1442キロバイトと欧州委員会のFP7のグラントPIRG08-GA-2010から276989、NEUREX、そしてドイツ研究財団助成SCHAにSS、教育研究連邦省（BMBF 01 EO 0803）にFazit財団大学院フェローシップでサポートされている/ CS 3-1〜著者らは相反する経済的利益を持っていません。

1。混合皮質細胞の単離及びメッキ星状膠細胞の培養のための混合皮質細胞の分離は、P4の仔マウスにP1を用いて行うことができる。適切なアストロサイト密度を達成するためには、T75組織培養フラスコあたり4マウス子犬皮質を使用する必要があります。したがって、次のプロトコル内のボリュームは4仔マウスを用いた細胞調製のために計算される。 <ol><li>解剖の手順を開始する前に、星状膠細胞培養培地のprewarm 30ミリリットル（DMEM、高グルコース+ 10％熱不活性化ウシ胎児血清+ 1％ペニシリン/ストレプトマイシン、表を参照してください）​​37℃ 37℃で1時間のための細胞培養グレードの水で50μg/ mlの濃度°CO 2インキュベーター中でCのポリ-D-リジン（PDL）の20mlで1コートT75フラスコ。 </li><li>解剖の手順については、必要なすべての試薬および材料を準備します。次のものが必要となります外科用ハサミ、滑​​らかな細かい鉗子、フラットチップピンセット、紙タオル、ゴミ袋、70％エタノール氷の上のd 2解剖皿（3.5 cmの直径）が2 mlのHBSSそれぞれでいっぱい。 </li><li>優しくホールドし、70％エタノールで仔マウスの頭と首をスプレーします。動物は、はさみを使用して断頭により屠殺されています。 </li><li>頭蓋骨を明らかにするために頭皮に沿って前方に後方正中切開し、実行します。 </li><li>首から鼻に慎重に頭蓋骨を切った。二つの追加カットは、脳へのさらなるアクセスを許可するために実行されます：最初のカットは、嗅球、頭蓋底から頭蓋を切断する小脳の劣る他の1の前方作られています。 </li><li>フラットチップピンセットを用いて、頭蓋のフラップはそっと側にフリップされ、脳を取り出し、HBSSで充填された第1解剖皿に置かれます。氷の上に戻って皿に置き、すべての4頭脳を収穫し続けています。 </li><li>解剖手順の残りの部分は実体顕微鏡下で行われる。まず、嗅球、小脳は罰金を使用して除去される解剖用ピンセット（ 図1B）。 </li><li> 、皮質を取得微鉗子による脳の後端をつかむためには、半球の間の正中切開を行い、作成した木立に鉗子の2番目のセットを挿入し、剥がれから皮質の板状構造脳。 </li><li>慎重に細かい鉗子（ 図1D &#39;）で引いて皮質半球から髄膜を切開する。このステップでは、髄膜細胞および線維芽細胞による最終アストロ文化の汚染を避けることができます。 HBSSで充填された第2の皿に準備された皮質半球を移し、氷の上にそれを返します。全4皮質とそれに応じて続行します。 </li><li>最後に、鋭い刃（約4〜8倍）を使用して小片に各皮質半球を切った。 </li><li>無菌条件下で、1 50mlのFalconチューブに皮質の部分を転送し、22.5ミリリットルの総量にHBSSを追加します。 </li><li> 2.5％トリプシン、ミックスの2.5 mlを加え、インキュベート37℃の水浴中で組織℃で30分間。 10分毎に揺れ、時折混和する。 </li><li>ペレット皮質組織片を300×gで5分間遠心します。 </li><li>慎重にデカンテーションにより上清を除去します。組織ペレットを失うことを避けるためには、機械的にそれはピペットを用いて保持することができる。組織片は単一の細胞（20〜30倍）に解離されるまで、10ミリリットルのプラスチックピペットを用いて10ミリリットルアストロメッキ媒体と活発なピペッティングを追加することによって、単一の細胞懸濁液に組織を解離する。アストロメッキ培地を用いて20mlに音量を調整します。あなたは、血球計数器を用いて計数することにより、単一細胞に証明皮質組織の解離をすることができます。 4マウス子犬皮質の一つ準備は10-15×10 6解離単一細胞が得られるはずです。 </li><li> T75培養フラスコからPDLを吸引し、プレートを37℃で解離単一細胞懸濁液とインキュベートし、CO 2インキュベーター内のC。 </li></ol> 2。 ENRを取得ichedアストロ文化<ol><li>その後混合皮質細胞と全3日間のめっき後の培地2日を変更します。 </li><li>星状膠細胞はコンフルエントとオーバーレイミクログリアアール星状膠細胞層に露出して座ったり、すでにアストロ層（ 図2）から切り離されて7から8日後、ミクログリアを削除するオービタルシェーカーで30分間180rpmでT75フラスコを振る。を含む上清を捨て、ミクログリアや、文化に希望とミクログリアを調べる場合は、それをスピンダウンと文化28,29用のプレート。 </li><li> 20ミリリットル新鮮なアストロサイトの培養液を追加し、オリゴデンドロサイト前駆細胞（OPC）を除去するために6時間240rpmでフラスコを振って続行します。いくつかのOPCは完全にアストロ層からデタッチされませんので、OPCの汚染を防止するために1分間手で激しく振り続けています。あなたは文化OPCの29にしたい場合、上清を捨てるか、またはそれをスピンダウンし、プレート。 </li><li>残りconflueをすすぐPBSで2回NTアストロ層、PBSを吸引除去し、37℃のCO 2インキュベーター中で5ミリリットルトリプシン-EDTAとインキュベートを追加℃に星状膠細胞の剥離を5分毎にチェックして、あなたの手の手のひら（2-3倍）に対してフラスコを押すことで、星状膠細胞の剥離を施行。 </li><li>星状膠細胞を培養フラスコから切り離された後、5分間180 xgで星状膠細胞の培養培地5ml、スピンセルを追加し、上清を吸引し、40 mlの新鮮なアストロメッキmediumを加えてください。一つT75組織培養フラスコは、最初の細胞分裂後、6×10の周りの細胞が得られるはずです。 37℃で2 T75培養フラスコとインキュベートで細胞をプレート°CO 2インキュベーター内のC。 2〜3日ごとに培地を変更します。 </li><li>最初の分割アストロサイトは、実験を行う前に、適切な細胞濃度は24〜48時間でメッキされて12から14日後。一つT75組織培養フラスコは、第2のセル分割後1.5から2×10 6個の細胞の周りが得られるはずです。 </li></ol>

