Name: live imaging of primary cerebral cortex cells using a 2d culture system
Uploaded: 2017-08-09T13:00:00.000+00:00
Duration: 10 min 12 s
Description: Watch this Scientific Journal Video about Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System at JoVE.com

この作業は、研究集会 (Conselho ナシオナル ・ デ ・ Desenvolvimento Científico e Tecnológico)、岬 (中・ デ ・ Aperfeiçoamento ・ デ ・ Pessoal ・ デ ・ Nível スーペリアー) によって支えられたおよび FAPERN (カルースト ・ デ ・ アンパロ Pesquisa はリオ ・ グランデは、ノルテを行う)。

All experiments involving live animals described in this protocol are conducted according to the National and International laws and were approved by the local University Animal Care and Use Committee (CEUA/UFRN), under the license 009/2014. The following protocol is performed in a sterile environment. Familiarity with basic cell culture is expected.
1. Dorsolateral Telencephalon Microdissection
<ol>
	<li>Prepare the dissection medium (100 mL): 98.5 mL Hank&#39;s Balanced Salt Solution (HBSS), 0.5 mL 5 M HEPES pH 7.4, 1 mL penicillin/streptomycin (10,000 units/mL and 10,000 &#181;g/mL).</li>
	<li>Filter-sterilize the dissection medium.</li>
	<li>Remove embryonic day 14 (E14) embryos from a pregnant mouse C57/Bl6 (Mus musculus) under anesthesia with isoflurane. 
	NOTE: Consider E0 as the day of vaginal plug detection. Dissection should be completed no later than 1 h after removal from the pregnant mouse. This protocol also applies for embryos E11-E17.</li>
	<li>Remove the embryos by hysterectomy under sterile conditions.</li>
	<li>Transfer the embryos to a Petri dish with cold dissection medium (4 &#176;C).</li>
	<li>Remove the brains from the skull by cutting along the longitudinal fissure. The number of embryos usually varies from five to ten. Each E14 brain yields approximately 2 x 106 cells, including progenitors and postmitotic neurons (1:1 ratio) 
	NOTE: Use the stereomicroscope for the following steps.</li>
	<li>Remove the meninges using dissection forceps. Split the telencephalon in the middle to separate the hemispheres. To isolate the dorsolateral telencephalon, cut along the dorsomedial curve and pallial-subpallial boundary using dissection forceps. Transfer the dorsolateral telencephalon to a 2 mL tube with cold dissection medium (4 &#176;C) until all brains are dissected.</li>
</ol>
2. Cell Dissociation and Plating
<ol>
	<li>Prepare the proliferation medium (50 mL of DMEM+10% FCS): 44 mL Dulbecco Modified Eagle&#39;s Medium (DMEM), 5mL Fetal Calf Serum (FCS), 0.5 mL 40% glucose, 0.5 mL penicillin/streptomycin (10,000 units/mL and 10,000 &#181;g/mL). Filter-sterilize the proliferation medium.</li>
	<li>Prepare differentiation medium (50 mL of DMEM+2% B27): 48 mL DMEM, 1 mL B27, 0.5 mL 40% glucose, 0.5 mL penicillin/streptomycin (10,000 units/mL and 10,000 &#181;g/mL). Filter-sterilize the differentiation medium.</li>
	<li>After micro-dissection of the dorsolateral telencephalon from E14 mice, centrifuge the tube with the collected tissue and cold dissection medium (for 5 min at 4 &#176;C, 340 x g) to precipitate the tissue.</li>
	<li>Remove the supernatant with a pipette and add 1 mL of pre-warmed (37 &#176;C) Trypsin-EDTA (0.05%) for chemical digestion. Incubate for 15 min at 37 &#176;C. Add 2 mL of proliferation medium to stop trypsin activity.</li>
	<li>Polish the tip of a glass Pasteur pipette over a gas burner or Bunsen burner for few seconds to slightly narrow the aperture. Wash the pipette by aspirating FCS to coat the tip. Avoiding bubbles, dissociate mechanically the cells with a fire-polished and FCS-coated Pasteur pipette.</li>
	<li>Centrifuge the cells (for 5 min at 4 &#176;C, 340 x g). Remove supernatant with a pipette. Add 1 mL of proliferation medium and resuspend the cells using a pipette. Repeat this step once.</li>
	<li>Prepare a 1:1 dilution of the cell suspension using a 0.4% Trypan Blue solution. Non-viable cells will be blue. Count the number of viable cells (unstained) using a Neubauer chamber.</li>
	<li>Dilute the cells in proliferation medium to get 106 cells/mL. Add 500 &#181;L of the cell suspension to each well of a 24 well tissue culture plate (approximately 2.5 &#215; 105&#160;cells/cm2). Incubate cells at 37 &#176;C and 5% CO2. 
	NOTE: Do not use glass coverslips, as they can move during the experiment and change the field of observation. Alternatively, use glass bottom tissue culture plates. 
	NOTE: If you are not using cell-culture treated multidished, it is important to pre-treat your plates with Poly-D-Lysin (50 &#181;g/ml) 2 h at 37 &#176;C. PDL-treated plates can be stored at 4 &#176;C after washing with distilled water.</li>
