著者は、独立賞 (K99CA201603) への国民の健康道機関国立がん研究所をご了承ください。

このプロトコルで使われる新生児ラット心筋細胞は次の機関動物ケアおよび Brigham および女性の病院の使用委員会によって承認された確立された手順56 2 日古い Sprague-dawley ラットから分離された.
1、Bioprinter の計測
<ol>
	<li>デュアルレイヤー、同心円状のマイクロ ヘッド; を構築するシースとして (例えば、18 G、1/2 インチ) 大きい鈍針の中心にコアとして (例えば、27 G、1 インチ) より小さい鈍針を挿入します。コア針が少し出ていることを確認してください (~ 1 mm) 外側シェル (図 1A) よりも長い。通常、先端で同心円配置を支援するためにバレルの側面内側/外側の針の間に適切なサイズのスペーサーを必要ことができます一時的挟ま場合が手動で、配置が調整されます。エポキシ接着剤でバレルの接合部をシールし、該当する場合の先端側からアライメント スペーサーを削除します。</li>
	<li>逆方向の中央の針のバレルに別の針 (23 G) を挿入します。 外側の針のバレルの側面にある穴を生成し、穴に、エポキシ接着剤でシールの順にサイズの一致の金属製のコネクタを挿入します。</li>
	<li>2 つの塩ビ管を bioink 及び架橋液の注入のデュアル チャネル シリンジ ポンプに印字ヘッドの入り江を個別に接続します。Poly(methyl methacrylate) は、プラスチック製のホルダーを使用して bioprinter の頭の上に押出機をマウントします。 注: Bioprinter 選択は、可用性に依存します。ここで私達は正常にいくつかの市販の bioprinters にこのセットアップをテストしました。ただし、x ・ y ・ z 電動ステージを備えて bioprinter 必要があります、原則として、有効にこのマイクロ プリント ヘッドの統合。</li>
</ol>
2. バイオプリンティングを用いた血管床
<ol>
	<li>アルギン酸 (4 w/v%、低粘度)、ゼラチン (ゲルマ、1-2 w/v%) メタクリロイル57,58,:dic Irgacure 2959 の混合物を使用して bioink を作る (0.2 - 0.5 wt.%) 25 mM 2-[4-(2-hydroxyethyl) piperazin-1-イルに溶解] エタン スルホン酸 (HEPES 緩衝液 pH 7.4) 含む 10 vol.% ウシ胎児血清 (FBS)。</li>
	<li>10 vol.% 架橋キャリア流体として FBS を含む HEPES バッファーで 0.3 M CaCl2溶液を作ります。</li>
	<li>バイオプリンティングの直前に 5-10 分の治療を 0.05 w/v% トリプシンによるフラスコからひと臍帯静脈血管内皮細胞 (Huvec) を切り離して、5-10 × 106セル/mL の濃度で bioink で細胞を再懸濁します。均一な分布を確実にゆっくりと 5 から 10 倍の懸濁液をピペットします。</li>
	<li>5 μ L/mL の同じ流量でデュアル チャネルのシリンジ ポンプを使用して bioink/架橋液の注入を開始します。フローは、彼らが安定するまで最大 1 分間継続的に実行する許可されます。その後、約 4 mm/s (図 1B) の堆積速度で bioprinter を制御することで印字ヘッドの移動を開始します。これらの速度は微調整が必要な場合があります最適バイオプリンティングを確保するため各新しいセットアップとチューニングします。バイオプリンティング プロセスは通常室温 (21-25 ° C) で行なわれるが、この温度を変更可能性があります。アルギン酸コンポーネントの高速イオン性ゲル化とを用いた足場 (図 1B) の沈着バイオプリンティング処理できるようにします。</li>
	<li>足場は、bioprinted が、した後は 20-30 s (図 1C) さらに光架橋約 5-10 mW/cm2の紫外線 (360-480 nm) で、ゲルマ成分による化学ゲル化を達成するため。</li>
	<li>バイオプリンティング架橋続いて、リン酸緩衝生理食塩水 (PBS) 過剰 CaCl2を削除すると足場を軽くすすいでください。培養内皮細胞成長培地 (EGM) まで 16 日間の 37 ° C および 5 vol.% CO2でインキュベーターで媒体で Huvec 満載を用いた足場少なくとも 2 日ごとに変更。培養期間中に顕微鏡下で比較の形態を監視します。</li>
</ol>
3. 血管柄付き組織を構築します。
<ol>
	<li>比較は、ルーメンのような構造 (図 1D) を形成する足場に超極細の周辺に移行して、一度足場を取得し、優しく (例えばpolymethylsiloxane [PDMS] のスラブ) 疎水性表面上に配置します。フィルター滅菌の紙切れを使用して、毛管力と足場の間質性スペースからすべてのメディアを慎重に取り外します。</li>
	<li>すぐに培地での足場は、足場 (図 1E) の間質性スペース全体に潜入する必要があります上に 1-10 × 106セル/ml の密度で二次電池の種類 (例えば、心筋細胞) の懸濁液の滴 (約 20-40 μ L) を追加します。0.5 - 個々 の超極細に付着する細胞を許可するように 2 h のためのインキュベーター (37 ° C, 5 vol.% CO2、相対湿度 95%) でこのような構成を孵化させなさい。顕著な蒸発が観測されないように期間液滴サイズを監視します。</li>
	<li>目的の血管柄付きの組織が形成されるまで関連媒体の非付着性のセルおよび文化構成を削除する PBS 風呂で揺することによって足場は水洗い。</li>
</ol>

