The work was funded by the University of Zurich. The authors would like to thank Estrela Neto and Dr. Meriem Lamghari for helping in establishing the co-culture conditions.

全てのマウスは、チューリッヒ、スイスの動物福祉法に、州立獣医局の規制に準拠してに従って維持し、取り扱いました。 解剖素材、文化メディア、マイクロ流体デバイスの作製<ol><li>オートクレーブマイクロ解剖ピンセットやハサミ（121℃、滅菌時間：20分）、滅菌容器に保管してください。 </li><li> 37℃でオービタルシェーカー上で24時間、1 M HCl中でそれらをインキュベートすることにより、ガラスカバースリップ（24ミリメートル×24 mm）を滅菌します。 99％エタノールで滅菌蒸留H 2 O 3回とそれらを3回洗浄します。乾燥後、37℃または滅菌流フードの下でカバースリップ。最後に、滅菌を完了するためにUV光（30分）にカバースリップをオートクレーブまたは露出します。カバースリップは、次いで、70％エタノールに保存することができます。 </li><li>滅菌ピンセットを使用してパッケージから慎重AXIS軸索分離デバイ​​スを取り外し、滅菌ペトリ皿に置きます。 </LI&gt; <li>滅菌生検パンチ（φ：1ミリメートル）を使用すると、培養室に対応して（ 図1）で培養するサンプルあたり一つの穴を作成します。 注：それらが適用された圧力によって損傷する可能性があるとして、微小溝に近すぎるパンチしないでください。 </li><li> 70％エタノールでそれらを浸すことにより、AXIS軸索分離デバイ​​スを滅菌します。完全無菌流フードの下で乾燥した後、AXIS軸索分離デバイ​​スとカバーガラス。先に進む前に、3時間以上の間隔をあけてください。 注：不完全な乾燥は、マイクロ流体デバイスの組立不良になります。 </li><li> 6ウェルプレート内で35ミリメートルペトリ皿にまたはウェルに各カバースリップを置きます。 </li><li>絶縁装置とカバーガラスとの間に完全な接着を可能にするために曲がった端部を有するピンセットで慎重にしっかりとカバーガラスとプレスにAXIS軸索遮断装置を配置します。 </li><li>ポリ-D-リジンの各培養室では、ピペットを150μl（滅菌蒸留H 2で0.1 mg / mlのO）。培養チャンバからすべての空気を除去するために、5分間真空下でのマイクロ流体デバイスを置きます。 </li><li>空気は、依然としてチャンバへの再ピペットポリ-D-リジン溶液、チャンバ内見られる場合。 </li><li> 37℃で、ポリ-D-リジンO / Nを有するデバイスをインキュベートします。 </li><li>滅菌蒸留H 2 Oで室を3時間を洗います</li><li> 150（PBS中のシグマアルドリッチ、5μg/ mlの、または無血清培地）μlのラミニン作業溶液とチャンバーを記入し、37℃でO / Nインキュベート。 </li><li> 48ミリリットルたNeurobasal培地、1mLのB-27、100 U / mlペニシリン/ストレプトマイシン、2mMのL-グルタミン、5 ngの/ mlの神経成長因子（NGF）：三叉次のように構成される神経節培養37のための培地50mlを準備、0.25のPMシトシンアラビノシド。 </li><li> 40ミリリットルDMEM-F12 10mlのウシ胎児血清（FBS、最終濃度：20％）を以下のように構成され、歯胚培養37培地50mlを調製し、100 U / mlペニシリン/ストレプトマイシン、2mMのL-グルタミン、150 / mlのアスコルビン酸。 </li></ol> 2.マウスの胚の発生と解剖<ol><li>膣プラグ（膣プラグ：開発0.5、E0.5の胚日）に応じて、胚の年齢を判断し、形態学的基準を介して確認します。