容積内の高細胞密度のシーディング及び成長を可能にする非常に高い表面積（2×10 7 / mlで）（ 図2B）で約500μmの結果の細孔径を有する足場の高度に多孔性の構造。さらに、足場の高気孔率は、延長時間枠を超える高密度の細胞培養物の優れた生存性が得られ栄養素や老廃物の無制限の拡散を可能にします。神経細胞を播種時に迅速かつ均一に足場孔の表面に付着します。 細胞接着が完了し、細胞が絹基板上に平衡化された後、スポンジ足場は、軸索ネットワーク形成（ 図3では 、3D環境を提供するために、柔らかいコラーゲンマトリックスが充填されています</strong&gt;）。ニューロンが唯一の2次元面（ 図3B）で見られるような混合ネットワークでは、得られた細孔の表面に拡張を成長するにつれてコラーゲンマトリックスが存在しない場合には、軸索および細胞体の区画化することはできません。これとは対照的に、コラーゲンゲルは、神経細胞体はシルク足場（ 図3C）に付着したままながら、3Dで大規模な軸索 ​​伸長をサポートしている網目構造を提供します。この成長パターンは、細胞体および純粋な軸索 ​​ネットワーク、脳内の皮質の灰白質と白質に似ている（ 図3D）の区画につながります。 顕微鏡から離れて、構築物は、実験の背後にある問題に応じて、他の方法18の様々な評価することができます。例えば、構築物からのDNA、RNAおよびタンパク質の単離を容易に市販のキット（ 図4）を用いて行うことができます。核酸およびタンパク質PEの収率R構築物は、最初は絹の足場に播種した細胞の数によって異なります。より多くの細胞を播種したDNA、RNAおよびタンパク質の収率が高いです。 <img alt="図1" src="/files/ftp_upload/52970/52970fig1.jpg" /> シルクスポンジ足場調製のために使用される図1のPTFE金型寸法：直径10cm、2cmの高さ<a href="https://www.jove.com/files/ftp_upload/52970/52970fig1large.jpg" target="_blank">、この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/52970/52970fig2.jpg" /> 図2. 3D脳のような組織モデル。（A）は、多孔質シルクスポンジ足場。前コラーゲン（緑生細胞、赤死細胞）を埋め込 ​​むシルク足場に播種時の1日目でのニューロンの（B）生/死染色。右側のパネルは、MAを表示赤いフレームでキャプチャエリアのgnification。 <a href="https://www.jove.com/files/ftp_upload/52970/52970fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/52970/52970fig3.jpg" /> 図3.神経3D脳のような組織モデルで区画さ伸長（A）足場コンパートメント：赤細胞体コンパートメント、青軸索コンパートメント。 （B）コラーゲン埋め込 ​​み前に播種時の1日目に神経成長パターン。 （C）。細胞体コンパートメントに播種時の7日目の神経突起伸長。 （D）軸索の区画に播種時の7日目の神経突起伸長。 βIIITubulin -グリーン。 <a href="https://www.jove.com/files/ftp_upload/52970/52970fig3large.jpg" target="_blank">これの拡大版を表示するには、こちらをクリックしてください</a>図。 <img alt="図4" src="/files/ftp_upload/52970/52970fig4.jpg" /> （A）RNAおよびDNA、および足場ごとに播種された細胞の量に関連した構造物あたり（B）は、タンパク質の図4の期待利回り（±SD、n = 5）であった。 <a href="https://www.jove.com/files/ftp_upload/52970/52970fig4large.jpg" target="_blank">この図の拡大版を表示するには、こちらをクリックしてください。</a>

Watch this Scientific Journal Video about Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue at JoVE.com

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

偏神経組織のエンジニア3Dシルクコラーゲンベースモデル

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

engineered-3d-silk-collagen-based-model-of-polarized-neural-tissue

Research

JoVE Journal

Bioengineering

12.4K ビュー.  Tufts University. Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

ビデオ: Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

2Dと3D環境での神経前駆細胞の電界制御監督移行

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

生物工学

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

トライリーフレットポリマーバルブの相対的な流体力学的評価のためのプロトコル

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

人間の3次元の組織工学<em&gt; in vitroで</em&gt;腫瘍テストシステム

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

マウスモデルにおける組織工学血管の生成とグラフト

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

生産、特性評価および3D組織工学ヒト食道粘膜モデルの使用可能性

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

操作された複合三次元組織<em&gt;インビトロ</em&gt; /<em&gt;インシリコ</em具体的な突然変異背景に薬物の有効性を予測するための&gt;肺腫瘍モデル

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

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 ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

シードとマウス モデルにおける合成組織設計気管移植片の移植

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

ラットにおける円周食道再建のための組織工学型移植片

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

ニューラル様組織の工学的なマウス皮質アストロサイトの3Dバイオプリンティング

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...