</ol>
3. Retroviral-mediated Transfection
NOTE: This is a simple method to transfect only progenitor cells. However, other viral vectors or chemical/electrical transfection can be used to insert genes of interest in cultured cells.
<ol>
	<li>To manipulate gene expression and label cells with fluorescent reporter proteins, add retroviruses carrying plasmids with genes of interest 2 h after cell plating. 
	NOTE: Retroviral vectors only integrate into the genome of dividing cells. Therefore, it is important to infect cells when proliferation is high. 
	NOTE: We used a plasmid containing the Beta-actin/CMV fusion sequence (CAG) and an internal ribosomal entry site (IRES) followed by the coding sequence for the green fluorescent protein (GFP), designated pCAG-IRES-GFP44. Genes of interest could be cloned between the CAG promoter and IRES sequence37.</li>
	<li>Dilute the retrovirus in differentiation medium to obtain the desired number of particles per mL. Add 500 &#181;L of differentiation medium with retrovirus to each well. 
	NOTE: The amount of retroviruses used is dependent on its titer and should be calculated to obtain 50 to 100 infected cells per well. This titer allows the identification of isolated GFP-expressing clones, whose relationship can be further confirmed by tracking phase contrast images (see section 6). The titration of retrovirus should be performed using the same cell population45.</li>
</ol>
4. Time-lapse Video-microscopy
NOTE: This step requires an inverted fluorescence microscope with incubation chamber (see Table of Materials).
<ol>
	<li>After the retrovirus infection, place the tissue culture plate into an incubator connected to temperature and CO2 controllers. 
	NOTE: Preheating the chamber for 3 h prior to starting imaging could help minimize drift and defocus during the acquisition. 
	NOTE: The chamber should maintain the cells at constant conditions of 37 &#176;C and 5% CO2.</li>
	<li>Select a position in the XY axis to serve as a zero-point (XY = 0). This will allow a reference to find the positions again in the future, for instance after post-imaging immunocytochemistry (see section 5). 
	NOTE: It is advisable to draw a sign in the plate that can be used as a reference to determine the zero-point.</li>
	<li>Select the positions to be imaged at 10X magnification. 
	NOTE: Select 10-15 positions per well to increase the likelihood to observe retrovirally-transfected cells. 
	NOTE: Long-distance high magnification objectives (20X or 40X) can be used to gain higher resolution images. Depending on the working distance of objectives, glass-bottom plates may be required.</li>
	<li>Acquire phase contrast images every 5 min and fluorescence images every 3 h to decrease phototoxicity. Check and adjust the focus in the first 3 h of experiment, as it may change while the temperature equilibrates. Check and adjust the focus daily. 
	NOTE: We observe that the focus becomes stable after the first 24 h. However, it is advisable to monitor the focus.&#160;Alternatively, use a software-based autofocus system.</li>
	<li>After 7 days, stop acquisition and proceed to post-imaging immunocytochemistry (see section 5).</li>
	<li>After immunocytochemistry, replace the plate in the microscope incubator, find the zero-point, reset XYZ = 0 and reload the experiment with saved positions. Acquire the fluorescence images of each position using appropriate fluorescence filters.</li>
</ol>
5. Post-imaging Immunocytochemistry
<ol>
	<li>After 7 days in culture, fix the cell culture with 4% Paraformaldehyde (PFA) fixative for 15 min at room temperature. 
	CAUTION: PFA is toxic and should be handle in a chemical fume hood.</li>
	<li>Rinse with 0.5 mL PBS 0.1 M for 5 min. Repeat 3 times.</li>
	<li>Incubate in primary antibodies overnight at 4 &#176;C in 0.5% triton and 10% of normal goat serum in PBS 0.1 M. 
	NOTE: Use anti-Microtubule Associated Protein 2 (MAP2) antibody to confirm the neuronal phenotype (mouse IgG1, 1:1,000) and anti-GFP to label the transduced cells (chicken antibody, 1:500). Other antibodies can be used to detect progenitor or glial cells.</li>
	<li>Rinse with 0.5 mL PBS 0.1 M for 5 min. Repeat 3 times.</li>
	<li>Dilute secondary antibodies in 0.5% triton and 10% of normal goat serum in PBS 0.1 M and incubate for 2 h at room temperature. Recommended dilution: 1:1,000.</li>
	<li>Rinse with 0.5 mL PBS 0.1 M for 5 min. Repeat 3 times.</li>
	<li>For nuclei staining, incubate the cells for 5 min with 0.1 &#181;g/mL 4&#8242;6&#8242;-diamino-2-phenylindone (DAPI) in PBS 0.1 M.</li>
	<li>Rinse with 0.5 mL PBS 0.1 M for 5 min. Repeat 3 times.</li>
	<li>Keep the cells in PBS 0.1 M and protect from light (e.g., wrap the plate with foil).</li>
</ol>
6. Cell Tracking
NOTE: Here, we briefly describe the main steps to analyze time-lapse video-microscopy data using the software The Tracking Tool (tTt), which is available at the link provided in Table of Materials.
NOTE: If the image acquisition is performed with other software, import the image data with tTt Converter available in the tTt installer (link provided in Table of Materials). Only png, tif or jpg image formats are accepted. Follow the steps shown at Figure 1 A - B after starting the tTt Converter.
<ol>
	<li>Start the software tTt and choose a user name. Click on &#34;add user&#34; and &#34;continue&#34; button (Figure 2A). Browse and select the &#34;TTTWorkFolder&#34; (Figure 2B). Lineages trees, statistics and exported images will be saved in this folder. Next, select the experiment to be analyzed and load it (Figure 2B).</li>
	<li>If the images were converted previously by tTt Converter, click on &#34;LogFileConverter&#34; (Figure 3A) and specify the number of seconds between two consecutive time points in the LogFileConverter window. Click on &#34;Convert log files for whole experiment&#34; (Figure 1C).</li>
	<li>In a list of images, which appear in the tTt window, select the desired images that by loading manually or using the option displayed in the program. Load images (Figure 3).</li>
	<li>Select File &#62; Open &#62; New colony. Click on &#34;Tracking&#34;, followed by &#34;Start Tracking&#34; to start the tracking in a chosen position.</li>
	<li>A movie window will appear (Figure 4). Select the cell using the tracking circle and press the key 0. The software will go to the next frame. Keep on positioning the tracking circle around the tracked cell using the mouse and click 0 in each frame to add a track mark on the cell.</li>
	<li>Use the keys 1 and 3 to move to the previous or next frame, respectively. To delete a track mark, just click on the correct position to mark and press 0.</li>
	<li>If the cell divided, select the button &#34;division&#34; (red rectangle at Figure 4). Continue tracking one daughter cell by pressing Shift+D. To track the second daughter cell, select it in the cell editor and press F2.</li>
	<li>If the cell died during the video microscopy, select &#34;apoptosis&#34; (blue rectangle at Figure 4). 
	NOTE: Although the software labels the event as &#34;apoptosis&#34;, other mechanisms may be responsible for the cell death.</li>
	<li>If the cell leaves the field of observation or intermingles with neighboring cells precluding tracking, select &#34;lost&#34; (green rectangle at Figure 4).</li>
	<li>To stop tracking and continue later click on &#34;interrupt&#34; (purple rectangle at Figure 4). 
	NOTE: In the Cell editor window a tree will be generated during the tracking. To save the lineage tree, select File &#62; Save &#62; Current tree (in the Cell editor window).</li>
	<li>To start tracking a different cell, click on the cell using the right mouse button or select the cell using the left mouse button and click on Tracking &#62; Start Tracking.</li>
	<li>To export the movie with the tracking data, the software should not be in &#34;tracking mode&#34;. To turn off the tracking mode, execute step 6.10. In the Movie window select Export &#62; Movie. Set up the format, range of frames, number of frames per second and bitrate. Click on &#34;Start export&#34;.</li>
</ol>
7. Quantifications
NOTE: Several measurements can be performed using video microscopy data46. Here, we describe three possibilities that are exemplified later in the &#34;representative results&#34; section.
<ol>
	<li>Quantify the cell survival for each cell lineage by dividing the number of cells alive at different time points by the total number of cells generated before these time points were reached within individual clones37.</li>
	<li>Quantify the proportion of symmetric progenitor (SP, both daughter cells continue to proliferate), asymmetric (A, one daughter cell continues to proliferate and the other becomes postmitotic), or symmetric terminal (ST, both daughter cells become postmitotic)15,38.</li>
	<li>Measure the cell cycle length by calculating the time span of proliferating cells between their generation and division15.</li>
</ol>

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主要な大脳皮質細胞のリアルタイム観察では、細胞増殖の解析、細胞分裂、細胞周期、細胞分化、細胞生存3,14,15,37のモードをことができます。もっと重大に、それは NSCs からニューロン3への進行の間に制定された中間相の同定につながる単一細胞系統の研究を許可します。最?...

神経幹細胞 (NSC) は、大脳皮質の開発中にニューロンと macroglial 細胞を生成します。初期-内因、NSCs 対称の細胞分裂のいくつかのラウンドを受けるし、前駆プールを展開します。その後、NSCs は中間1を介して直接または間接的にニューロンを生成する非対称的に分割します。だけで半ば-に後半-内因、前駆細胞のアストロ サイト、オリゴデンドロ サイトの2,3、4を生成するスイッチします。しかし、ユニークな種類のニューロンや macroglial 細胞の世代に運命制限前駆細胞の寄与と同様、細胞の増殖と分化を制御する完全なメカニズムは、激しい議論4,5,6の問題を続けます。神経細胞、アストロ サイト、オリゴデンドロ サイトを生成する個々 の皮質 NSCs の可能性は広範囲に研究生体外および生体内で無数の技術を使用して、されている: 単一細胞培養7,8,9,10、11,12,13; イメージング ライブライブ イメージングの高密度文化文化3,14,15;ライブ イメージング スライス文化16,17,18;ウイルス媒介性遺伝的ラベリング処理による高密度培養14,15,19,20,21; のクローン解析クローン解析体内レトロ ウイルス22,23,24,25,26,27,28,29,30,31,32,33; を使用してそして体内のクローン解析トランスジェニック動物の34を使用しています。
これらのテクニックは、長所と短所がそれぞれ。たとえば、トレースする血統体内一括と分割エラー3、個々 の大脳皮質前駆細胞の潜在性について矛盾する結論に至る。また、両方in vitroとin vivoの研究が早い時点で前駆細胞の分類に基づいて、35系統進行中に細胞死の検出されない発生による細胞系譜の事後分析を受けます。したがって、単一 NSCs の可能性の分析に適したシステムは生成されると、すべての細胞の同定だけでなく、系統内での細胞運命の適切な特性を考慮しなければなりません。一次電池文化とライブ イメージングの組み合わせでは、この設定を提供します。単一細胞培養とタイムラプス ビデオ顕微鏡を用いた、寺らは gliogenesis11神経新生から個々 の大脳皮質前駆細胞の血統のスイッチを示しています。後で、彼らはニューロンの異なったタイプが12の単一の大脳皮質前駆細胞から生成されることを示す同じシステムを使用しました。しかし、このシステムには重要な注意事項: 初期内因で分離された大脳皮質前駆細胞の 1% は 4 つ以上の子孫9のクローンを生成するだけです。FGF2 の付加の後で 4 つ以上のセルを生成する細胞の頻度は 8-109に増加します。それにもかかわらず、この数はこの段階で、ほとんどすべての大脳皮質前駆細胞が増殖を考慮した小さすぎます。また、36運命仕様に FGF2 の潜在的な影響は否定できません。(Pax6 を発現) 心室の両方の増殖をサポートする高密度細胞培養を用いてこれらの制限を回避するために、脳室下帯 (Tbr2 表現) 大脳皮質前駆細胞15。また、これらの文化のリアルタイム観察は NSC 系統進行のいくつかの機能、3、15細胞分裂、細胞周期の延長、ニューロンとグリアなどを生成する単一細胞の潜在性のモードなど、これらの条件下で再現を示しています。最近では、我々 も CREB シグナルがマウス37で未熟な大脳皮質ニューロンの細胞の生存に影響を与えることをこのシステムを使用しています。したがって、プライマリ マウス大脳皮質のタイムラプス ビデオ顕微鏡を考えて高密度成長のセルは細胞周期の進行のメカニズムを解明、細胞分裂、細胞生存率、細胞運命の指定のモードを研究する強力かつアクセス可能なツール。後者ができますリアルタイム38,39,40の特定細胞運命の識別またはポスト各種3,38,41,42をイメージングの使用許可、トランスジェニック動物を使用しています。
ここでは、NSCs の増殖とニューロンと macroglial 細胞の次の世代を支える主要な大脳皮質細胞培養を準備する手順を追ってプロトコルを提供します。識別およびコマ撮りビデオ顕微鏡を用いた単一細胞レベルで追跡することができます個々 の細胞の遺伝子を操作する遺伝子をレトロ ウイルス媒介の使用についても述べる。齧歯類、内因の端に最初から主要な大脳皮質細胞の研究にこのプロトコルを使用することができますが、ステージ14によるといくつかの調整が必要があります。二次元文化のタイムラプス ビデオ顕微鏡を用いた、NSCs 他のソースから分離を検討ことができますが、適切な培養システムは細胞挙動の in vitroとin vivo38,43を比較することによって決定する必要があります。