このプロトコルのために、私たちは一般的にE14.5-E17.5マウス胚を使用しています。 </li><li>解剖エリアとエタノール70％でステレオスコープを清掃してください。 </li><li>頚椎脱臼を経由して妊娠中の母親を生け贄に捧げます。グリッド上に第一および第二指でマウスの首をブロックし、決定に尾を引きます。 </li><li>下腹部の周りの皮膚を切開し、はさみを使って腹部を開きます。子宮の位置を確認します：妊娠のような後期の段階で、子宮が豊富に腹腔を埋めます。 </li><li>氷上でPBSで満たされたチューブ内の子宮と場所を解剖。氷の上で、組織はいくつかの時間のために残すことができます。機関のガイドラインに従って、母の死体を捨てます</li> &lt;LI&gt;子宮から胚を解剖し、それらの胚体外組織からそれらを解放。氷上でPBS中で胚を置きます。 </li><li>はさみを使用して胚を刎ねると、マイクロ解剖ハサミ（ 図2A）を使用して、ヘッドの残りの部分から下顎を分離します。三叉神経節を損傷することなく、正確に下顎を外します。後者は下顎に近接して局在しているように、それらの偶発的な損傷が可能です。氷上で、冷PBS中で下顎と頭の残りの部分を保持します。 </li><li> TGを分析するには、頭を取ると、以前に冷PBSで満たされた解剖ガラスシャーレ上に置きます。鉗子を使用して、皮膚や頭蓋骨を削除します。終脳とリフトの下に鉗子を配置することにより、次に終脳と小脳を削除します。終脳と小脳が露出頭蓋骨の底部を残して、一緒に反転します。 </li><li> （ 図2Bに示されている）三叉神経節をローカライズ。鉗子を使用してください三叉神経からTGを分離します。ナイフのように解剖針を使用して、三叉神経突起の残党を排除します。冷PBSで満たされたペトリ皿に解剖TGを配置し、氷上で保管してください。 </li><li> 、胚の歯を解剖下顎を配置するには、以前に冷PBSで満たされた解剖ガラスシャーレ上に、頭蓋骨から分離。ナイフのように解剖針を使用して、舌と顎の周囲の皮膚を取り除きます。顎の正中線に沿って切断することにより、左右の半顎を分離します。 図1Cに示すように、歯胚は、簡単に可視化されます。解剖針を使用して歯胚を分離し、非歯の組織の過剰を取り除きます。冷PBSで満たされたペトリ皿に解剖歯胚を置き、氷上で保管してください。 </li></ol> 3.マイクロ流体共培養<ol><li>解剖後、マイクロ流体デバイスからラミニンを削除します。 respecti200μlのチャンバーを埋めますメディアVEの。 </li><li>ピンセットで、（ 図1D）を打ち抜いて作成した穴の中に静かに解剖し、TGと歯胚を転送します。歯胚が浮いていないことを確認し、彼らがカバーガラスに連絡するまで、彼らは沈むこと。 </li><li>培養を37℃、5％CO 2のインキュベーター内のサンプル。 </li><li>培地を48時間ごとに変更します。完全にチャンバーを空にしないでください、培養室に直接ピペットではありません。チャンバの完全な排出はチャンバ内の気泡の形成をもたらします。チャンバ内への直接ピペッティングは軸索損傷をもたらすであろう。これらの問題を回避するために、井戸の外側に向かってピペットポインティング培地を除去し、同様に、チャンバの反対側に位置井戸の側に新鮮な培地をピペット。 </li><li>培養期間中、同時培養を簡単タイムラプス顕微鏡によって画像化することができます。共培養物を10日間維持することができます。 </li><li>培養後のペリオD、室につき1つのウェルにPBSを150μlをピペットし、チャンバーを3回PBSを流すことにより、チャンバーを洗浄します。 </li><li> PBSを除去し、チャンバー当たり1つのウェルに（PBS中）パラホルムアルデヒド150μlの4％をピペットでサンプルを修正。室温で15分間インキュベートします。 </li><li> 3.6で説明したようにPBSで2回チャンバーを洗浄します。 </li><li>さらに分析を進めます。 </li></ol>