著者は、彼らは競合する金銭的な利益があることを宣言します。

コーアクシャルのプリント ヘッドの構造は、コアと鞘から架橋剤からの両方の bioink の同時配信を可能にするのに成功したマイクロ バイオプリンティングに向けての重要なステップを表します。このプロトコルでは例のプリント ヘッドはシェルとコアと 18 G 針 27 G 針を使用して作成された中、は、さまざまな針のサイズが異なるを使用して組み合わせを容易に拡張可能性があります。ただ?...

組織エンジニア リング目標生成機能組織代用置換、復元、またはそれらの怪我や病気、人体1,2,3,4、生体材料7,8,9,10生理活性分子5,6、目的のセルの種類の組み合わせを頻繁に拡張するために使用できます。最近では、モデルを生成する体外組織と器官用医薬品の開発、従来の単純化しすぎられる平面内細胞文化11,12,13,14,15,16,17,18,19の交換など、生体内で同等の重要な機能を模倣した組織工学技術がますます採用されても。両方の状況で複雑なマイクロアーキテクチャーと人間の組織の階層構造を要約する能力が重要なエンジニア リング組織10の機能を有効にして特に、エンジニア リング組織に血管のネットワークを統合する方法は、需要が血管新生でフィールド20,21,22,23の最大の課題の 1 つは、ため。
日には、様々 なアプローチはさまざまな程度の成功8のエンジニア リングの組織構造に血管の構造を構築する試みにこの点で開発しました。たとえば、自己血管内皮細胞のことができます血管ネットワーク24; の世代のため血管増殖因子の配信2625、持続的な血管新生を誘導します。血管系前駆細胞を使用し、ペリサイト容易に血管内皮細胞の増殖およびアセンブリ24,27;足場・ プロパティの設計により、新生28,29; の正確な変調また、細胞シート テクノロジーにより、血管層30の便利な操作のため。それにもかかわらず、これらの戦略は、設計された組織構造およびこうして限られた再現性内で血管がランダムに分布につながる血管の空間パターン形成を制御する能力を授けるないです。過去数年間バイオプリンティングは、自動または半自動の方法31,32,33高再現性と再現性で複雑な組織パターンの彼らの比類のない汎用性のため、このような課題の解決に向けた技術を有効にするクラスとして浮上しています。いけにえバイオプリンティング34,35,36,37,38、埋め込みバイオプリンティング39,40,41, と中空構造バイオプリンティング/立体組織自動製造42,43,44,45,46,47,48,49,50,51,52,53が血管や血管柄付き組織を生成する可能性が説明されています。
またを用いた足場を作製するマイクロ流体バイオプリンティング戦略が最近開発されている、ハイブリッド bioink から成るアルギン酸、ゼラチン メタクリロイル基 (ゲルマ) 同心印字ヘッドと塩化カルシウム (CaCl2) ソリューションのコアを介して配信されたプリント ヘッド54,55の外側シースの流れを行った。後続の光架橋多層膜を用いた足場の永続的な安定を確保しながらマイクロファイバー形成を有効にするアルギン酸コンポーネントの即時の物理的な架橋の許可 2 つのフローの共押出。注記のうち、bioprinted 超極細内にカプセル化血管内皮細胞が増殖し、血管床54,55を真似たルーメンのような構造を仮定してマイクロファイバーの方に移住する見つかりました。これらの bioprinted endothelialized 血管ベッドその後を表示でしたさらに55血管柄付き組織を構築する二次電池タイプを希望します。このプロトコルは、したがって組織エンジニア リングと organoid モデリングへの応用可能性の血管組織の便利な加工を保証する同心円状ノズルの設計で有効にするようなマイクロ流体バイオプリンティングの戦略の詳細な手順を提供します。