<table><tbody><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Invitrogen Life Technologies</td><td>14175129</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375-25G</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Dulbecco Modified Eagle&#039;s Medium (DMEM)</td><td>Gibco</td><td>12400-024</td><td></td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Gibco</td><td>10437028</td><td></td></tr><tr><td>Glucose</td><td>Gibco</td><td>A2494001</td><td></td></tr><tr><td>B27</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>trypsin-EDTA (0.05%)</td><td>Gibco</td><td>25300054</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>16005</td><td></td></tr><tr><td>Goat serum</td><td>Sigma-Aldrich</td><td>69023</td><td></td></tr><tr><td>Triton X-100</td><td>VWR International Ltd.</td><td>306324N</td><td></td></tr><tr><td>Isoflurane</td><td>Sigma</td><td>792632</td><td></td></tr><tr><td>anti-MAP2, mouse</td><td>Sigma</td><td>M4403</td><td></td></tr><tr><td>anti-GFP chicken</td><td>Aves</td><td>0511FP12</td><td></td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td></td></tr><tr><td>Goat anti-mouse alexa 594</td><td>Invitrogen</td><td>A11005</td><td></td></tr><tr><td>Goat anti-chicken alexa 488</td><td>Invitrogen</td><td>A11039</td><td></td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td></td></tr><tr><td>tTt</td><td>ETH Zurich</td><td></td><td></td></tr><tr><td>Cell observer microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Pasteur pipette</td><td></td><td></td><td></td></tr><tr><td>PBS</td><td></td><td></td><td></td></tr><tr><td>The Tracking Tool (tTt) software</td><td></td><td>https://www.bsse.ethz.ch/csd/software/ttt-and-qtfy.html</td><td>download link</td></tr></tbody></table>

live imaging of primary cerebral cortex cells using a 2d culture system

大脳皮質の開発中には、前駆細胞は新しい前駆細胞または後の神経細胞を生成する対称および非対称細胞分裂のいくつかのラウンドを受けます。その後、いくつかの前駆細胞は、アストロ サイト、オリゴデンドロ サイトの人口への追加幹運命に切り替えます。主要な大脳皮質細胞培養のタイムラプス ビデオ顕微鏡を使用すると、細胞分裂のモードと前駆細胞の細胞周期のパラメーターを制御する細胞・分子メカニズムを研究することが可能です。同様に、セル固有蛍光レポーター蛋白質を使用してまたは後免疫細胞化学をイメージング、後細胞の運命は調べることができます。もっと重要なは、これらすべての機能は、特定のセル型を生成することを約束の前駆細胞の同定を許可する単一細胞レベルで分析できます。また細胞自律的・細胞自律的現象の研究を許可するウイルスを介する遺伝子導入を用いた遺伝子発現の操作を実行できます。最後に、蛍光の融合蛋白質を使用区分と娘細胞運命との対比の中に選択した蛋白質の対称および非対称分布に関する研究。ここでは、数日に及ぶ主要な大脳皮質マウス細胞を画像、細胞分裂、細胞周期の長さと新しく生成された細胞の運命のモードを分析してタイムラプス ビデオ顕微鏡法について述べる.興味のある遺伝子を操作する、または単にレポーター蛋白質と細胞をラベルに適用できる前駆細胞を使って簡単な方法について述べる。

Primary cultures of cerebral cortex cells isolated from embryos E14 contain both progenitor and neuronal cells. During the period of imaging, progenitors undergo several rounds of cell division, increasing the number of cells (Figure 5 and Video Figure 1).
Retroviral-mediated transfection of a few progenitor cells facilitates the identification of cell clones (F...

Watch this Scientific Journal Video about Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System at JoVE.com

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

ライブ画像は、リアルタイムで携帯電話の行動を研究するための強力なツールです。ここでは、タイムラプス ビデオ-顕微鏡検査のため主要な大脳皮質細胞の主な神経幹細胞から分化したニューロンとグリア系統進行中に成立段階の詳細な検査を可能にするプロトコルについて述べる。

2次元培養系を用いた主な大脳皮質細胞のイメージングをライブします。

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

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Neuroscience

8.0K ビュー.  Federal University of Rio Grande do Norte. ライブ画像は、リアルタイムで携帯電話の行動を研究するための強力なツールです。ここでは、タイムラプス ビデオ-顕微鏡検査のため主要な大脳皮質細胞の主な神経幹細胞から分化したニューロンとグリア系統進行中に成立段階の詳細な検査を可能にするプロトコルについて述べる。

