Name: isolation and culture of primary neurons and glia from adult rat urinary bladder
Uploaded: 2020-05-23T14:45:00.000+00:00
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Description: Watch this Scientific Journal Video about Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder at JoVE.com

本研究は中国国立自然科学財団(グラント81673676)と東莞科学技術局(グラント2019622101002)によって支援されました。著者らは、メアリーローズ・サリバン博士(ハーバード大学医学部外科助教授)に技術コンサルティングをしてくれたことに感謝している。

すべての実験プロトコルと動物の手順は、国家研究評議会の倫理原則ガイドラインに準拠しています。
1. 材料の準備
<ol><li>実験を行う前に、オートクレーブを使用してすべての機器とddH2Oを殺菌します。器械は外科用はさみ、眼科用はさみ、鉗子、スプーン核の分け器、ガラス皿(直径60-100 mm)、およびガラスブレーカに限定されない。</li>
	<li>以下のようにクレブス溶液を調製する(表1):使用前にddH2Oと一緒にすべての化学物質を溶解します。CaCI2 を別々に溶解し、沈み込みを避けるために攪拌しながら混合溶液にゆっくりと加えます。</li>
	<li>10%の牛血清(FBS)と1%の抗生物質/抗ミコティック(100x)でF12培地で構成されるリンス培地を準備します。F12培地44.5mLにFBS5 mLと抗生物質/抗菌薬0.5mLを加えます。</li>
	<li>2%B-27、1%FBS、1%L-グルタミン、1%抗生物質/抗ミキ酢(100x)、および0.1%グリア由来神経栄養因子(GDNF)を有する神経基底A培地を調製する。B-27の200 μL、FBSの100 μL、L-グルタミンの100 μL、抗生物質/抗抗抗ミキティックの100 μL(100x)、および9.5mLのGDNF(10 μg/mL)の10 μLを神経基底A培地に加える。 注:B-27、L-グルタミンおよびGDNFの鮮度を確保するために使用の1週間以内にニューロンメディアAを準備してください。</li>
	<li>ニューロンメディアAの調製と同じ手順を用いてニューロンメディアBを準備するが、FBSを加えることなく。</li>
	<li>20mgのコラゲナーゼII型、6mgの牛血清アルブミン、200μLの抗生物質/抗ミキサン(100x)を酸素安定クレブス溶液10mLに溶解して消化液1を調製した。</li>
	<li>0.25%トリプシンの1 mLをハンクのバランスの取れた塩溶液の4 mLで希釈して消化液2を調製する。</li>
	<li>コーティングされたカバーリップでプレートを準備します。<ol>
<li>ガラスカバーリップを48ウェルの培養プレートに入れる場合は、層流のベンチで鉗子を使用します。</li>
		<li>ポリD-リジンストックとして0.1 mg/mLの濃度にddH2OでポリD-リジンを希釈します。在庫を–20°Cで保管し、使用前に解凍してください。</li>
		<li>各カバースリップの上にポリD-リジンストック40 μLを加え、室温で10分間インキュベートします。 注:異なる基底領域を持つ別のプレートの場合は、4 μg/cm2に濃度を調整します。たとえば、24ウェルプレートからの1ウェルの基底面積が2cm2の場合、80μLのポリD-リジンストックをウェルに移す必要があります。各cm2 は4μgのポリD-リジンを含んでいる。</li>
		<li>48ウェルプレートでポリD-リジンを取り除き、ddH2O thriceでカバーリップをすすいでください。</li>
		<li>プレートが無水であることを確認するために、少なくとも30分間ラミナーフローフードでプレートを乾燥させます。</li>
		<li>ラミニンコーティングの前に4°Cでプレートを保管してください。プレートを−20°Cで保管することは、長期保存に適しています。</li>
		<li>ラミニンを4°Cで解凍する。 ラミニンを薄い50 μg/mLの濃度にddH2Oで希釈します。在庫を-20°Cで保管し、使用前に4°Cで解凍してください。</li>
		<li>ピペットを使用して、希釈したラミニンを各カバースリップの上部に100 μL、4°Cで1時間インキュベートラミニンを1時間移動します。 注:異なる基底領域を持つ別のプレートの場合は、5 μg/cm2に濃度を調整します。例えば、24ウェルプレートからの1ウェルの基底面積が2cm2の場合、希釈したラミニンを200μLのウェルに移します。各cm2 は5μgのラミニンを含んでいる。</li>
