この作品は、リチャード·トーマス白血病基金、レディタタメモリアルトラスト、ノースウィックパーク病院の白血病研究基金と保健研究（NIHR）の国立研究所、英国によって資金を供給された。

1。足場の足場製造·バイオ機能化<ol><li>ペトリ皿ディスクの形でPU足場を（孔径100〜250ミリメートル、気孔率90〜95％）を作製するには、凍結とその後に続くポリマー溶液（ジオキサン中の5重量％）を調製することにより、熱誘起相分離13プロセスを使用して溶剤昇華（ 図1A）。 </li><li> ECMタンパク質（ 図1B）でコーティングする前に0.5×0.5×0.5 mmの立方体に足場ディスクを切断します。 </li><li> 1分間エタノールでそれらを浸漬することによってプリウェット足場（70％）、続いてリン酸塩への転送は20分​​間緩衝生理食塩水（PBS）。その後、3630 xgで10分間遠心し、タンパク質溶液を追加します。 </li><li> 20分間1420×gで蛋白質溶液中で足場を遠心分離します。 </li><li>表面の毛穴のブロックを解除し、足場の奥深くに浸透して播種した細胞を可能にするために、10分間910×gで1より多くの時間を足場遠心PBSインチ</li><li>エタノール（70％）で2時間浸漬して、UVランプを0.14 230 V、50 Hzで、少なくとも8分露出の組み合わせを使用して足場を殺菌。その後、30％ウシ胎児血清（FBS）および1％ペニシリンおよびストレプトマイシン（P / S）を添加したイスコフ改変ダルベッコ培地（IMDM）を追加する前に、PBSで2回足場を洗う。 ℃、5％CO 2は、使用前に37℃で3日間の加湿インキュベーター中で足場を置きます。無菌の足場は、24ウェル組織培養プレート（ウェル当たり1足場）に配置されます。 </li></ol> 2。単核細胞の単離と足場シード<ol><li>をFicoll-Paque密度勾配遠心分離（1500 rpmで35分）（ 図1C）を使用して 、臍帯血または白血病骨髄サンプルから人間のMNCを抽出します。 </li><li> IMDMに再懸濁し、MNCは、30％FBSおよび1％P / Sを含む</li><li>マイクロピペットを用いて足場の上にMNC懸濁液の100μlの種子（2×10 6細胞/足場）。 </li><li>足場に解決するためにセルの10分間、5％CO 2下で37℃で播種足場をインキュベートします。 </li><li> 37℃インキュベーターで30％FBSおよび1％P / Sと場所ウェルプレートを含むIMDM 1.5 mlを各ウェルアップトップ℃、実験期間中5％CO 2。 </li><li>シード足場は、毎日すべてのメディアの変化を受けています。 </li></ol> 3 In Situ細胞増殖と形態、次の操作を行います 。MTS、SEMとCytospins <ol><li> MTSアッセイ：培養1非シードとクリーンな新しい24ウェルプレートに2シードの足場と場所から削除します。 37メディアと3時間MTS溶液とインキュベートし、200μlの1 mlの℃、5％CO 2を追加します 。 <ol><li>マイクロプレートリーダーを用いて490nmで96ウェルプレートおよび測定の吸光度で行われ、上清から8×100μlのサンプルを取る。 </li></ol></li><li> 走査電子顕微鏡（SEM）：番目の異なる時点でeの文化は、メディアからのMNCを接種した足場を削除して、4時40分°C、2.5％PBS-緩衝グルタルアルデヒド溶液で固定し、PBSで2回洗浄する。 <ol><li>エタノールの段階的シリーズ（25、50、70、80、90、95、および100％）で脱水足場、各4時間の無菌環境で10分間、乾燥した。 </li><li>セクションの標本とし、前のSEM評価に2分間アルゴン雰囲気中での金のスパッタコートして、20 kVの加速電圧を使用しています。 </li></ol></li><li> Cytospins：文化の異なる時点で足場から細胞を吸引することにより2.0×10 4細胞/スライドを収集します。 <ol><li> 1500 rpmで3分間のガラススライド上に細胞を遠心分離します。 </li><li>ライト·ギムザ染色と変更された光学顕微鏡を用いて観察してスライドを染色。 F-VIEWソフトイメージングシステムは、写真を撮るために使用することができます。 </li><li>顕微鏡観察の前にスライドを洗浄します。 </li></ol></li><li> M<html> ultiphoton分析：さらに分析まで-20℃でエタノール蒸気（70％）一晩乾燥した、店舗°Cで別の時点で修正足場。 30μmの厚さのスライドに多光子顕微鏡のセクション足場と可視化の前に、PBSでぬれた。 <ol><li>室温で30分間、10％ウシ胎児血清を含むPBS中に播種足場をブロックします。 </li><li>マウス抗ヒトCD71を：°C暗闇の中で主要なモノクローナル抗体の1:50希釈し4℃で足場は、一晩インキュベートします。 </li><li>サンプルをPBSで3回洗浄し、二次抗体で染色：暗闇の中で、室温で1時間インキュベートした抗マウスのAlexa Fluor 488。 </li><li> PBSでサンプルを3回洗浄し、水の排出X60対物レンズを用いた共焦点顕微鏡で観察します。 </li><li>蛍光体のAlexa Fluor 488拍レーザーで488nmで励起されています。 Volocity 5.3.2ソフトウェアは、その後の画像解析に使用することができます。 </li></ol></li></ol>jove_title細胞集団の &quot;&gt; 4。