The research leading to these results has received funding from the European Research Council under grant agreement ERC-2010-StG-260991.

体積ハンドヘルド光音響プローブを動作させるための詳細な手順については後述する。この手順は、動物およびヒトの実験に関する承認された制度的規制に従って行われる。 1.システムの準備<ol><li>出力光ビームを安定化させるための操作前〜15分のウォームアップ期間のためのレーザーのスイッチを入れる。 </li><li>皮膚（ 図1）と接触している隔離膜で一部を囲む水を置きます。 NOTE：（皮膚接触）分離膜とトランスデューサ（球形プローブの中心）の最大感度を有する領域との間の距離は、有効撮像深さを確立する。 </li><li>ポンプを用いて脱イオン水を用いて単離した膜と変換器の表面との間に約100mlの全容積を満たす。 </li><li>水漏れでも気泡がpresaの複数形されていないことを確認NT。あるいは、水の循環を提供することにより、気泡を避ける。 </li><li>室温で実験を実施し、この温度に維持される結合媒体（水）を確保する。 </li></ol> 2.イメージングの準備<ol><li>人間の撮影準備。 </li><li>画像内の望ましくないバックグラウンドを避けるために、脱毛ローション付きツー·画像化することが部分から毛を削除します（このステップはオプションです）。 </li><li>効果的な音響結合を提供するために、画像化される領域の周りの皮膚に超音波ゲルを適用する。関心領域内の光音響プローブを配置します。気泡が超音波カップリングゲル中に存在しないことを確認してください。 </li><li>動物の撮影準備。 </li><li>動物とのケアと実験手順は、制度や政府の規則や規制と一致していることを確認してください。 </li><li>シェービングローションで画像化される領域での動物の毛皮を削除します。で動物の目を保護超短パルスレーザ放射への暴露から乾燥や損傷を防止獣医軟膏、。 </li><li> 0.9リットルでケタミン/キシラジン体積（100 mg / kgをkgのケタミン+ 5mg / kgのキシラジンKG）は、実験に先立って、または使用イソフルラン麻酔（2-3％（）の腹腔内注射（IP）を使用して動物を麻酔/実験中の最小ガス流量）。動物の後肢の反射をチェックすることにより、麻酔を確認します。 </li><li>効果的な音響結合を提供し、関心領域内の光音響プローブを配置するために画像化される領域の周りの皮膚に超音波ゲルを適用する。気泡が超音波カップリングゲル中に存在しないことを確認してください。 </li></ol> 3.プリビュー動作モード<ol><li> 690 nmおよび900 nmおよび10および50 Hzのパルス繰り返し率と撮像波長（複数可）を確立する。音響データ収集システムのためのパラメータを選択します - 1MΩ入力インピーダンス。第12ビットの垂直分解能あたり40メガサンプルのサンプリングレートで各レーザパルス2030のサンプルを取得する。レーザーのQスイッチ出力と取得をトリガーする。 </li><li>オペレータと患者の両方が光励起波長（群）に適合した保護メガネを使用していることを確認してください。人体実験19の安全曝露限界を満たすために及び動物における熱ひずみおよび皮膚損傷を防止するために、組織表面での光のフルエンスは、近赤外波長について、実験中は20mJ / cm 2程度以下に維持されるように、レーザパワーを設定する。 </li><li>レーザーのパルス繰り返しレートに対応するフレームレートで三次元画像を可視化できるようにする処理アルゴリズムのGPU実装とプリビューソフトウェアを起動します。 </li><li>プローブおよび/または可視化の性能を最適化し、目的の構造を局所化するために、撮像対象を移動させる。 </li></ol> 4.データ収集<ol><li> （ハンドヘルド）モードを走査するためのデータ収集。 </li><li>必要であれば、関心領域のコントラストを豊かにするための取得に先立って造影剤を注入する。 注：私たちの実験では、我々は造影人間イメージングを行っていない。しかし、様々な造影剤は、潜在的にこの目的に使用することができる。インドシアニングリーン（ICG）は、成人の体重を2mg / kgでの最大推奨用量でのコントラストの強調のために使用することができる臨床的に承認された光学造影剤の一例である。 </li><li>プレビューソフトウェアの実行を維持し3.1で説明したパラメータを持つデータ取得のためのハードウェアを起動します。静かに関心のある構造を追跡するために、画像化領域の周囲にプローブを移動させる。 注：複数のレーザ波長で画像を同時に取得されると、ハンドヘルドモードでのプローブの運動の速度は、（好ましくは2mm /秒未満に著しく減少されなければならないスペクトル的に混合されていない画像の動きに関連するアーチファクトを回避するために、50Hzのレーザパルス繰返し率）である。 </li><li>定常モードのためのデータ収集。 </li><li>ホルダーに、撮像対象（ 例えば 、動物）とハンドヘルドプローブをマウントし、プレビューソフトウェアの実行を維持し3.1で説明したパラメータを使用して取得を開始します。 </li><li>関心領域内の動的な生物学的事象を可視化するために、実験中の同じ位置における光音響プローブと撮像部を保持する。 </li><li>関心領域内の動的配布を追跡するために造影剤を注入する。 注：我々のマウス実験では、インドシアニングリーン（ICG）はコントラスト強調のために使用した。一般的なガイドラインとして、ICGの10ナノモル、または0.4〜10mg / kgの量は、in vivoでのマルチスペクトルoptoacousticsで検出可能なコントラストを作成するために、マウスの循環中に導入されなければならない。 注：造影剤は、でなければなりませんそれぞれの機関によって、ヒトおよび/または動物の使用のために承認した。 </li></ol> 5.実験ファイナライズ<ol><li>レーザーを停止します。 </li><li>撮像された領域から光音響プローブを除去。動物実験では、麻酔の供給を停止する。 </li><li>暖かいそれを維持し、それが完全に麻酔から回復するまで他の動物との接触を防ぐために、赤外線ヒーターの下に動物を配置します。麻酔からの回復中に無人の動物を放置しないでください。 </li></ol> 6.オフラインデータ処理<ol><li>データ処理に使用されるソフトウェアアプリケーションで取得した光音響信号を含むファイルを読み込む。 </li><li>各フレームおよび各波長に対する光吸収のボリュメトリック画像に対応する3次元マトリクス配列を得るために再構成アルゴリズムを使用する。 注：再構成のために、そのようなheterogeneなどの要因を歪ませるためのアルゴリズムのアカウンティングを使用することが好ましい吸収されたエネルギー分布のより定量的な表現を得るために、撮像対象でITIES減衰、最終的な帯域幅と検出素子と光フルエンス変動の幾何学的形状の影響。 </li><li>各多波長フレーム、試料中に存在する各吸収性物質の光吸収を表す3次元マトリックスアレイの新しいセットから、取得するアンミキシングアルゴリズムを使用する。 </li><li>必要に応じて、さらなる生物学的に関連するパラメータの可視化および読み取りを容易にするために、光吸収分布を表す行列アレイを処理する。 </li></ol>