		<li>ラミニン溶液を取り出し、ddH2Oでカバーリップを一度すすい取ります。スクレイピングを避けるために、カバースリップの端にこの操作を実行します。 注:コーティングされたカバーリップは、せいぜい2週間4°Cで保管することができます。</li>
	</ol>
</li>
</ol>2. 膀胱の収穫
<ol><li>5週齢のスプレイグ-ドーリーラットを入手する。</li>
	<li>安定した酸素状態とpHレベルに到達するために、氷浴中の少なくとも30分間、クレブス溶液にカルボゲン(95%酸素、5%CO2)を注入します。</li>
	<li>頸椎脱臼による安楽死後、ラットを殺菌のために30sの75%エタノールに浸す。</li>
	<li>殺菌した外科用タオルの上にラットを置き、彼らの腹部を露出させる。腹腔を開き、はさみと鉗子のセットで膀胱を明らかにする。</li>
	<li>膀胱をそっと持ち上げ、膀胱頸部から膀胱を別のハサミと鉗子で切り取り、クロスコンタミネーションを避けます。細胞の生存率を向上させるために、冷たい酸素安定クレブス溶液に膀胱を迅速に置きます。 注:膀胱を取り除いたら、ニューロン生存の見通しを改善するために、次の操作を迅速に行います。</li>
	<li>3つのガラス皿とガラスブレーカにクレブス溶液を加えます。</li>
	<li>ガラス皿とガラスブレーカを氷浴でクレブス溶液で準備して、予冷します。</li>
	<li>これらのコンテナに 1 ~ 3 の番号を付けて、混乱を防ぎます。</li>
	<li>各ガラス皿を鉗子とスプーン核仕切りと組み合わせる。</li>
	<li>ガラス皿1では、眼科用はさみで膀胱を切り開き、鉗子とスプーン核仕切りで開きます。</li>
	<li>ガラスブレーカ1で膀胱をすすいで、ガラス皿2に入れる。</li>
	<li>ガラス皿2の鉗子および眼科用はさみを使用してティッシュ表面の付着脂肪を除去する。</li>
	<li>ガラスブレーカ2で膀胱をすすい、ガラス皿3に入れる。</li>
	<li>鉗子とスプーン核仕切り器をガラス皿3に使用して膀胱を静かにこすり、外因性の付着物を取り除く。</li>
	<li>ガラスブレーカ3で膀胱をすすい、14 mLの冷たいクレブス溶液を用いた15mL遠心管に膀胱を移し、356 x g および4°Cで1分間サンプルを回す。</li>
	<li>上記の手順を、他の 2 つのチューブで 2 回、他の 2 つのチューブで 2 回繰り返して、汚染を減らします。</li>
</ol>3. 二段階膀胱消化
<ol><li>1 mLの消化液1を含む2mLバイアルに遠心管から膀胱を移す。眼科用はさみを使用して、膀胱を溶液中の小片(1mm未満)に切ります。</li>
	<li>膀胱溶液を9 mLの消化液1と滅菌細胞培養皿(直径100mm)で混合する。5%CO2、37°C、および200rpm下で1時間の振盪インキュベーターでステップ1消化を行います。</li>
	<li>ステップ1の消化後、溶液を4°Cで356xgで8分間遠心分離する。</li>
	<li>消化液2を予熱用の37°C水浴に入れる。</li>
	<li>遠心分離後、消化液1を含む上清を除去し、細胞沈降物を収穫する。残りの液体の一部は許可されています。溶液の完全な除去は、細胞の損失を促進する可能性があります。</li>
	<li>細胞沈積物と温かい消化液2を15 mL遠心分離管に混ぜ、37°Cの水浴で5分間消化しながら混合物を振る。ステップ2消化またはニューロンの7分を超えないで滅びる。</li>
	<li>消化後、10 mLのコールドリンス培地を混合したトリプシンを直ちに非アクティブ化する。 メモ:0°C~4°Cで以下の手順を実行します。 氷浴はそのような状態を提供することができます。</li>
	<li>遠心分離後の細胞沈込を4°Cで4°Cで8分間収穫します。残りのトリプシンは細胞増殖に有害であるため、できるだけ多くの培地を取り除きます。</li>
	<li>3 mLのニューロン培地で沈澱を緩やかに中断する。高い生存率のために細胞を含む溶液中に気泡が生成されないようにしてください。</li>
	<li>70 μmのセルストレーナーを介して、50 mL遠心分離チューブに混合培地をフィルターします。</li>
	<li>30rpmのシェーカーで30分間氷浴に入れ、濾過液を保管します。この手順は必須ではありませんが、推奨されます。</li>
	<li>4°Cで356xgで8分間遠心分離して細胞を採取し、ニューロン培地Aの1mLで細胞ペレットを静かに再懸濁する。</li>
	<li>調製した48ウェルプレートの各ウェルに500μLの細胞混合物を加えます。</li>
	<li>培養細胞は、37°Cおよび5%CO2で培養器中に含まれる。</li>
	<li>すべての培地を1時間でニューロン培地Bに交換し、無血清培養を行う。</li>
	<li>ニューロン培地Bの半分を3日ごとに変更します。 注意:ニューロンは、培養5~7日後に免疫細胞化学実験を行う準備ができています。</li>
</ol>