フローサイトメトリー分析<ol><li>フローサイトメーター（FC）の細胞分析の前に、検出に対応する免疫抗体で目的の細胞にラベルを付けます。サンプル数は、検出のために選択された抗体に応じて用意されています。 MNC検出用抗体の次の組み合わせが使用されています。CD45-FITC/CD71-PE/CD235a-PE-Cy5。 </li><li>異なる時間ポイントと遠心で足場から細胞を吸引します。 100μlのFC緩衝液（PBS + 0.1％アジ化ナトリウム）で6×10の周りの細胞の細胞ペレットを溶解し、各抗体の蛍光色素の10μLを追加します。 4℃で30分間細胞をインキュベートし、PBSで2回洗浄し、最終的にFCの緩衝液に再懸濁する。 </li><li>アイソタイプコントロール抗体発現の &quot;否定&quot;を設定し、フローサイトメーターの制御と検出チャネルを校正するためで染色しフローサイトメトリーにサンプルをロードします。 </li><li> wを染色したサンプルを読む<html> i番目の3種の異なる抗体、最後にWinListソフトウェアを使用してデータを表します。 </li></ol> 5。代表的な結果外因性の成長因子を添加せずに、造血細胞の増殖動態の例を図2に示されています。造血の不均質な性質のために、二つの異なる細胞：正常と異常な造血細胞が示されている。 図2Aにおいて、ヒトCBMNCの細胞増殖は、培養28日後に明らかである。 図2Bは、ヒトの主要な白血病細胞を用いた擬態の成長速度を示しています。細胞増殖は、吸光度との関係で細胞の代謝活性を測定するMTSアッセイを用いて評価されています。両方のケースでは、細胞は増殖し、モデルに設立された。成長速度の違いが観察されています。文化を確立し、正常造血細胞より速い白血病細胞より。 外因性増殖因子の不在下でさえ文化の28日後に採取した細胞の形態が正常な造血細胞（ 図3A）および白血病細胞（ 図3B）の典型的だった。_content &quot;は&gt;足場の中央のセクションは、後のSEMで分析した足場は、培養液から除去し、クラスターおよび&quot;ニッチのような&quot;構造（ 図3A &#39;＆B&#39;）の自分自身を確立し、足場全体に播種細胞の拡散を示した。 図Cは、シーディングの細孔サイズや分布を示した足場は、コントロールとして使用されます。多光子顕微鏡は、28日はその場で 3D足場内の細胞の分布を明らかにするために使用され、それがマーカーの発現により足場の中央セクション（ 図4）赤芽球島の存在を示した後赤芽球で陽性である。これは、成熟した成熟細胞デュの重要性を証明するCD71リング赤血球。最後に、細胞のフローサイトメトリーのグラフ前の播種は 、 図5Aは、正常な造血細胞を表して造血細胞の表現型の違いを示しています。人間CBMNCsと図5Bは、異常な造血細胞を示しています。主要な白血病細胞。 CD235a赤血球と白血球+とCD45 +対応のレベルはそれぞれ白血病のhemoblastic性質を強調する白血病に比べて通常のサンプルでは高くなっています。 <img alt="図1" src="/files/ftp_upload/3654/3654fig1.jpg" /> 図1：PU足場製造·バイオ機能化に関与するプロセスのイラスト。 A）PUはジオキサン（5重量％）に溶解し、熱誘起相分離過程とその後の溶剤昇華によって足場はSafinia ら 13で説明したように、生成されます。 B）足場ディスクは、次にint切断されoののキューブ0.5×0.5細胞外マトリックス（ECM）タンパク質と、遠心分離により被覆された×0.5 mmとして。 C）MNCは、ヒト臍帯CBから、またはマイクロピペットで密度勾配遠心分離およびPU足場に播種し（2×10 6細胞/足場）を使用して、BM吸引から抽出されます。 <img alt="図2" src="/files/ftp_upload/3654/3654fig2.jpg" /> 図2細胞の増殖はMTSアッセイを用いて測定しました。）は、ヒト臍帯血多国籍企業を使用して、B）ヒト初代白血病細胞を用いた。列には、外因性のサイトカインを添加せずにPU足場に播種時間をかけて細胞の成長を示しています。 <img alt="図3" src="/files/ftp_upload/3654/3654fig3.jpg" /> 図3：cytospinsを使用して足場の周りの細胞の形態と分布、scanningの電子顕微鏡写真を示す。 （AB）培養28日後にPUの足場から収集された）臍帯血単核細胞の代表的なライト·ギムザ染色したcytospins、AとB）骨吸引白血病細胞は、培養14日後にPU足場から収集された。両方の実験は、サイトカイン遊離状態で行った。 （A&#39;-B &#39;））シード臍帯血多国籍企業とB&#39;）を28日間培養された後、白血病細胞、無細胞とC）コントロール足場のPU足場SEM写真の代表的な中央のセクション &quot;。 <img alt="図4" src="/files/ftp_upload/3654/3654fig4.jpg" /> 図4赤血球マーカーCD71との文化やステンドグラスの28日後に臍帯血多国籍企業のPU足場シードの多光子顕微鏡写真。 <img alt="図5" src="/files/ftp_upload/3654/3654fig5.jpg" /> 図5：フローサイトメトリー3Dのドットプロットは、CD45、CD71及びCD235a表面発現マーカーを染色した。得られたアイソタイプコントロールはまた、相対的な蛍光強度の比較のために提示されます。パネルは、上記のマーカーのヒト臍帯血単核細胞が陽性を示し、パネルBは、ヒト初代白血病細胞を示しています。