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The authors have nothing to disclose.

小動物研究に光音響イメージング技術によって提供されるユニークな利点は、 例えば想定診断と治療モニタリングアプリケーションの数と、臨床の実践に技術を変換するための強力な動機を作成している。、乳癌および皮膚癌、炎症または末梢血管疾患で。しかし、効果的な断層像取得ジオメトリを作成するための照明源と検出素子の十分な数で囲むことができるマウス又は小さな動...

光音響（光音響）イメージング技術1-5が提供するユニークな利点を活用する新しいアプリケーションのさまざまな出版物を網羅増え続けることで明示されるよう、生物学および医学研究コミュニティからの関心の高まりを魅了しています。具体的には、はるかに光の拡散限界を超えた深さで高時空間解像度の画像スペクトル的に独特の光吸収剤の能力は、機能と分子イメージング6-10前例のない機能を開きます。 実際、臨床診療への光音響技術の翻訳は多くの疾患の診断および治療のモニタリングに有望な見通しが付属しています。しかし、光学的に散乱された光子を吸収組織や光音響現象に関連した一般的に弱い応答の限られた伝播は、この方法の適用可能な深さを制限。結果として、ハンドヘルドoptoa内視鏡システムは、自然のオリフィス13を介してそれらを挿入することによって、身体内から画像を提供するために使用されている間cousticプローブボディ11,12の外部からアクセス可能な画像部分に試みられてきた。そのような女性の胸のような人間の体の一部の低吸収部も、断層光音響スキャナ14,15でアクセスできます。それは同様に超音波検査を、大きな汎用性を可能にするので特に興味深いのは、手持ち式のアプローチである。ここでは、光音響イメージングのための一般的な超音波リニアアレイプローブの適応は、主に超音波およびoptoacoustics間の断層画像化要件の根本的な違いにより、依然として厳しい。標準的な超音波検査の高いフレームレートはkHzの範囲で高いパルス繰り返し周波数を用いた送受信シーケンシャル方式で有効にされているが、リアルタイムの三次元光音響イメージングは​​、単一のintから体積断層撮影データを同時に収集することにより達成されるレーザーパルスをerrogating。これにより、高品質の光音響イメージングは​​、撮像対象の周りに可能な限り大きな立体角から3次元データの取得を意味する。 最近、我々はリアルタイムで16を 3次元（体積）イメージングのための最初のハンドヘルド光音響プローブを導入しました。システムは、90°の角度をカバーする球面上に配置された圧電素子256（ 図1A中の青色のドット）の二次元配列に基づいている。約3×3mmの2の個々の要素の大きさ、ならびにそれらの配向および周波数帯域幅（約2-6メガヘルツ）は、 図中の球体の中心（黒立方体を囲むセンチメートルスケールのボリュームから有効な信号収集を保証する1A）。任意の波長SUSCように、撮像領域の光学的励起は、アレイの中央の円筒形キャビティを通って挿入繊維束を備えて​​いる繊維束を透過するのeptibleは、イメージングのために使用することができる。光ファイバ束と共にトランスデューサのアレイの実際の画像は、 図1Bに示されている。レーザーのパルス繰り返し周波数で決まるフレームレートでリアルタイムイメージングは​​さらに、グラフィックスを用いて有効にされるように、効率的な励起および信号の同時検出は、シングルショット励起（一方のレーザーパルス）と深部組織イメージングを可能にする処理ユニット（GPU）再建手続き17の実装。透明なポリエチレン膜（ 図1C）を有する円筒状ケーシングは、音響的に送信する液体媒体（水）を囲むように、トランスデューサアレイに接続されている。膜はさらに音響ゲルを用いて組織に結合されている。手持ち式の動作モードで使用されているような光音響プローブの画像が図1Dに示されている。 実証THR臨床診断および潜在的な用途の数の重要な利点が付属してリアルタイム機能イメージング能力と組み合わせるeeの次元ハンドヘルド光音響イメージングは​​、例えば、末梢血管疾患などの種々の適応症、リンパ系障害、乳癌、皮膚病変のために想定されている炎症または関節炎18。さらに、高速撮像能力は静止位置に配置されたプローブを用いた動的な生物学的事象の可視化を可能にします。高速波長同調光パラメトリック発振器（OPO）レーザー技術と組み合わせて、このアプローチは、光吸収剤の生体内分布のリアルタイムイメージングを可能にする。これにより、新たな可能性は等しく、例えば 、小動物イメージング用途に出現することができる。、組織血行動態を研究する、 インビボでの細胞追跡、薬物動態、臓器灌流の視覚化は、腫瘍及び心臓血管系、または神経画像の分子イメージング標的。