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著者らは、大きな利益相反を宣言していない。

プレートの準備 免疫蛍光またはカルシウムイメージング実験用の6-、12、または48ウェル培養プレートでのガラスカバーリップの使用は、経済的でサンプル節約的な操作です。細胞は、一次細胞培養の調製中にカバースリップのないプレートでよく成長する。そのため、カバーリップは、ウェスタンブロットまたはポリメラーゼ連鎖反応などの実験で欠かせない。さらに、コー?...

下部尿路には、尿の定期的な保存と、1のミチュリンの2 つの機能があります。下部尿路神経系(LUTNS)はこれらの機能を制御し、繊細で多くの神経障害の影響を受けやすく、先天性(ポルフィリン症)、後発性疾患状態(糖尿病性膀胱障害)、薬物誘発(出血性膀胱炎)、または外傷(脊髄損傷)2,3,4,6,7, 7, 7, 7, 7, 7, 7,7, 7.生理学的/病理学的研究では、生体内およびインビトロ実験も同様に重要である。LUTNSに関するインビボ研究は、しばらくの間、臓器、細胞、および分子レベルで行われてきたが、尿膀胱からの一次ニューロンに関するインビトロ研究はほとんど存在しない8,9である。今回の研究は限られていますが、他の研究者が改善できるように、この分野の研究を開拓したいと考えています。このようにして、この共培養は、膀胱ニューロン機能不全などの表現型における生理学的機能不全の細胞的理解につながる可能性がある。
離散層への筋肉細胞の明確な方向性を持つ腸筋とは対照的に、膀胱の筋肉は組織化されていない10である。したがって、膀胱の外層を剥離する代わりに、この方法は、手術困難を低減し、高い細胞生存率のための前処理時間を短縮するために膀胱全体を消化することを提案する。
この方法に従って、ニューロンと他の細胞の混合培養を得ることができる。その存在は生体内環境11を模倣するので、他の細胞は不可欠である。また、このような細胞は、培地で利用できない物質を提供する。
この方法は、消化のための2つのステップを伴う。まず、コラゲザーゼII型は、コラーゲンを加水分解するために使用され、続いてトリプシンが、組織を細胞10に解化する。このようにして、膀胱組織は単一細胞に分散され、その後比較的独立して成長する。ニューロンの培養が成熟すると、ニューロンは、イメージングまたは機能的アッセイに使用することができます。

<table><tbody><tr><td>0.25% trypsin</td><td>Gibico</td><td>15050065</td><td>Enzyme digestion</td></tr><tr><td>48-well culture plate</td><td>Corning</td><td>3548</td><td>Coating dish</td></tr><tr><td>antibiotic/antimycotic</td><td>Gibico</td><td>15240062</td><td>Culture media/Rinse media</td></tr><tr><td>Anti-Glial Fibrillary Acidic Protein Antibody</td><td>Santa Cruz</td><td>sc-33673</td><td>ICC</td></tr><tr><td>B-27</td><td>Gibico</td><td>17504044</td><td>Culture media</td></tr><tr><td>BSA Fraction V</td><td>Gibico</td><td>332</td><td>Enzyme digestion</td></tr><tr><td>Choline Acetyltransferase Antibody</td><td>Abcam</td><td>ab18736</td><td>ICC</td></tr><tr><td>CO2 Incubator</td><td>Heraeus</td><td>B16UU</td><td>Cells culture</td></tr><tr><td>Collagenase type II</td><td>Sigma</td><td>2593923</td><td>Enzyme digestion</td></tr><tr><td>DMEM/F-12</td><td>Gibico</td><td>11330032</td><td>Rinse media</td></tr><tr><td>DYNLL2 Antibody</td><td>Santa Cruz</td><td>sc-13969</td><td>ICC</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibico</td><td>10100147</td><td>Culture media/Rinse media</td></tr><tr><td>Forceps</td><td>Shanghai Jin Zhong Medical Devices</td><td>1383</td><td>10 cm; Sterile operation</td></tr><tr><td>Glass breakers</td><td>Huan Qiu Medical Devices</td><td>1101</td><td>50 ml; Sterile operation</td></tr><tr><td>Glass coverslips</td><td>WHB Scientific</td><td>WHB-48-CS</td><td>Coating dish</td></tr><tr><td>Glass dishes</td><td>Huan Qiu Medical Devices</td><td>1177</td><td>100 mm; Sterile operation</td></tr><tr><td>Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 488</td><td>Southernbiotech</td><td>3050-30</td><td>ICC</td></tr><tr><td>Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 555</td><td>Southernbiotech</td><td>3050-30</td><td>ICC</td></tr><tr><td>Hoechst 33342</td><td>BD</td><td>561908</td><td>ICC</td></tr><tr><td>Laminar flow bench</td><td>Su Jie Medical Devices</td><td>CB 1400V</td><td>Sterile operation</td></tr><tr><td>Laminin</td><td>Sigma</td><td>L2020</td><td>Coating dish</td></tr><tr><td>L-glutamine</td><td>Gibico</td><td>25030081</td><td>Culture media</td></tr><tr><td>MAP-2 Antibody</td><td>Affinity</td><td>AF5156</td><td>ICC</td></tr><tr><td>Murine GDNF</td><td>Peprotech</td><td>AF45044</td><td>Culture media</td></tr><tr><td>Neurobasal-A Medium</td><td>Gibico</td><td>10888022</td><td>Culture media</td></tr><tr><td>Ophthalmic scissors</td><td>Shanghai Jin Zhong Medical Devices</td><td>J21010</td><td>12.5 cm; Sterile operation</td></tr><tr><td>Pipettes</td><td>Eppendorf</td><td>3120000240</td><td>100-1000 ul; Reagent and sample pipetting</td></tr><tr><td>Pipettes</td><td>Eppendorf</td><td>3120000267</td><td>10-100 ul; Reagent and sample pipetting</td></tr><tr><td>Poly-D-lysine</td><td>Sigma</td><td>P7280</td><td>Coating dish</td></tr><tr><td>Refrigerated centrifuge</td><td>Ping Fan Instrument</td><td>TGL-16A</td><td>Enzyme digestion</td></tr><tr><td>Shaking incubator</td><td>Haimen Kylin-Bell Lab Instruments</td><td>T8-1</td><td>Enzyme digestion</td></tr><tr><td>SLC17A9 Antibody</td><td>MBL International</td><td>BMP079</td><td>ICC</td></tr><tr><td>Spoons nucleus divider</td><td>Shanghai Jin Zhong Medical Devices</td><td>YZR030</td><td>12 cm; Sterile operation</td></tr><tr><td>Substance P Antibody</td><td>Santa Cruz</td><td>sc-58591</td><td>ICC</td></tr><tr><td>Surgical scissors</td><td>Shanghai Jin Zhong Medical Devices</td><td>J21130</td><td>16 cm; Sterile operation</td></tr><tr><td>Surgical towel</td><td>Fu Kang Medical Devices</td><td>5002</td><td>40 x 50 cm; Sterile operation</td></tr><tr><td>Synapsin-1 Antibody</td><td>CST</td><td>5297T</td><td>ICC</td></tr><tr><td>Tubulin beta Antibody(&amp;beta;-III-tubulin)</td><td>Affinity</td><td>AF7011</td><td>ICC</td></tr></tbody></table>

isolation and culture of primary neurons and glia from adult rat urinary bladder

下部尿路は、2つの主要な機能、すなわち、定期的な尿貯蔵およびミチュリションを有する。これらの機能は、中央および末梢神経調節を介して仲介されます。低尿路神経系に関する広範な研究が行われているが、ほとんどの研究は、原発性文化に焦点を当ててきた。このプロトコルは、スプレイグ-ドーリーラットからの膀胱ニューロンとグリアの分離と培養のための方法を導入します。この方法では、ニューロンおよびグリアを37°C、5%CO2インキュベーターで5〜7日間インキュベーターでインキュベートした。その結果、それらは関連する後続の免疫蛍光実験に適した成熟した形状に成長した。細胞は光学顕微鏡を用いて形態学的に観察した。ニューロン、シナプス小胞、およびグリアは、それぞれβ-III-管状およびMAP-2、シナプシン-1、およびGFAP染色によって同定された。一方、免疫細胞化学は、コリンアセチルトランスビセラーゼ、DYNLL2、SLC17A9などのいくつかの神経伝達物質関連タンパク質に対して行われました。

一次細胞培養の過程で、獲得した細胞は、付着状態の前に明るく明確な境界で丸くした。ニューロンが成長するにつれて、樹状突起と軸索は明確になり始めました。5~7日間培養した後、ニューロンは長い投影で成熟した形に達し、イメージングや機能研究に最適でした。不純物および細胞デブリの大部分は、メディアの変化により除去することができたが、ポリD-リジンおよびラミニンコー?...

Watch this Scientific Journal Video about Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder at JoVE.com

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

このプロトコルは、一次ニューロンとラット膀胱からのグリア分離のための反復可能なプロトコルを確立し、さらなる細胞実験を試みる。

成人ラット尿膀胱からの一次ニューロンとグリアの分離と培養

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

isolation-culture-primary-neurons-glia-from-adult-rat-urinary

Research

JoVE Journal

Neuroscience

3.8K ビュー.  Guangzhou University of Chinese Medicine. このプロトコルは、一次ニューロンとラット膀胱からのグリア分離のための反復可能なプロトコルを確立し、さらなる細胞実験を試みる。

ビデオ: Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

成体ラットおよびヒト脊髄の脳室周囲領域から神経幹/前駆細胞の単離

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

発生生物学

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

新生仔ラットの脳組織の混合グリア細胞培養物から主要なミクログリアの分離

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

免疫・感染学

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 ...

神経科学

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

ラット胚神経細胞の分離と文化：クイックプロトコル

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

胚性齧歯類の脳から単離、プライマリ脳ドーパミンニューロンの文化と長期メンテナンス

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

成体マウスの脊髄組織からの初代混合グリア培養の調製

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

伝達物質の放出を勉強する後根神経節細胞の分離と培養

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

成人ヒト脳組織からのヒトミクログリアの取得

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

Isolation and Culture of Primary Neurons and Glia from a Rat Urinary Bladder

Source: Wang, R. et al., <a href="https://app.jove.com/v/61177">Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder.</a> J. Vis. Exp. (2020).
This video demonstrates a technique for generating a primary culture of neurons and glia from a rat&#39;s urinary bladder. It outlines the steps involved in tissue preparation, enzymatic digestion to obtain single cells, and culturing the cells in a neuron-specific medium to establish a primary neuronal and glial culture.

ラット膀胱からの初代ニューロンとグリアの分離と培養

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

神経細胞培養技術

一次神経細胞培養

Isolation of Neural Stem Progenitor Cells from the Spinal Cord Periventricular Region of an Adult Rat

Source: Mothe, A., et al., <a href="https://app.jove.com/v/52732">Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord.</a> J. Vis. Exp. (2015).
This video presents a method for isolating neural stem/progenitor cells from the periventricular tissue of the spinal cord in adult rats. The technique includes incubating the tissue fragments in a solution containing proteolytic enzymes to disrupt the extracellular matrix and release the cells. Subsequently, the released cells are plated in a medium containing specific growth factors that enhance neural stem/progenitor proliferation and promote aggregation into three-dimensional neurospheres.

成体ラットの脊髄脳室周囲領域からの神経幹前駆細胞の単離

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparation of Dissection ...