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我々は、開示することは何もありません。

ここで紹介するex vivoでの三次元培養システムは、外因性のサイトカインの元BMアーキテクチャと、細胞の表現型が独立して反復することが造血の3Dバイオミミクリーを確立することを可能にします。 3Dモデルの構造と正常および異常な造血細胞が in vivo で遭遇したと同様の条件で増殖することができます微小環境を提供しています。 高分子足場材料の...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue No</td>
		</tr>
		<tr>
			<td>Dioxan</td>
			<td>Invitrogen</td>
			<td>D20,186-3</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-094</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Invitrogen</td>
			<td>12440-053</td>
		</tr>
		<tr>
			<td>Ficoll-Paque</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
		</tr>
		<tr>
			<td>MTS</td>
			<td>Promega</td>
			<td>G3580</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Fluka Biochemika</td>
			<td>49624</td>
		</tr>
		<tr>
			<td>Wright-Giemsa</td>
			<td>Sigma-Aldrich</td>
			<td>WG32</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10108-165</td>
		</tr>
		<tr>
			<td>CD71</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-32272</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11001</td>
		</tr>
		<tr>
			<td>CD45-FITC</td>
			<td>BD Pharmigen</td>
			<td>74895</td>
		</tr>
		<tr>
			<td>CD71-PE</td>
			<td>BD Pharmigen</td>
			<td>555537</td>
		</tr>
		<tr>
			<td>CD235a-PE-Cy5</td>
			<td>BD Pharmigen</td>
			<td>555570</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S-8032</td>
		</tr>
	</tbody>
</table>

