著者らは、心臓血管研究のための基金基金とメリーランド幹細胞研究基金（2016-MSCRFI-2735）という資金源を認めています。

1.心筋細胞の調製<ol><li>記載されているように、基底膜マトリックスでコーティングされた6ウェルプレート上で、ヒト誘導性多能性幹細胞（hiPSC）を生成および培養する。10 。 </li><li>以前に記載された方法11,12を用いてhiPSCをhiPSC由来心筋細胞（hiPSC-CM）に分化させる。 </li><li>分化後19日目に、室温で5分間、各ウェルで2mLのトリプシン/ EDTA 0.05％を用いて心筋細胞を単離する。 </li><li>心筋細胞を光学顕微鏡下でモニターし、細胞の解離を観察する。 </li><li> 2mLのトリプシンインヒビター0.0125％を用いてトリプシンを中和する。 </li><li>単離された心筋細胞をプールし、モーター付きピペットフィラーを使用して1つの50mLコニカルチューブに移す。 </li><li>室温で250xgで5分間細胞懸濁液を遠心分離してペレットを得る。 </li><li>ペレットをロズウェルパーク5mLに再懸濁するB-27（RPMI / B-27細胞培地）を補充したMemorial Institute（RPMI）細胞培地。 </li><li>細胞培地に20μLの細胞をピペットで入れ、同量の0.4％Trypan Blue溶液で染色します。 </li><li>自動細胞計数器または手動血球計算器を使用して、細胞懸濁液の濃度および細胞生存率を数え、得る。 </li></ol>繊維芽細胞の調製<ol><li>ヒト心臓線維芽細胞（HCF）（成人心室型）細胞系を開始する。 </li><li>継代し、HCF培養プロトコールに従ってそれらを単離する。 </li><li>細胞培地中の20μLの細胞をピペットで等量のTrypan Blue溶液0.4％で染色する。 </li><li>新しい細胞懸濁液の濃度および細胞生存率を数え、得るために、自動細胞カウンターまたは手動血球計算機を使用する。 </li></ol> 3.内皮細胞の調製<ol><li>人間のumbを開始する記載されているように、静脈内皮細胞（HUVEC）系統14 。 HUVEC培養プロトコールに従い、それらを単離する（ 14） 。 </li><li>細胞培地に20μLの細胞をピペットで入れ、同量の0.4％Trypan Blue溶液で染色します。 </li><li>自動細胞計数器または手動血球計算器を使用して、細胞懸濁液の濃度および細胞生存率を数え、得る。 </li></ol> 4.共培養： <ol><li> RPMI / B-27細胞培地中で3種類の細胞型（hiPSC-CM、HCF、およびHUVEC）を混合して、hiPSC-CM：HCF：HUVEC細胞比50mLコニカルチューブに混合細胞溶液のストックを生成する。 70:15:15およびmL当たり165,000細胞の濃度。 </li><li>混合細胞溶液を、マルチチャネルピペットを使用してウェル当たり200μLまたは1ウェルあたり33,000細胞の超低付着96ウェルU底プレートに分配する。 </li><li> 96ウェルプレートを3日間インキュベートする（37℃、5％の炭素二酸化炭素湿度95％）。 </li><li>光学顕微鏡下で個々のウェルの中央と底に混合細胞多細胞スフェロイドの存在を検査する。 注：2次元の顕微鏡投影では、これらの回転楕円体は円形に見えます （ 図1 ）。スフェロイドは、細胞播種後、24時間以内に極低温付着96ウェルプレートに形成されるべきである。スフェロイドは96ウェルプレートに細胞を播種した後、48時間以内に鼓動を開始するはずです。鼓動していないスフェロイドは、最終的な3Dバイオプリントされた心臓パッチが機能することを保証するために3Dバイオプリントに使用すべきではありません。コネキシン43（Cx43）およびトロポニンのスフェロイド免疫蛍光法を用いて、球状細胞の組成と品質を評価することができます15 。 </li></ol> 5.足場のない心臓組織の3Dバイオプリント<ol><li>スフェロイドを含むプレートを、足場のない回転楕円体ベースの3Dバイオプリンタのマガジンにロードします。 </li><li> 3Dバイオプリンタの電源を入れ、3Dバイオプリントソフトウェアを実行します。 <ol><li> 「補助」、「操作準備」、「検査モード」、「原点復帰」の順にクリックします。 </li><li> 「スタート」の下にある緑色のアイコンをクリックして回転楕円体検査を開始します。 </li><li>画面上の &quot;（2）Dia（μm）&quot;欄の下に回転楕円体の直径が表示されていることを確認します。 </li></ol></li><li>平均回転楕円体の直径を100μmに近づけるには、「初期設定」をクリックし、平均回転楕円体直径をStack Zピッチ（μm）に入力します。 </li><li> 「合計」をクリックすると、「レイヤー」の下の数字が自動的に再調整されます。 [適用]をクリックして設定を確定します。 </li><li> 「ニードルアレイマップ」をクリックし、「レイヤー番号」に移動して、3Dバイオプリント用に必要なレイヤーを選択し、個々の回転楕円体の位置を選択して、画面の左側のマップに目的の3Dバイオプリントデザインを描画します。 Click「適用」をクリックして3Dバイオプリントデザインを確認します。 </li><li> 「ニードルアレイチェック」をクリックしてニードルアレイ検査を開始します。ニードルアレイにピントが合っていない場合は、0〜500μmの範囲の値を &quot;OffsetZ（μm）&quot;に入力して、500μmから100μmまで減少するようにカメラのフォーカスを調整します。 </li><li> 「検査パラメータ」、「タイプ1」をクリックし、「直径（μm）」を希望の直径範囲に設定します。 「真円度（％）」と「平滑度（％）」を希望のパーセンテージに設定します。 [適用]をクリックして設定を確定します。 注：ここで回転楕円体の直径は450μm〜550μmに設定されています。 「真円度（％）」および「滑らかさ（％）」は、個々の回転楕円体のコンピュータ計算による評価です。ここでは、「真円度（％）」は60％、「平滑度（％）」は70％です。 </li><li> 「存在」をクリックし、「直径（μm）」を同じ設定に更新します前の手順では、 [適用]をクリックして設定を確定します。 </li><li> 「Stack Mode」をクリックし、「Start」の下にある緑色のアイコンをクリックして、3Dバイオプリンタのノズルを真空吸引し、針アレイの特定の位置にスフェロイドを配置して、回転楕円体のピックアップを開始します。 注：81個の回転楕円体（9個の回転楕円体と9個の回転楕円体）からなる心臓パッチの場合、3Dバイオプリントには約20〜30分かかります。 </li><li>バイオプリント後、滅菌ピンセットを使用して、3Dバイオプリントパッチを含む針アレイをピックアップし、150mLのRPMI / B-27細胞培地を含む250mL滅菌ビーカーに移す。 </li><li> 3日間（37℃、5％二酸化炭素、95％湿度）、3Dバイオプリントパッチを針アレイとインキュベートする。 </li></ol> 6.ニードルアレイからの3D Bioprintedパッチの除去とパッチ成熟<ol><li> 3日間のインキュベーションの後、一方の手は針アレイの基部に、他方のものはプラスチックカバー上のt貼付し、プラスチック製のカバーを静かにスライドさせて、3Dバイオプリントパッチをニードルアレイから外す。 注意：リン酸緩衝生理食塩水（PBS）による潤滑は、必要に応じて、このステップの直前のニードルに適用して、スムーズで簡単に取り外すことができます。 </li><li>一対の無菌鉗子を使用して、その上に3Dバイオプリントパッチを入れたプラスチックカバーをピックアップし、パッチとプラスチックカバーをRPMI / B-27細胞培地を含む35mmディッシュに移す。 </li><li>ピンセットをプラスチックカバーに固定した状態で、RPMI / B-27細胞培地のパッチでプラスチックカバーを静かに振って、パッチを培地に放出させます。 </li><li> 3Dバイオプリントされたパッチが光学的に検査され、3Dバイオプリントされたパッチが機能しているかどうかを確認しているかどうかを視覚化する。 </li><li> 3日以上（37℃、5％二酸化炭素、95％湿度）、35mmディッシュ内の3Dバイオプリントパッチをインキュベートする。 注：3日後、回転楕円体が融合し、針があったパッチはもはや見えなくなるはずです。 </li></ol>

