資金は、NIH MH - 64611とNARSAD若手研究者賞によって提供されていました。また、作者は彼らの技術支援のためにアダムビーグリー、マークDikopf、とベンスミスに感謝します。

SU - 8金型製作マスターの準備<ol><li>シリコンウエハ基板上にSU - 8のマスターは、2段階の標準的なネガ型レジストリソグラフィプロセスを用いて調製される。 </li><li>シリコンウエハ上のアライメントマークは、（ウェーハの外周に沿って位置する）これらの構造体の高さとカミソリの刃を使って削除している実際のデバイス構造以上のものです。 </li><li>シリコンウエハは、イソプロピルアルコールで洗浄し、N 2の流れで乾燥させる。最も高いデバイスの構造よりも小さい厚さでテープで作られた支柱は、ウェハの四辺上のアライメントマークを交換してください。 注：支柱の高さが最も高いデバイス構造を超えている場合は、スルーホールPDMSモールドに形成されていません。 </li><li>マスターのシリコンウエハは、室温でホットプレート上に置かれる。 </li></ol> PDMSの溶液の調製<ol><li>ポリジメチルシロキサン（PDMS）ソリューションの4グラムは十分に硬化剤1部とシリコーンエラストマーの10部を混合することによって調製される。 注意 ：2つの溶液をPDMSモールド全体に類似した物性を得るために均一に混合されていることを確認します。 </li><li>混合プロセス中にPDMSの溶液中で生成された気泡は、真空デシケーターを使用して削除されます。 </li></ol> PDMSコーティングおよび硬化<ol><li>泡のないPDMSの溶液を徐々に気泡がディスペンシング工程中に生成されていないことを確認してSU - 8マスター上に分配される。 </li><li>書き込みの透明フィルムの一端は、ホットプレートの上に置き、ゆっくりと均等にSU - 8マスターにPDMSを広めるためにPDMSのソリューションに配置されます。このプロセス中に生成される気泡は、プローブを使用して削除する必要があります。 [注：複数の気泡の発生を防ぐために透明性を持ち上げないでください。] </li><li> borofloatのスラブは、均一な圧力を適用する透明シートの上に配置されます。穏やかな圧力は、透明性と円形のSU - 8表面の間に挟まれた、最小限、あるいはまったくPDMSがあるような背の高い円形の構造物の上面（チャネル長の中間に位置し、チャネルの幅に沿って中央）に適用されます。構造。 </li><li>さらに3つのborofloatのスラブは、前に円形の開口部の構造物の上面にし、硬化プロセスの間に一定の圧力を適用するには、シリコンウエハーborofloatスラブサンドイッチの上に配置されます。 </li><li>ホットプレートの温度は、75に増加して℃、PDMSは、1時間この温度で​​硬化させる。ホットプレートを50℃の温度にしています</li><li>スラブが削除され、透明性を穏やかにマスターし、その上にコーティングされた薄いPDMSシートを残して削除されます。 </li></ol>マイクロ流体デバイスの構築マスターからのPDMSシートの除去<ol><li>灌流チャンバーの外側の境界は、SU - 8マスター上のポートと灌流チャンバーにポートを合わせて後にカミソリの刃を使用して、PDMSシートに出刻まれています。 </li><li> PDMSシートは、ゆっくりとPDMSの引き裂き防止するためのチャネルの長さに沿って引っ張って、マスターから削除されます。 PDMSシートは上向きに微小流体ネットワークの面を持つ透明シート上に配置されます。 </li><li>入口および出口ポートは、コルクボーラーを用いて作られています。 </li></ol>ガラスカバースリップにPDMSシートの接着<ol><li> PDMSシート（表面はマイクロ流体ネットワークを含む）、ガラスのカバースリップの接合面は、その後、静かに透明のシートに配置、3Mのスコッチテープを使用して洗浄し、最後にO 2プラズマチャンバ内に配置されます。 </li><li>接合面は10秒165ワットのプラズマで処理されていますガラスのカバースリップのプラズマ処理表面はすぐにPDMSシートのプラズマ処理表面に接着されている。 注意：接着面の間に閉じ込められた気泡を除去するためにガラスの表面に穏やかな圧力を与える。 </li><li> 5分PDMSとガラスの間に良好な接合を得ることができます。 5分後に軽くPDMSシートを接合プロセスのために置かれた透明性を取り除く。 </li><li>マイクロ流体デバイスは、チャネルは親水性にするための酸素プラズマシステムに配置されます。プラズマ処理は、1分間165ワットで行われます。 </li></ol>マイクロ流体デバイスと灌流チャンバーの統合灌流チャンバーの準備<ol><li>既製の灌流チャンバーを命じられた、と入口と出口ポートが結合しているときにマイクロ流体デバイスとチャンバー、チャンバーと、デバイス上のポートが整列されるように掘削された。灌流チャンバーの底面は、Pが塗布されているDMS室とマイクロ流体デバイス間の密封度を得るために。 </li><li>書き込み上の透明シートは、室温でホットプレート上に置かれる。 </li><li>一グラムPDMSのソリューションは、前述と同様のプロセスで調製される。泡のないPDMSの溶液は、次いで徐々に気泡の発生を回避透明シート上に分配される。 </li><li>灌流チャンバーは、PDMSのソリューションに配置されます。 </li><li> borofloatのスラブは、均一な圧力を適用すると、チャンバーの底面にPDMSの薄いコーティングを得るためにチャンバーの上部に配置されます。 </li><li>ホットプレートの温度は、75に増加して℃、PDMSは、1時間この温度で​​硬化させる。 </li></ol>マイクロ流体デバイスと灌流チャンバーの接合<ol><li>スラブが削除され、透明性を穏やかに灌流チャンバーと、その底部の表面にコーティングされた薄いPDMSシートを残して削除されます。不要なPDMSは、アクセスポートからPDMSの除去のためのカミソリの刃と鋭い先端のプローブを用いて除去される。 </li><li>マイクロ流体デバイスのPDMS表面とチャンバーのPDMSの底面は、静かに、3Mテープを使用してきれいに透明のシートに配置し、最後に酸素プラズマチャンバ内に配置されます。 </li><li>表面は10秒165ワットのプラズマで処理されていますチャンバーのPDMSコーティングされた表面は、すぐにマイクロ流体デバイスのPDMSの表面に接着されている。 注意：接着面の間に閉じ込められた気泡を除去するためにガラスの表面に穏やかな圧力を与える。 </li><li>使用直前に、デバイスは、チャネルが親水性にするための酸素プラズマシステムに配置されます。プラズマ処理は、1分間165ワットで行われます。 </li></ol>マイクロ流体デバイスを用いた神経化学的微小環境に脳スライスを公開<ol><li> （マイクロ流体デバイス）の親水性マイクロチャネル - （灌流チャンバー）の組み合わせが標準ACSF（人工脳脊髄液）溶液を充填されます。 </li><li>入口ポートでACSF液の小滴を分注し、ソリューションは、最大のチャネルに邪悪になることができます。出口ポートからシリンジを使用して残った気泡を取り除きます。口からマイクロ流体デバイスの出口ポートへの流体の受動的なポンピングを可能に出口ポートでACSF液の大粒を分注する。 注意：注意はパッシブポンピング法を用いた流体の流れを可能にするためにチャネルからすべての気泡を除去するために取られるべきである。 </li><li>プレーンプラットフォームの（灌流チャンバー）コンボと顕微鏡アダプタにプレーンプラットフォームを修正 - （マイクロ流体デバイス）を修正しました。 </li><li>標準の入口と出口（吸引）標準ACSF液と脳スライスの連続的な灌流のための灌流チャンバーへの配管を接続します。 ACSF液を連続して95％O2 - 5％CO 2で吸引される。 注：灌流チャンバーの槽内のACSFの一定レベルを維持するために、吸引チューブの位置を調整します。 </li><li>灌流チャンバーはACSF液で満たされると、スポイトを使用して灌流チャンバ内の脳のスライスを配置します。プローブを使用して、マイクロ流体デバイスの円形の開口部の上に脳スライスを配置します。脳のスライスが円形の開口部の所望の位置になると、脳のスライスを固定化するスライスのアンカーを使用してください。 </li><li>マイクロ流体デバイスは、現在、流体のポンピングパッシブを使用して様々な神経伝達物質に脳スライスを（灌流チャンバー内およびマイクロ流体ネットワークの上に置かれた）公開するために使用することができます。 </li></ol>