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W., Vahidi, B., Taylor, A. M., Rhee, S. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nature protocols. 1 (4), 2128-2136 (2006).</li><li>Hosmane, S., Tegenge, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll/interleukin-1+receptor+domain-containing+adapter+inducing+interferon-&#946;+mediates+microglial+phagocytosis+of+degenerating+axons.">Toll/interleukin-1 receptor domain-containing adapter inducing interferon-&#946; mediates microglial phagocytosis of degenerating axons.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 32 (22), 7745-7757 (2012).</li><li>Delamarche, E., Tonna, N., Lovchik, R. D., Bianco, F., Matteoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology+on+microfluidics:+multimodal+analysis+for+studying+celll-cell+interaction.">Pharmacology on microfluidics: multimodal analysis for studying celll-cell interaction.</a> Current opinion in pharmacology. 13 (5), 821-828 (2013).</li><li>Neto, E., Alves, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+neurons+and+osteoblasts:+close+partners+in+a+microfluidic+environment.">Sensory neurons and osteoblasts: close partners in a microfluidic environment.</a> Integrative Biology. , (2014).</li><li>Pagella, P., Neto, E., Jim&#233;nez-Rojo, L., Lamghari, M., Mitsiadis, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+co-culture+systems+for+studying+tooth+innervation.">Microfluidics co-culture systems for studying tooth innervation.</a> Frontiers in physiology. 5 (August), (2014).</li><li>Connor, R., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+maxillary+factor,+a+maxillary+process-derived+chemoattractant+for+developing+trigeminal+sensory+axons.">Identification of maxillary factor, a maxillary process-derived chemoattractant for developing trigeminal sensory axons.</a> Neuron. 24, 165-178 (1999).</li></ol>

The authors declare that they have no competing financial interests.

歯の神経支配のインビトロ研究における前は三叉神経節及び歯の組織または細胞26,28,29の従 ​​来の共培養に基づいていました。これらの研究は主に感覚軸索38にこれらの細胞または組織の魅力的な効果を調査するために行きました。フィールドに大きな進歩をもたらしたが、いくつかの技術的な問題が提起されました。歯胚を培養37の数日後に退化し始...

神経支配は、臓器や組織1,2の発達、ホメオスタシスおよび再生に重要な役割を果たしています。 5 -さらに、神経支配は、幹細胞の増殖、分化及び動員3の調節に関与します。実際には、口腔顔面複合体の組織で実現最近の研究は、上皮前駆細胞は唾液腺6,7の開発と再生中の機能のために副交感神経が必要であることを示しています。 11 -同様に、神経支配が味蕾8の開発及び維持のために必要であることが実証されています。したがって、このような歯のような他の重要な口腔顔面器官および組織の発達における神経支配のまだ無視役割を分析することが重要です。 大人の歯の豊富な神経支配にもかかわらず、ボディ、develo他のすべての臓器や組織とは対照的に、pingの歯は、最も初期の出生後の段階で神経支配され始めます。歯は口腔外胚葉および頭蓋神経堤由来の間葉間シーケンシャルと逆数の相互作用の結果として発生します。これらの相互作用は、それぞれ12、上皮由来エナメル芽細胞とエナメル質や象牙質の形成に関与している間葉由来の象牙芽細胞を生じさせます。 15 -上頸神経節から三叉神経節および交感神経からの感覚神経は大人の歯13を支配します。胚発生時には、神経線維は、現像歯胚に向けて三叉神経節プロジェクトから発せられると次第にそれらを囲むが、彼 ​​らは歯乳頭間葉13に浸透しません。神経線維は、象牙芽細胞分化と象牙質マトリックスの沈着16と相関より高度な発達段階に歯髄間葉を入力してください。歯髄神経支配はCOMPLですすぐに口腔13内の歯の噴火後にeted。 19 -これまでの研究では、様々なセマフォリンおよびニューロトロフィンは、歯牙16の間に神経支配の調節に関与することを明らかにしました。以前の研究は明らかに神経支配が魚20で歯の形成のための前提条件であることを実証しました。より最近の研究は、ソニックヘッジホッグ（SHH）21の分泌を介して感覚神経によって調節されるマウス切歯歯科間充織幹細胞の恒常性を示しました。 24 -それにもかかわらず、歯の開始、開発、再生中の神経支配の役割は、哺乳類22にまだ非常に論争です。 インビボ研究の過多は、様々な動物モデル13,25,26の開発および修復プロセス中に歯の組織の神経支配のパターンに関する重要な情報を提供しています。しかし、これらのほとんどは、アプロ痛みは、神経線維と標的器官と組織との間の相互作用の分子的基礎を強調するために最適ではありません。 29 -共培養は、制御され、隔離された環境26に神経線維と歯の間の相互作用を調査し、操作するための貴重な方法を構成しています。同時に、共培養は、種々の技術的な調整を受けます。 32 -例えば、神経および特定の歯の組織（ 例えば、歯髄、歯小嚢、歯科上皮）は、多くの場合、時間は30の長期間のための組織の生存を保証するために、異なる培地を必要とします。 29 -過去数十年間で、同じ培地を用いた従来の共培養は、感覚神経線維27の口腔および歯の組織を開発するの魅力や反発効果を調査するための非常に短い期間（ 例えば 、2日）のために行われています。しかし、培養期間の延長は、歯の形態形成および細胞分化における神経支配の影響を調査するために、および標的器官内の分岐神経線維の動態を研究するために必要とされます。したがって、非連続的な共培養は、神経、歯の組織の相互作用の研究を実行することがより適切であろう。 マイクロ流体システムは、適切な培養培地中のニューロンと異なる細胞型の共培養を可能にします。それらの標的組織33を含む区画に向かってマイクロチャネルを介して神経細胞体からの軸索の成長を可能にしながら、これらのデバイスでは、歯の組織およびニューロンは、異なる区画に分離されます。マイクロ流体デバイスは、すでにがんや血管新生35で細胞の相互作用へのニューロンとミクログリア34,35との間の相互作用、ならびに細胞を研究するために使用されてきました。さらに、これらのシステムは、ドースの間の相互作用を研究するために使用されていますアル根神経節および骨芽細胞36。 我々は最近、三叉神経節（TG）と歯が時間共培養した場合、マイクロ流体デバイス37での長期間生存することができることを実証しています。また、我々は、異なる発達段階から歯がこれらのin vitroの条件彼らは生体内 37 に示す三叉神経支配で同じ反発や魅力的な効果に維持することを実証しました。このプロトコルは、単純な強力かつ柔軟な方法する共培養神経節/神経と標的組織と制御され、隔離された環境でのこのような相互作用の特定の分子の役割を研究するための情報を提供します。

<table><tbody><tr><td>AXIS Axon Isolation Devices</td><td>Millipore</td><td>AX15010-TC</td><td>Microchannels of different lenght are available</td></tr><tr><td>Laminin</td><td>Sigma Aldrich</td><td>L2020</td><td></td></tr><tr><td>Neurobasal</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>B27</td><td>Gibco</td><td>17504</td><td></td></tr><tr><td>Recombinant Mouse beta-NGF</td><td>R&amp;amp;D Systems</td><td>1156-NG-100</td><td>Human and Rat beta-NGF (R&amp;amp;D Systems) are equivalent</td></tr><tr><td>DMEM-F12</td><td>Gibco</td><td>11320-033</td><td></td></tr></tbody></table>

analysis of developing tooth germ innervation using microfluidic co-culture devices

神経支配は、臓器や組織の発達、ホメオスタシスおよび再生に重要な役割を果たしています。しかし、これらの現象の根底にあるメカニズムはまだよく理解されていません。具体的には、歯の発生と再生における神経支配の役割は無視されます。 in vivo試験でのいくつかは、様々な動物モデルの開発および修復プロセス中に歯の組織の神経支配のパターンに関する重要な情報を提供しています。しかし、これらのアプローチのほとんどは、神経線維と標的器官と組織との間の相互作用の分子的基礎を強調するために最適ではありません。 共培養物を制御し、隔離された環境で神経線維と歯の間の相互作用を調査し、操作するための貴重な方法を構成しています。過去数十年間で、同じ培地を用いた従来の共培養物は、非常に短い期間に行われている（ 例えば 、2日）感覚神経線維上の口腔や歯の組織の開発の魅力や反発効果を調査します。しかし、培養期間の延長は、歯の形態形成と細胞分化の神経支配の影響を調査するために必要です。 マイクロ流体システムは、適切な培養培地中のニューロンと異なる細胞型の共培養を可能にします。我々は最近、三叉神経節（TG）と歯が長期間生存することができることを実証している時に共培養マイクロ流体デバイスで、それらは、これらの条件で、それらが生体内で示す同一の神経支配のパターンを維持します。 これに基づき、我々は単離する方法を説明し、共培養マイクロ流体共培養system.Thisプロトコルで三叉神経節と歯の細菌の開発は神経節/神経および標的組織培養共同すると役割を研究するために、シンプルで柔軟な方法について説明しますCONTRでこのような相互作用の特定の分子のolledと隔離された環境。

これらの結果は、単離された三叉神経節は、単離された歯の細菌の開発は、マイクロ流体デバイスの他の区画内の長期間持続すること、加えて、マイクロ流体デバイスの一の区画で成長し得ることを示します。異なる培地は、二つの区画で使用されると、2つの区画間マイクログルーブは、現像歯胚に向かって三叉神経節からの軸索の伸長を可能にする。 図3マウスの共培養にお?...

Watch this Scientific Journal Video about Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices at JoVE.com

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

マイクロ流体共培養装置の使用歯胚神経支配の開発の分析

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

analysis-developing-tooth-germ-innervation-using-microfluidic-co

Research

JoVE Journal

Neuroscience

8.2K ビュー.  University of Zurich. Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

ビデオ: Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

マイクロ流体文化·プラットフォーム（MCP）ベースの神経軸索とグリア共培養系におけるグリア相互作用へのニューロンのイメージング解析

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

神経科学

Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to overcome the resistance to current treatments and find a new way of cure, modeling the disease as close as possible in an in vitro setting to test new drugs and therapeutic procedures is highly demanding. Studying their fundamental pathobiological processes, including glutamatergic neuron hyperexcitability, will be a real advance in understanding interactions between the environmental brain and pHGG cells. Therefore, to recreate neurons/pHGG cell interactions, this work shows the development of a functional in vitro model co-culturing human-induced Pluripotent Stem (hiPS)-derived cortical glutamatergic neurons pHGG cells into compartmentalized microfluidic devices and a process to record their electrophysiological modifications. The first step was to differentiate and characterize human glutamatergic neurons. Secondly, the cells were cultured in microfluidic devices with pHGG derived cell lines. Brain microenvironment and neuronal activity were then included in this model to analyze the electrical impact of pHGG cells on these micro-environmental neurons. Electrophysiological recordings are coupled using multielectrode arrays (MEA) to these microfluidic devices to mimic physiological conditions and to record the electrical activity of the entire neural network. A significant increase in neuron excitability was underlined in the presence of tumor cells.

Recent works uncover the neuronal impact on high-grade pediatric glioma (pHGG) cells and their reciprocal interactions. The present work shows the development of an in vitro model co-culturing pHGG cells and glutamatergic neurons and recorded their electrophysiological interactions to mimic those interactivities.

グルタミン酸ニューロンと小児の高品位グリオーマ細胞の微小流体デバイスへの共培養による電気的相互作用の評価

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to ...

がん研究

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

単セルの精度で神経共培養の調製

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

歯髄パラクリン信号に反応した神経突起の成長を研究する共培養法

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

発生生物学

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

歯科幹細胞の制御を研究するためのトロウェル型臓器培養の使用

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

外植片培養での歯胚の開発に続いてのためのスライス培養法

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

生物学

A Gut-on-a-Chip Model to Study the Gut Microbiome-Nervous System Axis

The human body is colonized by at least the same number of microbial cells as it is composed of human cells, and most of these microorganisms are located in the gut. Though the interplay between the gut microbiome and the host has been extensively studied, how the gut microbiome interacts with the enteric nervous system remains largely unknown. To date, a physiologically representative in vitro model to study gut microbiome-nervous system interactions does not exist. 
To fill this gap, we further developed the human-microbial crosstalk (HuMiX) gut-on-chip model by introducing induced pluripotent stem cell-derived enteric neurons into the device. The resulting model, 'neuroHuMiX', allows for the co-culture of bacterial, epithelial, and neuronal cells across microfluidic channels, separated by semi-permeable membranes. Despite separation of the different cell types, the cells can communicate with each other through soluble factors, simultaneously providing an opportunity to study each cell type separately. This setup allows for first insights into how the gut microbiome affects the enteric neuronal cells. This is a critical first step in studying and understanding the human gut microbiome-nervous system axis.

neuroHuMiX is an advanced gut-on-a-chip model to study the interactions of bacterial, epithelial, and neuronal cells under proximal and representative co-culture conditions. This model allows unravelling of the molecular mechanisms underlying the communication between the gut microbiome and the nervous system.

腸内細菌叢-神経系軸を研究するためのGut-on-a-chipモデル

The human body is colonized by at least the same number of microbial cells as it is composed of human cells, and most of these microorganisms are located ...

Establishing a Co-Culture of Dental Pulp Cells and Trigeminal Neurons for Cross-Communication

Source: Barkley, C. et al., <a href="https://app.jove.com/v/60809">A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals.</a>&#160;J. Vis. Exp. (2020).
This video showcases a technique for establishing a co-culture of trigeminal neurons and primary dental pulp cells using a transwell filter. In this physically separated condition, mesenchymal cells obtained from dental pulp tissue secrete paracrine signaling molecules that traverse through the pores, promoting the differentiation and migration of neurons towards the mesenchymal cells establishing a co-culture system.

歯髄細胞と三叉神経細胞の共培養を確立し、クロスコミュニケーションを実現

1. Plate Preparation
NOTE: Be sure the plate lid is on during all incubation and rinsing steps outside of the sterile tissue culture hood to prevent ...

JoVE Encyclopedia of Experiments

神経細胞培養技術

共培養と相互作用の研究

Generating Neuromuscular Junctions Using a Microfluidic Device

Source: Stoklund Dittlau, K. et al., <a href="https://app.jove.com/v/62959">Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices.</a>&#160;J. Vis. Exp. (2021)
The video demonstrates the formation of neuromuscular junctions using a microfluidic device. Neural precursor cells and muscle progenitor cells are seeded into different wells within the device and left to mature into motor neurons and myotubes, respectively. The creation of a volume and chemical gradient leads to the development of active neuromuscular junctions.

マイクロ流体デバイスを用いた神経筋接合部の生成

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
All reagents and equipment used in ...

高度な神経細胞モデル

Analyzing Tooth Germ and Trigeminal Ganglia Interactions Using a Microfluidic Co-culture System

Source: Pagella, P., et al. <a href="https://app.jove.com/v/53114">Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices.</a> J. Vis. Exp. (2015).
This video demonstrates the utilization of a microfluidic device to study the interactions between tooth germ pulp and trigeminal ganglia. It showcases the placement of tissues inside the microfluidic device and provides a detailed visualization of trigeminal neurite growth toward the tooth germ through the device&#39;s microgrooves. Additionally, it highlights the role of tooth-derived molecular factors in promoting or inhibiting neurite extension into the tooth germ pulp, which is crucial for ensuring efficient postnatal tooth innervation.