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+progress+in+3D+printing+for+cardiovascular+tissue+engineering.">Current progress in 3D printing for cardiovascular tissue engineering.</a> Biomed Mater. 10, 034002 (2015).</li><li>Chang, C. C., Boland, E. D., Williams, S. K., Hoying, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-write+Bioprinting+Three-Dimensional+Biohybrid+Systems+for+Future+Regenerative+Therapies.">Direct-write Bioprinting Three-Dimensional Biohybrid Systems for Future Regenerative Therapies.</a> J Biomed Mater Res. Part B, Appl Biomater. 98, 160-170 (2011).</li><li>Lee, J. M., Sing, S. L., Tan, E. Y. S., Yeong, W. 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著者は何も開示することはない。

It is important to use beating, functional spheroids for 3D bioprinting. If spheroids are not beating, continuing to use them will invariably result in a non-functional 3D bioprinted patch.
One benefit of this approach is the ability to manipulate the cell content of the patch by varying the total number of cells and the percentage of cardiomyocytes, endothelial cells, and fibroblasts in the spheroids. This allows for many different types of cardiac patches to be printed, with varying histolog...

3Dバイオプリント1,2,3の方法にはさまざまなものがあります。 3Dバイオプリントは、インクジェットバイオプリンティング、マイクロ押出バイオプリント、レーザーアシストバイオプリント、方法の組み合わせ、またはより新しいアプローチなどの例を用いて、印刷技術1によって分類されることが多い。 3Dバイオプリントは、スキャフォールドフリーまたはスキャフォールド依存法に分類することもできます4 。 3Dバイオプリントのほとんどの方法は、生体材料、 例えばバイオインキ5または足場6が必要な足場依存性である。しかしながら、足場依存性3Dバイオプリンティングは、足場材料の免疫原性、独自のバイオインキのコスト、分解産物の遅い速度および毒性など、多くの問題および限界に直面している4,7。 Scafスフェロイドを用いた無折返し心臓組織工学が試​​みられており、足場依存性組織工学のこれらの欠点を克服する可能性がある。しかし、この論文の著者が認めたように、生物工学の過程で、固定された場所でスフェロイドをしっかりと取り扱い、配置することは困難でした。 3Dバイオプリントと回転楕円体ベースの組織工学の併用は、これらの困難を克服する可能性を秘めています。このプロトコールでは、スフェロイドの形態の細胞のみを用いて、他の生体材料を用いずに心臓組織の3Dバイオプリントを説明する。 足場のない回転楕円体ベースの3Dバイオプリンタ9は、真空吸引を使用して個々の回転楕円体をピックアップし、それらを針アレイ上に配置する能力を有する。 3次元バイオプリンティングにおける針アレイ上のスフェロイドの位置づけの概念は、古代ジャパ（ Japa ）においてニードルアレイ（「 ケンザン （ kenzan ）」として知られている）花の配置、 生け花の nese芸術。このシステムは、スフェロイドを任意の構成に正確に配置することを可能にし、個々のスフェロイドを短時間で融合させて、3Dバイオプリント組織を作製する。したがって、この方法は、スフェロイドを容易に操作することを可能にし、足場を用いない器官のバイオファブリケーションの将来への潜在的な影響をもたらす。