<table width="1270">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="width:467px;height:21px;">Alginic acid sodium salt from brown algae</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:163px;">A0682</td>
			<td style="width:480px;">BioReagent, plant cell culture tested, low viscosity, powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:467px;height:21px;">Gelatin type A from porcine skin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:163px;">G2500</td>
			<td>Gel strength 300</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:467px;height:42px;">Irgacure 2959 (2-Hydroxy-4&#39;-(2-hydroxyethoxy)-2-methylpropiophenone)</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:163px;">410896</td>
			<td>98%</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:467px;height:21px;">HEPES buffer</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:163px;">H0887</td>
			<td>1 M, pH 7.0-7.6, sterile-filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:467px;height:42px;">Fetal bovine serum&#160;</td>
			<td style="width:160px;">Thermo Fisher Scientific</td>
			<td style="width:163px;">10438026</td>
			<td>Qualified, heat-inactivated, USDA-approved regions</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:467px;height:21px;">Calcium chloride dihydrate</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:163px;">C5080</td>
			<td>BioXtra, &#8805;99.0%</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:467px;height:42px;">Phosphate buffered saline</td>
			<td style="width:160px;">Thermo Fisher Scientific</td>
			<td style="width:163px;">10010023</td>
			<td>pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:467px;height:21px;">Human umbilical vein endothelial cells</td>
			<td style="width:160px;">Angio-Proteomie</td>
			<td style="width:163px;">cAP-0001</td>
			<td>Human Umbilical Vein Endothelial Cells (HUVECs)</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:467px;height:21px;">GFP-expressing human umbilical vein endothelial cells</td>
			<td style="width:160px;">Angio-Proteomie</td>
			<td style="width:163px;">cAP-0001GFP</td>
			<td>GFP-Expressing Human Umbilical Vein Endothelial Cells (GFPHUVECs)</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:467px;height:21px;">Endothelial cell growth medium</td>
			<td style="width:160px;">Lonza</td>
			<td style="width:163px;">CC-3162</td>
			<td>EGM-2 BulletKit</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:467px;height:42px;">Dulbecco&#8217;s Modified Eagle Medium&#160;</td>
			<td style="width:160px;">Thermo Fisher Scientific</td>
			<td style="width:163px;">12430054</td>
			<td>High glucose, HEPES</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:467px;height:42px;">Sylgard 184 silicone elastomer kit</td>
			<td style="width:160px;">Ellsworth Adhesives</td>
			<td style="width:163px;">184 SIL ELAST KIT 0.5KG</td>
			<td>Clear 0.5 kg Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:467px;height:21px;">UV curing lamp system</td>
			<td style="width:160px;">Excelitas Technologies</td>
			<td style="width:163px;">OmniCure S2000</td>
			<td>Spot UV Light Curing System with Intelligent UV Sensor</td>
		</tr>
	</tbody>
</table>