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特別な利害関係は宣言されません。

ここに概説された方法は、もともと1980年に27マッカーシーとde Vellisによって記述げっ歯新生児の脳からアストロサイトの培養準備に基づいています。生後P1からP4をマウス脳に皮質星状膠細胞の分離と培養の変法は速いここに提示された、純粋な一次星状膠細胞を生み出す、再現性の高いです。この手法は、簡単にこのようなラットやブタ由来や脊髄などの他の脳領域からなどの他の?...

アストロサイトは、中枢神経系（CNS）において非常に豊富な細胞型である。人間の大脳皮質のニューロン1あたり1.4アストロサイトが存在するのに対し、ニューロンへのアストロサイトの比率は、マウスおよびラットの大脳皮質で1:3である。アストロサイト機能に興味は近年劇的に増加している。アストロサイトの主な機能は、ニューロン2,3に構造的および代謝のサポートを提供する上で自らの役割である。アストロサイトのための新たに発見された役割は、機能の広いスペクトルをカバーしています。これらは、開発4月6日の間に開発の軸索と特定の神経芽細胞の遊走を誘導、シナプス伝達関数、神経回路7から9によるシナプス強度と情報処理、血液脳関門における役割（BBB）の形成10と整合性11月13日を含む脳血管トーン14と規制。アストロサイトのもう一つの大きな特徴は、傷害への応答です。 astrocyt病的条件下でES反応となり、さらに中間フィラメントグリア線維性酸性タンパク質（GFAP）と抑制性細胞外マトリックス（ECM）タンパク質15,16の発現をアップレギュレートする。反応性アストロサイトは、コンドロイチン硫酸プロテオグリカン（CSPG）家族のアストロ分泌ECMタンパク質、CNS損傷15から17の後に軸索の再生を阻害する主要な要因の主に構成されていますグリア瘢痕を形成することによって、健康な組織から損傷部位の境界を定める。 アストロサイトは後期胚発生および出生後の早い時期に放射状グリア（RG）の細胞に由来する。アストロサイトの仕様が発生した後、アストロサイト前駆体は、彼らは終末分化のプロセスを開始し、最終的な位置に移動する。 生体内では 、アストロサイトは彼らの典型的な形態18,19によって示されるように3〜4週間、出生後成熟したように見える。 RGの細胞の亜集団は、脳室下帯のアストロサイト（タイプB細胞）に変換します。 BOTH、RGとB型細胞はそれぞれ、開発中および成人におけるアストロサイトのような神経幹細胞（NSC）として機能します。アストロサイトと同様に、RGとB型細胞はまた、これらのマーカーはもっぱら特に成人星状膠細胞を標識するために使用できないことを示す、アストロサイト特異的グルタミン酸トランスポーター（GLAST）、脳脂質結合タンパク質（BLBP）、およびGFAPを発現している。健康な脳、RGとB型細胞では分裂しない大人実質アストロサイトとは対照的に、自己再生する能力のような幹細胞の可能性を示す。アストロサイトの異常は、アルツハイマー病20,21、ハンチントン病22、パーキンソン病23、レット症候群24とアレキサンダー病25を含む、数多くの病態に関与している。また、アストロサイトはアストロサイトの活性化とアストログリア瘢痕形成16,26につながる、CNSのすべての侮辱に反応します。以下の脳trを形成アストロサイトグリア瘢痕アウマまたは脊髄損傷は、神経再生15を防止プライム障壁であると考えられている。 細胞の精製された集団を分離し、維持するための信頼できる方法の開発は、神経系の我々の理解に不可欠となっています。マッカーシーとde Vellisによる先駆的な仕事は、新生児ラット組織27からアストロサイトのほぼ純粋な培養物を調製するためにこれまでに研究者を可能にします。多くはマウス大脳皮質アストロサイトを単離するためのわずかに修正された形でここに提示され、この方法を用いて、星状膠細胞生物学を学んだされています。 in vivo試験、アストロサイトと同様にin vitro培養で説明を使用して得られた馴化培地中で補完して、アストロサイト機能にさらなるゲイン洞察に貴重なツールです。

<table border="1"><tbody><tr><td> ワーキングソリューションの名前 </td><td> 会社 </td><td> カタログ番号 </td><td> 最終濃度 </td></tr><tr><td></td><td></td><td></td><td> 星状膠細胞培養培地 </td></tr><tr><td> DMEM、高グルコース</td><td>ライフテクノロジーズ</td><td> 31966-021 </td><td></td></tr><tr><td>熱不活性化FBS、 </td><td>ライフテクノロジーズ</td><td> 10082-1​​47 </td><td>最終濃度：10％ </td></tr><tr><td>ペニシリン - ストレプトマイシン</td><td>ライフテクノロジーズ</td><td> 15140-122 </td><td>最終濃度：1％ </td></tr><tr><td></td><td></td><td></td><td> 脳組織の発掘のためのソリューションestion </td></tr><tr><td> HBSS </td><td>ライフテクノロジーズ</td><td> 14170-088 </td><td></td></tr><tr><td> 2.5％トリプシン</td><td>ライフテクノロジーズ</td><td> 15090-046 </td><td>最終濃度：0.25％ </td></tr><tr><td></td><td></td><td></td><td> その他 </td></tr><tr><td> 70％（vol / vol）のエタノール</td><td>ロート</td><td> 9065.2 </td><td></td></tr><tr><td>ポリ-D-リジン</td><td>ミリポア</td><td> -003-E </td><td> 50μg/ mlの</td></tr><tr><td>水</td><td> PAA </td><td> S15-012 </td><td>細胞培養グレード</td></tr><tr><td> PBS </td><td> PAA </td><td> H15-002 </td><td>細胞培養グレード</td></tr><tr><td> 0.05％お試しくださいpsin-EDTA </td><td>ライフテクノロジーズ</td><td> 25300-062 </td><td></td></tr><tr><td> 0.45μmの滅菌フィルター</td><td>縫工筋</td><td> 16555 </td><td></td></tr><tr><td> 3.5センチメートルシャーレ</td><td> BDファルコン</td><td> 353001 </td><td></td></tr><tr><td> 15mlファルコンチューブ</td><td> BDファルコン</td><td> 352096 </td><td></td></tr><tr><td> 50mlのFalconチューブ</td><td> BDファルコン</td><td> 352070 </td><td></td></tr><tr><td> 75cm 2の組織培養フラスコ</td><td> BDファルコン</td><td> 353136 </td><td></td></tr><tr><td>細かい鉗子、 </td><td>デュモン</td><td> 2から1032まで、2から1033まで</td><td> ＃3cは、第5 </td></tr><tr><td>鉗子、フラットチップ</td><td> KLSマーティン</td><td> 12-120-11 </td><td></td></tr><tr><td> 13センチメートル手術用のハサミ</td><td> AESCULAP </td><td> BC-140-R </td><td></td></tr><tr><td>実体顕微鏡</td><td>ライカ</td><td></td><td> MZ7.5 </td></tr><tr><td>実体顕微鏡+カメラ</td><td>ライカ</td><td></td><td> MZ16F; DFC320 </td></tr><tr><td>顕微鏡+カメラ</td><td>ツァイス、キヤノン</td><td></td><td>プリモヴェール、のPowerShot A650 ISを</td></tr><tr><td>遠心</td><td>エッペンドルフ</td><td> 5805000.017 </td><td> Centrifuge5804R </td></tr><tr><td>オービタルシェーカー</td><td>サーモサイエンティフィック</td><td> SHKE 4450-1CE </td><td> MAXQ 4450 </td></tr><tr><td>水浴</td><td> JULABO </td><td></td><td> SW20、37℃ </td></tr></tbody></table>

isolation and culture of mouse cortical astrocytes

アストロサイトは、哺乳類の脳に豊富に細胞型で、まだ多くのそれらの分子的および機能的特性について学んだことが残っている。 体外アストロサイト細胞培養系では詳細にこれらのグリア細胞の生物学的機能を研究するために使用することができます。このビデオプロトコルは仔マウスの混合皮質細胞の単離および培養により純粋アストロサイトを取得する方法を示します。法は生存ニューロンの欠如およびアストロサイト、オリゴデンドロサイトおよびミクログリア、文化の中枢神経系の3つの主要なグリア細胞集団の分離に基づいています。文化の最初の日中に代表的な画像は、混合細胞集団が存在することを実証し、アストロサイトがコンフルエントになるとミクログリア、オリゴデンドロサイトから分離する必要があるとき、時点を示しています。また、純度と十分に確立されたために免疫組織化学染色を用いて培養アストロサイトのアストロサイトの形態を示し、新たにアストロサイトのマーカーを説明した。この培養系は簡単に星状膠細胞生物学の様々な側面を研究するための純粋なマウスのアストロサイトおよびアストロサイト馴化培地を取得するために使用できます。

完全なマウス脳（ 図1A）、小脳、嗅球の分離の際（ 図1B）が除去されなければならない。皮質は、マウス脳幹（ 図1C）と個々の皮質（ 図1D &#39;）の髄膜の剥離し慎重に（ 図1E）は削除されます。髄膜は、硬膜動脈系と髄膜の細胞や線維芽細胞による最終アストロサイト培養の汚染の除去が不完全の結果は明白です。 <p class="jov...

Watch this Scientific Journal Video about Isolation and Culture of Mouse Cortical Astrocytes at JoVE.com

Isolation and Culture of Mouse Cortical Astrocytes

アストロサイトは、脳の正常な発達と機能、および中枢神経系の修復に欠かせない基本的な生物学的プロセスに参加して多目的な細胞であると認識されてきた。ここでは、中枢神経系細胞のこの主要なクラスの生物学を研究するために純粋なマウスアストロサイトの培養液を得るための迅速な方法を提示します。

マウス大脳皮質アストロサイトの単離および培養

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

isolation-and-culture-of-mouse-cortical-astrocytes

Research

JoVE Journal

Neuroscience

90.8K ビュー.  University of Freiburg. アストロサイトは、脳の正常な発達と機能、および中枢神経系の修復に欠かせない基本的な生物学的プロセスに参加して多目的な細胞であると認識されてきた。ここでは、中枢神経系細胞のこの主要なクラスの生物学を研究するために純粋なマウスアストロサイトの培養液を得るための迅速な方法を提示します。

ビデオ: Isolation and Culture of Mouse Cortical Astrocytes

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofectionとマウス胎児海馬および皮質ニューロンの初代培養

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

神経科学

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

マウス胚の運動ニューロンのレクチンに基づく分離と文化

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

初代ラット皮質ニューロンとグリアのBilaminarの共培養

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

胎児マウスから海馬ニューロンの単離および培養

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

人間の茸状の味覚乳頭細胞の単離および培養

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

モデル星細胞腫発症機序<em&gt;体外</em&gt;と<em&gt;インビボ</em&gt;皮質アストロサイトまたは条件付き、遺伝子改変マウスから神経幹細胞を使用した

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately.

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia.

マウス海馬アストロサイトにおける性差後<em&gt;インビトロ</em&gt;虚血

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

マウス梁下ゾーンから成体神経幹細胞の単離および培養

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

マウス胚性幹細胞の皮質介在ニューロン前駆細胞への分化

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.