<table border="0" cellpadding="0" cellspacing="0" width="587">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Geltrex</td>
			<td style="width:157px;">Invitrogen&#160;</td>
			<td style="width:88px;">A1413202</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Trypsin/EDTA 0.05%</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">15400054</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Defined Trypsin inhibitor 0.0125%</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">R007100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:176px;">RPMI Cell Media</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:88px;">11875-093</td>
			<td style="width:167px;">RPMI supplemented with B27 constitutes HIPSC-CM culture media</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:176px;">B-27 Supplement</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">17504044</td>
			<td style="width:167px;">RPMI supplemented with B27 constitutes HIPSC-CM culture media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Countess Automated Cell Counter</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:88px;">C10227</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:176px;">Human cardiac fibroblasts (adult ventricular type)</td>
			<td style="width:157px;">Sciencell</td>
			<td style="width:88px;">6310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Human umbilical vein endothelial cells</td>
			<td style="width:157px;">Lonza</td>
			<td style="width:88px;">CC-2935</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:176px;">PrimeSurface ultra-low attachment 96-well U-bottom plates&#160;</td>
			<td style="width:157px;">Akita Sumitomo Bakelite Co.</td>
			<td style="width:88px;">MS-9096UZ</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Regenova Bio 3D Printer</td>
			<td style="width:157px;">Cyfuse Biomedical K.K.</td>
			<td style="width:88px;">N/A</td>
			<td>www.cyfusebio.com/en/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Troponin T Antibody</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">701620</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Connexin 43 (Cx43) Antibody</td>
			<td style="width:157px;">Chemicon</td>
			<td>MAB3068</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">ProLong Gold Antifade Mountant with DAPI</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">P36935</td>
			<td></td>
		</tr>
	</tbody>
</table>

creation of cardiac tissue exhibiting mechanical integration of spheroids using 3d bioprinting

このプロトコルは、細胞のみを使用して、生体材料を使用せずに心臓組織の3Dバイオプリントを記述する。心筋細胞、内皮細胞および線維芽細胞を最初に単離し、計数し、所望の細胞比で混合する。それらは、超低付着96ウェルプレート中の個々のウェルで共培養される。 3日以内にスフェロイドが鼓動する。これらの回転楕円体は、真空吸引を使用してノズルによってピックアップされ、3Dバイオプリンタを使用してニードルアレイ上に組み立てられる。スフェロイドは、次に、針アレイ上で融合することが可能となる。 3次元バイオプリンティングの3日後、スフェロイドは、すでに自発的に鼓動している無傷のパッチとして除去される。 3Dバイオプリントされた心臓パッチは、構成要素スフェロイドの機械的統合を示し、心臓組織の再生および心臓病の3Dモデルとして非常に有望である。

ステップ4.4（共培養）の終わりに、各ウェルの細胞は、超低付着96ウェルU底プレートの底に凝集して、重力によってスフェロイドを形成するはずである。これらのスフェロイドにはhiPSC-CM、HCFs、HUVECが含まれており、光学顕微鏡下で視覚的に検査することができ、2次元投影で円形に見えるはずです（ 図1 ）。ステップ6.3の終わりに、3Dバイオプ?...

Watch this Scientific Journal Video about Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting at JoVE.com

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

このプロトコルは、生体材料を使用せずに心臓組織の3Dバイオプリントを記述する。 3Dバイオプリントされた心臓パッチは、構成要素スフェロイドの機械的統合を示し、心臓組織の再生および心臓病の3Dモデルとして非常に有望である。

3D Bioprintingを用いたスフェロイドの機械的統合を示す心臓組織の創出

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

creation-cardiac-tissue-exhibiting-mechanical-integration-spheroids

Research

JoVE Journal

Bioengineering

9.9K ビュー.  Johns Hopkins Hospital. このプロトコルは、生体材料を使用せずに心臓組織の3Dバイオプリントを記述する。 3Dバイオプリントされた心臓パッチは、構成要素スフェロイドの機械的統合を示し、心臓組織の再生および心臓病の3Dモデルとして非常に有望である。 

ビデオ: Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

細胞ネットワークの作成のためのプラズマのリソグラフィ表面パターニング

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

生物工学

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting細胞化は、組織特異的ヒドロゲルBioinkを使用して構築します

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

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 ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

スキャフォールド フリー三次元心筋組織構築純金型ベース方式

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

ゼラチンメタクリロイルヒドロゲルベースバイオインクの3Dバイオプリンティングのプロトコル

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

均質な細胞分布のための三次元バイオプリンティングにおける多層ヒドロゲルバイオインクの使用

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

内皮ネットワーク上の3D多細胞乳房スフェロイドの直接バイオプリンティング

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

ヒトiPSC由来心筋細胞、心臓線維芽細胞、内皮細胞を用いた3次元心臓ミクロ組織アレイの作製

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

マイクロ流体プラットフォーム内での3D組織化ヒト心臓組織の開発

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

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 ...