microfluidic bioprinting for engineering vascularized tissues and organoids

血管の組織工学を構築し、organoids は歴史的に挑戦されています。ここで多層ヒドロゲル超極細をインター レースで足場を生成するマイクロ流体バイオプリンティングに基づく手法について述べる.滑らかな達成するためにバイオプリンティング、コアの流れとシース流によって運ばれる架橋ソリューションから押出複合 bioink 製剤を含む芯鞘マイクロ プリント ヘッドはように設計され、bioprinter の上に装着。アルギン酸ゼラチン メタクリロイル (ゲルマ) をブレンドすることにより多糖の存在下で瞬時のイオン架橋を受ける選択恒久的な安定化を達成するためにゲルマ成分の二次光架橋続いて二価のイオン、このバイオプリンティング方式を用いた足場を得ることができた。重要なは、血管内皮細胞の bioprinted 超極細内部にカプセルは 16 日文化にわたって血管系に似たルーメンのような構造を形成できます。Endothelialized を用いた足場はさらに、超極細の間質性スペースに二次電池型の後播種によって血管柄付き組織を構築する血管床として使用することがあります。マイクロ流体バイオプリンティング高忠実度で血管組織の便利な工学の一般的な戦略を提供します。

マイクロ流体バイオプリンティング戦略を用いた足場低粘度 bioinks54,55を使用しての直接押出バイオプリンティングことができます。6 × 6 × 6 mm のサイズを図 2A足場に示すように3を含む &#62; 超極細 30 層が 10 分以内の bioprinted をあることができます。CaCl2アルギン酸コンポ?...

Watch this Scientific Journal Video about Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids at JoVE.com

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

我々 は、二次電池の種類がさらに血管柄付き組織とオルガノイドを生成するには、このを用いた構造の間質性スペースに播種できるを用いた血管床をエンジニア リングのためのマイクロ流体バイオプリンティング戦略に基づく一般化されたプロトコルを提供します。

マイクロ流体工学バイオプリンティング血管組織や Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

microfluidic-bioprinting-for-engineering-vascularized-tissues

Research

JoVE Journal

Bioengineering

15.6K ビュー.  Harvard Medical School. 我々 は、二次電池の種類がさらに血管柄付き組織とオルガノイドを生成するには、このを用いた構造の間質性スペースに播種できるを用いた血管床をエンジニア リングのためのマイクロ流体バイオプリンティング戦略に基づく一般化されたプロトコルを提供します。

ビデオ: Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

タンパク質の折り畳みを研究するためのマイクロ流体ミキサー

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

生物工学

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

あらかじめ設計されたサイズと形状を用いるポリマー繊維およびバイオハイブリッド繊維のマイクロ流体製造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

マウス胚性幹細胞からの神経組織エンジニアリングのためのポリエレクトロ繊維状足場（グリセロール - ドデカン）

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

細胞外マトリックス内に埋め込まれたエンジニアリング三次元上皮組織

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

干渉のないマイクロ/ナノ粒子の細胞工学のためのマイクロ流体緩衝液交換

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

細胞の表面のガイダンスとBioprintingための自動ロボット調剤技術

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

血管新生および血管組織工学を研究するための小さな動物モデルにおける動静脈（AV）ループ

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

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 ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

組織工学用植物組織の Decellularization の 2 つの方法

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

新血管新生初期事象を模倣する微発性モデル

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...