<ol>
	<li>Blake, A. J., Pearce, T. M., Rao, N. S., Johnson, S. M., Williams, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+PDMS+microfluidic+chamber+for+controlling+brain+slice+microenvironment.">Multilayer PDMS microfluidic chamber for controlling brain slice microenvironment.</a> Lab on a Chip. 7, 842-849 (2007).</li><li>Walker, G. M., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+passive+pumping+method+for+microfluidic+devices.">A passive pumping method for microfluidic devices.</a> Lab on a Chip. 2, 131-134 (2002).</li></ol>

著者は生物学の様々な分野への実証マイクロ流体技術に関する共同研究に開放されています。

既存のマクロスケールやマイクロスケールの脳スライスの灌流チャンバーは、それらが神経伝達物質で脳のスライスを公開するために提供する空間分解能の点で制限されています。ここに示したマイクロ流体デバイス技術は、簡単なバイオMEMS技術を使ってこの制限を克服しています。それは、マイクロ流体デバイスの製作と、既存の電気生理学的セットアップと統合の容易さのシンプルさが実証デバイス技...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>RC-26GPL</td>
<td>Tool</td>
<td>Warner Instruments</td>
<td>W2-64-0236</td>
<td>Low Profile Large Bath RC-26GLP Recording Chamber</td>
</tr>
<tr>
<td>SHD-26GH/10</td>
<td>Tool</td>
<td>Warner Instruments</td>
<td>W2-64-0253</td>
<td>Stainless steel slice hold-down for RC-26G, 1.0 mm thread spacing </td>
</tr>
<tr>
<td>PDMS (polydimethylsiloxane)</td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>Sylgard 184</td>
<td>Silicone Elastomer Kit</td>
</tr>
<tr>
<td>Plasma Preen-II 862</td>
<td>Tool</td>
<td>Plasmatic Systems, Inc.</td>
<td>&nbsp;</td>
<td>Microwave plasma system</td>
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<td>Model P-1</td>
<td>Tool</td>
<td>Warner Instruments</td>
<td>W2-64-0277</td>
<td>Series 20 Plain Platform, Model P-1</td>
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<td>SA-NIK</td>
<td>Tool</td>
<td>Warner Instruments</td>
<td>W2-64-0291</td>
<td>Adapter for Nikon Diaphot/TE200/TE2000, SA-NIK</td>
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<tr>
<td>Oxygenated, heated ACSF (Artificial cerebro-spinal fluid)</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Exact composition will vary with application</td>
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brain slice stimulation using a microfluidic network and standard perfusion chamber

我々は、簡単に既存の電気生理学のセットアップに統合することができる2つのレベルのマイクロ流体デバイスの作製を実証している。 2レベルのマイクロ流体デバイスは、2段階の標準的なリソグラフィプロセス1をネガ型レジストを用いて製造されています。最初のレベルは、それぞれの終了時に入口と出口ポートを持つマイクロチャネルが含まれています。番目のレベルは、チャネル長の中間に位置し、チャネルの幅と一緒に中央にマイクロスケールの円形の穴が含まれています。受動的な揚水方法は、入口ポートから出口ポート2に流体をポンプするために使用されます。マイクロ流体デバイスは、既製の灌流チャンバーと統合され、電気生理学のセットアップとのシームレスな統合を可能にしています。流体は出口ポートに向かってマイクロチャネルを介して吸気ポートの流量で導入されており、また血流の浴中にマイクロチャネルの上部にある円形の開口部を通って脱出。このように灌流チャンバーのバスで、マイクロ流体デバイス上に置かれた脳のスライスの底面は、別の神経伝達物質で公開することができます。マイクロ流体デバイスのマイクロスケール厚さと材料の透過的な性質[ガラスカバースリップとPDMS（ポリジメチルシロキサン）]マイクロ流体デバイスは、脳スライスの顕微鏡を可能にするために使用。マイクロ流体デバイスは、脳スライスの微小環境に導入された化学的刺激の変調を（空間的および時間的の両方）が可能です。

Watch this Scientific Journal Video about Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber at JoVE.com

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

我々は、さまざまな神経伝達物質によく制御された方法で脳のスライスのマイクロスケールの表面を露出させる標準的な電気生理学的セットアップと統合可能なシンプルなマイクロ流体デバイスの作製を実証する。

マイクロ流体ネットワークを用いた脳スライスの刺激と標準灌流チャンバー

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The ...

brain-slice-stimulation-using-microfluidic-network-standard-perfusion

Research

JoVE Journal

Biology

10.8K ビュー.  University of Illinois, Chicago. 我々は、さまざまな神経伝達物質によく制御された方法で脳のスライスのマイクロスケールの表面を露出させる標準的な電気生理学的セットアップと統合可能なシンプルなマイクロ流体デバイスの作製を実証する。

ビデオ: Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

ニューロン相馬と軸索のコンパートメントのためのマイクロ流体デバイスの作製

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

生物学

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

マイクロ流体デバイスでコンパートメントのためのE18脳ラットのニューロンの準備

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

CD4 + Tリンパ球キャプチャHIVのための使い捨てマイクロ流体チップを用いた

Cell Capture Using a Microfluidic Device

マイクロ流体デバイスを用いた細胞のキャプチャ

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

アガロースゲルベースの​​マイクロ流体デバイスを用いた神経幹細胞運動の評価

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

哺乳動物細胞をトリプシン処理し、継代培養

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

マイクロ流体プローブ：局在表面処理のための操作と使用方法

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

マイクロ流体フローセルを使用して流れの下で血小板粘着と凝集

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

機能イメージングによるヒト聴覚皮質上のシータバースト刺激の後遺症のマッピング

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

軟骨アガロースゲルの機械的刺激

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...