<table border="0" cellpadding="0" cellspacing="0" width="1663">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Name of Material/ Equipment</td>
			<td style="width:389px;">Company</td>
			<td style="width:171px;">Catalog Number</td>
			<td style="width:777px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Optical parametric oscillator (OPO)-based laser</td>
			<td style="width:389px;">Innolas Laser GmbH, Krailling (Germany)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The laser provides laser pulses with a duration around 10ns and an energy up to 80mJ. 
			The wavelength is tunable between 680-950nm.</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:327px;">Spherical array of piezocomposite detectors</td>
			<td style="width:389px;">Imasonic SaS, Voray (France)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The array consists of 256 piezoelectric sensors distributed on a spherical surface. 
			Each element has dimensions 3x3mm2, a central frequency of 4MHz and a bandwidth of 100%.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Data acquisition system (DAQ)</td>
			<td style="width:389px;">Falkenstein Mikrosysteme GmbH, Taufkirchen (Germany)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The DAQ simultaneously acquires 256 signals at 40 megasamples per second and 2030 samples. 
			The input impedance is 1MW.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Fiber bundle</td>
			<td style="width:389px;">CeramOptec GmbH, Bonn (Germany)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The bundle consists of 480 individual fibers randomly distributed in the input and output. 
			The numerical aperture of each individual fiber is 0.22.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Athymic Nude mouse</td>
			<td style="width:389px;">Harlan Laboratories (The Netherlands)</td>
			<td style="width:171px;">Athymic nude - Foxn1nu</td>
			<td style="width:777px;">The mouse was 8 weeks old (adult) at the time of the experiment. 
			The ethical protocol was approved by the Bavarian goverment (number 55.2.1.54-2632-102-11)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Bepanthen cream</td>
			<td style="width:389px;">Bayer AG (Germany)</td>
			<td style="width:171px;">N/A</td>
			<td>Vet ointment to protect the eyes during anesthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Data processing software</td>
			<td style="width:389px;">Matlab (Mathworks, Natick, MA, USA)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The data processing software was devoped at our institute. 
			It allows reconstruction at each wavelength and multi-wavelength unmixing, as well as further data processing.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:327px;">Water-enclosing part</td>
			<td style="width:389px;">N/A</td>
			<td style="width:171px;">custom-made</td>
			<td>This part contains the water that acts as an acoustic coupling medium between skin and transducer elements</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Indocyanine green (ICG)</td>
			<td style="width:389px;">PULSION Medical Systems SE</td>
			<td style="width:171px;">N/A</td>
			<td style="width:777px;">ICG-PULSION (active ingredient: indocyanine green dye) is a drug used in cardiac, circulatory and micro-circulatory diagnostics, liver function diagnostics and ophthalmic angiography diagnostics.</td>
		</tr>
	</tbody>
</table>

universal hand-held three-dimensional optoacoustic imaging probe for deep tissue human angiography and functional preclinical studies in real time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

代表的な結果は、説明した容積ハンドヘルド光音響プローブの能力を実証し、このセクションで紹介されています。全ての場合において、皮膚表面上の光のフルエンスを20mJ / cm 2と19の安全曝露限界以下に維持した。 ヒト末梢血管系の追跡リア ​​ルタイムでプローブの性能は、図2に紹介されている。この実験の過程で、プローブをゆっく?...

Watch this Scientific Journal Video about Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time at JoVE.com

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

ディープティシュ人間血管造影および機能前臨床研究のためのユニバーサルハンドヘルド三次元光音響イメージングプローブリアルタイムで

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

universal-hand-held-three-dimensional-optoacoustic-imaging-probe-for

Research

JoVE Journal

Bioengineering

10.6K ビュー.  Helmholtz Zentrum München. We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

ビデオ: Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三次元光学分解能光音響顕微鏡

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

生物工学

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

導電性インクの平面および三次元印刷

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

生理フローの状況の下で循環細胞のケモカイン指向溢出を研究する新規三次元フローチャンバーデバイス

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

三次元インクジェット印刷技術を用いて、ヒト軟骨組織の作製

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三次元の全層皮膚等価物と自動傷つけ処理の発生

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

微小重力下で増殖させたヒト腸粘膜の多細胞三次元器官型モデルの開発

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

三次元組織配置に発達メカニズムを要約し、神経系の再生を容易にするアストロ サイト ネットワーク

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

組織工学的応用の制御可能な形態の三次元 Poly(ε-caprolactone) 足場のエレクトロスピニング記述を溶かす

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

手持ちの臨床音響小動物の非侵襲的な実時間イメージングによる画像計測

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

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

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

三次元ペプチド足場で哺乳動物の細胞を培養

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