ビデオ: Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

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

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

開発神​​経上皮における細胞挙動の高解像度のライブイメージング

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

初代培養海馬ニューロンにおける高密度コア小胞のライブイメージング

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

生物学

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

器官型脳スライスと癌細胞の3Dスフェロイドとの共培養システム

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

医学

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

オリゴデンドロサイトダイナミクスと髄鞘形成を研究するための器官型スライス培養

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

2Dおよび3Dヒト人工多能性幹細胞ベースのモデルにより、新皮質発生中の原発性繊毛の関与を解剖

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

発生生物学

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

ライブ イメージング モニター細胞生物学および複数の神経集団の血統で、進行を追跡 1 つのセルに続いて

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) axons form branches and synapses in specific layers of the cerebral cortex. Despite the obvious spatial correlation between axon branching and synapse formation, the causal relationship between them is poorly understood. To address this issue, we recently developed a method for simultaneous imaging of branching and synapse formation of individual TC axons in organotypic cocultures.
This protocol describes a method which consists of a combination of an organotypic coculture and electroporation. Organotypic cocultures of the thalamus and cerebral cortex facilitate gene manipulation and observation of axonal processes, preserving characteristic structures such as laminar configuration. Two distinct plasmids encoding DsRed and EGFP-tagged synaptophysin (SYP-EGFP) were co-transfected into a small number of thalamic neurons by an electroporation technique. This method allowed us to visualize individual axonal morphologies of TC neurons and their presynaptic sites simultaneously. The method also enabled long-term observation which revealed the causal relationship between axon branching and synapse formation.

This protocol describes a method for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. Individual thalamocortical axons and their presynaptic terminals are visualized by a single cell electroporation technique with DsRed and GFP-tagged synaptophysin.

視床皮質軸索分岐とシナプスの可視化脊髄共培養系における形成

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6&#8210;8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC)&#160;transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

ヒト人工多能性幹細胞由来皮質およびドーパミン作動性ニューロンの統合されたライブ細胞モニタリングによる自動生産

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are ...

Isolation and Culturing of Cells from Mouse Embryo Dorsolateral Telencephalons

Source: Landeira, B. S., et al. <a href="https://app.jove.com/v/56063">Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System.</a> J. Vis. Exp. (2017)
This video demonstrates a protocol for isolating and culturing primary cerebral cortex cells from the dorsolateral telencephalon of mouse embryos.

マウス胚背外側末脳からの細胞の単離と培養

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

2次元培養系を用いた主な大脳皮質細胞のイメージングをライブします。

要約

さらに動画を探す

この動画の章

初代ラット皮質ニューロンとグリアのBilaminarの共培養

開発神経上皮における細胞挙動の高解像度のライブイメージング

初代培養海馬ニューロンにおける高密度コア小胞のライブイメージング

器官型脳スライスと癌細胞の3Dスフェロイドとの共培養システム

オリゴデンドロサイトダイナミクスと髄鞘形成を研究するための器官型スライス培養

2Dおよび3Dヒト人工多能性幹細胞ベースのモデルにより、新皮質発生中の原発性繊毛の関与を解剖

ライブイメージングモニター細胞生物学および複数の神経集団の血統で、進行を追跡 1 つのセルに続いて

視床皮質軸索分岐とシナプスの可視化脊髄共培養系における形成

ヒト人工多能性幹細胞由来皮質およびドーパミン作動性ニューロンの統合されたライブ細胞モニタリングによる自動生産

マウス胚背外側末脳からの細胞の単離と培養

2次元培養系を用いた主な大脳皮質細胞のイメージングをライブします。

要約

さらに動画を探す

この動画の章

初代ラット皮質ニューロンとグリアのBilaminarの共培養

開発神​​経上皮における細胞挙動の高解像度のライブイメージング

初代培養海馬ニューロンにおける高密度コア小胞のライブイメージング

器官型脳スライスと癌細胞の3Dスフェロイドとの共培養システム

オリゴデンドロサイトダイナミクスと髄鞘形成を研究するための器官型スライス培養

2Dおよび3Dヒト人工多能性幹細胞ベースのモデルにより、新皮質発生中の原発性繊毛の関与を解剖

ライブ イメージング モニター細胞生物学および複数の神経集団の血統で、進行を追跡 1 つのセルに続いて

視床皮質軸索分岐とシナプスの可視化脊髄共培養系における形成

ヒト人工多能性幹細胞由来皮質およびドーパミン作動性ニューロンの統合されたライブ細胞モニタリングによる自動生産

マウス胚背外側末脳からの細胞の単離と培養

開発神経上皮における細胞挙動の高解像度のライブイメージング

ライブイメージングモニター細胞生物学および複数の神経集団の血統で、進行を追跡 1 つのセルに続いて