ex vivo mimicry of normal and abnormal human hematopoiesis

造血幹細胞は、血液細胞の形成1を維持するためにユニークな微環境を必要とします。骨髄（BM）は、複雑な3次元（3D）ティッシュ式中、造血である空間的に組織化、細胞微小環境によって制御されているニッチ2-4と呼ばれる。 BMニッチの組織が ​​正常または悪性BM 5の機能または機能不全のために重要です。したがって、ex vivoでの擬態を用いたin vivo微小環境でのより良い理解は、私たちは、白血病6の分子、細胞と微小環境の決定要因を解明に役立つだろう。 現在、造血細胞のいずれか同種または異種間質細胞との共培養または外因性のサイトカインの8の加算を必要とする2次元（2D）の組織培養フラスコ/ウェルプレート7にin vitroで培養されています。これらの条件は、人工的であり、 生体内での異なる</eM&gt;彼らが3D携帯電話のニッチを欠いており、分化多能性と9,10が失われる可能性が異常に高いサイトカイン濃度に細胞をさらすという点で微小。 ここで、我々 は 、in vivo 3次元成長環境でシミュレートし、外因性成長因子の存在下で多系統造血をサポートして新たな3D骨髄培養系を提示します。ポリウレタン（PU）で作られたこのシステムで使用される非常に多孔性の足場は、2Dの11の従来の単層培養よりも高い比表面積にわたって高密度の細胞増殖を促進します。私たちの仕事は、このモデルはヒト臍帯血（CB）単核細胞（MNC）外因性のサイトカインの存在下で12および主要な白血病細胞の成長を支えたことを示した。この小説の3D擬態は、造血を勉強すると白血病の新しい治療法を探求する人間の実験モデルの開発のための実行可能なプラットフォームを提供します。

Watch this Scientific Journal Video about Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis at JoVE.com

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

造血のための3D培養系は、ヒト臍帯血および白血病骨髄細胞を用いて記述されています。方法は、細胞外マトリックスタンパク質でコーティングされた多孔質のポリウレタン合成足場の使用に基づいています。この足場は、細胞の広い範囲に対応するために適応可能である。

正常と異常ヒト造血のex vivoでの擬態

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

ex-vivo-mimicry-of-normal-and-abnormal-human-hematopoiesis

Research

JoVE Journal

Bioengineering

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

15.4K ビュー.  Imperial College London. 造血のための3D培養系は、ヒト臍帯血および白血病骨髄細胞を用いて記述されています。方法は、細胞外マトリックスタンパク質でコーティングされた多孔質のポリウレタン合成足場の使用に基づいています。この足場は、細胞の広い範囲に対応するために適応可能である。

ビデオ: Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

三次元ヒト皮膚は、モデルを再構築：ノーマルスキンとメラノーマ進行を勉強するツールを

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

生物工学

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

のための周期的圧力のバイオリアクターの設計生体外で</em大動脈心臓弁の>研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

機械的に適応型ポリマーナノコンポジットの環境制御のMicrotensileテスト<em&gt; ex vivoで</em&gt;キャラ

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

使用プラ​​センタアクロス異物およびナノ材料の輸送速度の決定<em&gt; ex vivoで</em&gt;ヒト胎盤灌流モデル

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

変性コラーゲンストランドを画像化する<em&gt;インビボ</em&gt;と<em&gt;生体外</em&gt;リテーナ入りコラーゲン模倣ペプチド写真·トリガブリダイゼーション経由

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a key hemodynamic parameter related to cerebral oxygen supply. Resting state low frequency fluctuations based on oxygenation contrast have been shown to provide correlations between functionally connected regions. The presented protocol uses optical diffuse correlation spectroscopy (DCS) to assess blood flow-based resting state functional connectivity (RSFC) in the human brain. Results of CBF-based RSFC in human frontal cortex indicate that intra-regional RSFC is significantly higher in the left and right cortices compared to inter-regional RSFC in both cortices. This protocol should be of interest to researchers who employ multi-modal imaging techniques to study human brain function, especially in the pediatric population.

This protocol demonstrates how to measure resting state functional connectivity in the human prefrontal cortex using a custom-made diffuse correlation spectroscopy instrument. The report also discuss practical aspects of the experiment as well as detailed steps for analyzing the data.

脳血流ベースの安静状態機能性コネクティビティ(光学拡散相関分光法)

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

心臓トラベキュラEx Vivoの同時ブライトフィールド、蛍光、光コヘレンス断層イメージング

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

All-optical Mechanobiology Interrogation of Yes-associated Protein in Human Cancer and Normal Cells using a Multi-functional System

Long-term multi-functional imaging and analysis of live cells require streamlined, functional coordination of various hardware and software platforms. However, manual control of various equipment produced by different manufacturers is labor-intensive and time-consuming, potentially decreasing the accuracy, reproducibility, and quality of acquired data. Therefore, an all-in-one and user-programmable system that enables automatic, multi-functional, and long-term image acquisition and is compatible with most fluorescent microscopy platforms can benefit the scientific community. This paper introduces the complete operating protocols of utilizing a novel integrated software system that consists of (1) a home-built software program, titled "Automatic Multi-functional Integration Program (AMFIP)," which enables automatic multi-channel imaging acquisition, and (2) a suite of quantitative imaging analysis and cell traction computation packages. 
This integrated system is applied to reveal the previously unknown relationship between the spatial-temporal distribution of mechano-sensitive Yes-associated protein (YAP) and the cell mechanics, including cell spreading and traction, in CRISPR/Cas9-engineered human normal cells (B2B) and lung cancer cells (PC9). Leveraging this system's capability of multi-channel control and readout, the result shows: (1) B2B normal cells and PC9 cancer cells show a distinct relationship between YAP expression, traction, and cell dynamics during cell spreading and migration processes; and (2) PC9 cancer cells apply noticeable peri-nuclear forces on substrates. In summary, this paper presents a detailed stepwise protocol on how to utilize an integrated user-programmable system that enables automatic multi-functional imaging and analysis to elucidate YAP mechano-sensitivity. These tools open the possibility for detailed explorations of multifaceted signaling dynamics in the context of cell physiology and pathology.

This paper presents a detailed stepwise protocol on how to utilize an integrated multi-functional and user-programmable system that enables automatic multi-channel imaging and mechanobiological analysis to elucidate the mechano-sensitivity of Yes-associated protein (YAP).

多機能システムを用いたヒトがんおよび正常細胞における有形関連タンパク質の全光学機構調査

Long-term multi-functional imaging and analysis of live cells require streamlined, functional coordination of various hardware and software platforms. ...

灌流中の血管のex vivo培養のための新しい3Dプリントバイオリアクター

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious surgical and pharmacological interventions to prevent and treat vascular disease are urgently needed. In part, the shortage of translational models limits the understanding of the cellular and molecular processes involved in vascular disease. Ex vivo perfusion culture bioreactors provide an ideal platform for the study of large animal vessels (including humans) in a controlled dynamic environment, combining the ease of in vitro culture and the complexity of the live tissue. Most bioreactors are, however, custom manufactured and therefore difficult to adopt, limiting the reproducibility of the results. This paper presents a 3D printed system that can be easily produced and applied in any biological lab, and provides a detailed protocol for its setup, enabling users&#39; operation. This innovative and reproducible ex vivo perfusion culture system enables the culture of blood vessels for up to 7 days in physiological conditions. We expect that adopting a standardized perfusion bioreactor will support a better understanding of physiological and pathological processes in large animal blood vessels and accelerate the discovery of new therapeutics.

著者スポットライト:EasyFlow - 大血管培養のための経済的で適応性の高い灌流バイオリアクター 

This protocol presents the setup and operation of a newly developed, 3D printed bioreactor for the ex vivo culture of blood vessels in perfusion. The system is designed to be easily adopted by other users, practical, affordable, and adaptable to different experimental applications, such as basic biology and pharmacological studies.

Ex Vivo(エクス・ビボ )3Dプリントされたバイオリアクターでの大血管の灌流培養

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious ...