Name: culturing in vivo-like murine astrocytes using the fast, simple, and inexpensive awesam protocol
Uploaded: 2018-01-10T13:00:00.000+00:00
Duration: 7 min 56 s
Description: Watch this Scientific Journal Video about Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol at JoVE.com

我们承认来自欧洲研究理事会 (FP7/260916)、亚历山大·洪堡-基金会 (Sofja Kovalevskaja) 和德意志 Forschungsgemeinschaft (DE1951 和 SFB889) 的资助。

这里描述的所有动物实验都是按照德国动物福利指南进行的。
注意: 协议的时间轴和主要步骤如图 1所示。
1. 准备工作
<ol><li>
准备以下解决方案。
	<ol>
<li>准备 DMEM + 为培养多边形 MD 星形胶质细胞: DMEM 10% FCS, 100 U 青霉素和100µg/毫升链霉素。</li>
		<li>为培养星状胶质细胞制备 NB + H: neurobasal 培养基, 5 ng/毫升 HBEGF, 1x B-27 补充, 1x 谷氨酰胺补充剂, 100 U 青霉素和100µg/毫升链霉素。 注: 等分浓度较高的 HBEGF 可在需要时在-20 ° c 下添加到培养基中。</li>
		<li>准备解剖培养基 (10 毫米 HEPES 在 HBSS)。</li>
		<li>在15毫升管中制备6-7 毫升0.05% 胰蛋白酶-edta 和6-7 毫升0.25% 胰蛋白酶-edta 的等分。这些可以被存放在-20 ° c 和解冻在37° c 水浴。</li>
	</ol>
</li>
	<li>在开始该协议的前一天, 10 厘米的组织培养处理的菜肴与5毫升的0.04% 亚 (PEI) 每一个。</li>
	<li>第二天, 去掉 PEI, 用去离子洗碗, 蒸馏水两次。在37° c 和 5% CO2中, 将12毫升的 DMEM 加到每个板和 pre-equilibrate 中, 至少在电镀电池前30分钟。</li>
	<li>准备解剖区域: 在层流解剖罩, 放置两个10厘米的培养皿, 填充在冰上的解剖介质。对于要隔离的每个脑区 (例如, 皮质, 海马), 准备一个15管, 填充14毫升解剖培养基, 并保持冰。喷雾解剖工具 (细剪刀, #3 和 #5 钳) 与70% 乙醇和地方的解剖显微镜旁边的遮光罩。</li>
</ol>2. 大鼠或小鼠脑解剖
注: 小鼠和老鼠都可以使用。年龄比 P8 的动物也可以使用 (我们测试了 P12), 但去除脑膜将变得越来越困难。此外, 细胞的产量和健康将会减少, 因为来自较年长动物的脑细胞将形成复杂的过程和连接, 在组织分离过程中可能破裂, 导致细胞死亡增加。皮质制剂在这里描述, 但其他大脑区域也可以使用。
<ol><li>在37° c 水浴中放置0.05% 胰蛋白酶-EDTA。</li>
	<li>安乐动物 (为 E19 幼崽, 或 P0-P8 动物) 通过缓慢提高 CO2浓度, 并隔离大脑。以下步骤需要无菌技术。</li>
	<li>
如果使用胚胎组织, 首先将胚胎从子宫中取出, 然后打开胚胎囊, 然后立即用剪刀切断头部。当所有的头被切断, 转移到预解剖介质。
	<ol>
<li>一旦头部被淹没在介质中, 打开皮肤和头骨 (两个都是非常柔软的幼小动物) 与钳子和掐掉小脑。小心地把大脑的其余部分从头骨上抬起来。 注: 两到四 E19 胚胎在解剖当天为每道50万细胞的皮层星形胶质细胞至少八10厘米的食物提供足够的组织。</li>
	</ol>
</li>
	<li>对于新生儿或年长的幼崽, 用剪刀切断头部, 通过内侧从头部后部到鼻尖的一条直线切割皮肤来隔离大脑。把皮肤拉到一边, 小心地把头骨向左和向右, 然后抬起头骨。切断小脑, 并把大脑的其余部分从头骨中取出。 注: 两到三 P0-P8 幼崽的组织至少八10厘米的皮质星形胶质细胞在解剖的时候每道50万细胞上都有一组。</li>
	<li>将大脑皮层与底层的中脑结构分开。把镊子放在两半球之间的纵裂处, 然后将镊子移到一个半球的皮质和中脑结构之间 (垂直于纵裂), 以释放每个半球。</li>
	<li>使用镊子, 从每个半球 (及其海马) 中取出所有脑膜, 然后分离海马。如果需要海马星形胶质细胞, 将每个海马体移动到一个15毫升的管内, 在冰上填满14毫升的解剖介质。</li>
	<li>如果需要特定区域 (例如, 额叶皮质), 则从每个半球的皮层移除更多区域。切割任何皮质组织, 用于将星形胶质细胞生成不大于 1 mm3的碎片。收集所有的皮质组织在一个单独的15毫升的管中填充解剖介质冰。</li>
</ol>3. 组织消化和分离
<ol><li>一旦所有的组织碎片收集, 等待他们定居到底部的管, 然后吸尽可能多的培养基。</li>
	<li>用血清学吸管加2-3 毫升0.05% 胰蛋白酶-EDTA, 在37° c 的水浴中孵育20分钟的试管。 注: 将所有胰蛋白酶-EDTA 从吸管中迅速释放, 混合组织块并允许再次沉淀。</li>
	<li>小心地抽出胰蛋白酶-EDTA, 将组织块留在尽可能少的液体中, 在试管的底部。</li>
	<li>加入5毫升的预剥离培养基, 冲洗剩余的胰蛋白酶-EDTA。吸管在管子上部的一侧, 以达到混合的组织件。</li>
	<li>吸取解剖介质, 并尽可能少地将组织块留在液体中。重复这个洗涤步骤与预解剖介质两次以上。</li>
	<li>最后的洗涤步骤后, 添加1毫升的 DMEM + (温暖到室温) 使用1000µL 吸管尖端, 研制的组织由移上下不引入气泡。 注意: 避免产生气泡是很重要的, 因为这可能会改变溶液的 pH 值, 而星形胶质细胞是非常敏感的。</li>
	<li>在50毫升管的开口处放置一个100µm 的细胞过滤器, 并湿4.5 毫升的预 DMEM。</li>
	<li>将1毫升的游离组织悬浮液移到细胞过滤器上, 再添加4.5 毫升预 DMEM, 通过细胞过滤器清洗细胞。</li>
</ol>4. 传代星形胶质细胞富集的电池电镀
<ol><li>计算细胞,例如, 通过稀释10µL 的细胞悬液与10µL 台盼蓝和移混合到例。</li>
	<li>板50万细胞在12毫升 DMEM + 每10厘米的菜。 注: DMEM + 有助于星的生长, 比神经元或小胶质细胞更重要。在摇晃过程中, 在井内的玻璃片的物理力比在10厘米的盘子上的作用要强得多 (没有片)。如果细胞在24井板上震动, 它们就会变得不健康或死亡。只有板星形胶质细胞在 24-井板震动后。</li>
	<li>保持细胞培养在孵化器在37° c 和 5% CO2七天。</li>
	<li>在6天, 在震动和传代细胞之前的前一天, 准备在步骤5中描述的 PEI 涂层培养基。</li>
</ol>5. 传代细胞
<ol><li>
前一天传代, 外套细胞培养板材与 0.04% PEI。
	<ol>
<li>对于免疫细胞化学, 病毒转导, 或质粒转染, 使用24井或6井板 (每一个包含一个灭菌12毫米或25毫米玻璃片, 分别)。</li>
		<li>将70% 乙醇中的玻璃片消毒至少1小时, 然后在去离子、蒸馏水中清洗三次, 然后将片放入井中。</li>
		<li>增加至少70µL 0.04% pei 每12毫米片在24井板材和600µL 0.04% pei 每25毫米片在 6-井板材。</li>
		<li>对于西方的印迹或 RNA 序列分析, 10 厘米的菜肴已经包含了细胞解剖后可以继续使用。盘子只需要摇晃, 然后用 1x PBS 洗净, 然后用 NB + H (不要转移到新的菜肴) 处理。</li>
	</ol>
</li>
	<li>在天体外(DIV) 7 在解剖后, 将10厘米的碗碟与细胞在 DMEM + 在孵化器的实验室振动筛和动摇的菜在 110 rpm 为6小时。</li>
	<li>用去离子、蒸馏水两次冲洗片。将 NB + H 介质添加到板上 (每井500µL 24 井板和6井板2毫升)。预平衡的板在孵化器在37° c 和 5% CO2 , 而 10 cm 菜是震动, 或至少30分钟。</li>
	<li>20-30 分钟前 6 h 震动, 预热 1x PBS, DMEM +, NB + h, 和0.25% 胰蛋白酶-EDTA 到37° c 在水浴。</li>
	<li>经过6小时的震动, 采取了培养皿关闭振动筛和立即取代培养基 (包括 shaken-off 神经元, 突和小胶质细胞) 与10毫升 5ml 1x PBS 每道菜。 注意: 重要的是要立即吸取旧培养基中含有分离细胞, 以避免 non-astrocytic 细胞重新。</li>
	<li>取出 PBS, 加入3毫升5ml 胰蛋白酶-EDTA 每道菜。孵育每道菜4分钟在孵化器在37° c 和 5% CO2。 注: 如果准备样品为西方印迹或 RNA-Seq, 不添加胰蛋白酶-EDTA。取而代之的是, 用12毫升5ml 的铌 + H 取代 PBS, 然后将培养基放回孵化器中。</li>
	<li>在每道菜中加入5毫升 5ml DMEM + 3 毫升胰蛋白酶-EDTA, 用5毫升的血清吸管将细胞从盘子中移出, 并在50毫升管中收集细胞悬浮液。 注: 从盘子底部直接吸管--当细胞分离时, 盘子的地板会变得更清晰。只使用含血清的培养基 (代替 DMEM +), 作为血清流感胰蛋白酶。</li>
	<li>离心的细胞悬浮在 3220 x g 在20° c 为 4 min。</li>
	<li>取出上清和重1毫升5ml 的细胞颗粒。</li>
</ol>6. 最后的星形胶质细胞培养
<ol><li>计算单元格数。</li>
	<li>在6井板 (2 毫升铌 + h 介质) 或24井板 (500 µL 铌 + h 介质) 中, 每井板2万细胞。</li>
	<li>保持细胞培养在孵化器在37° c 和 5% CO2直到进一步治疗或分析。</li>
	<li>每周交换一半的培养基。 注意: 在 low-nutrient 的环境中, 星形胶质细胞停止增殖, 但任何残留的小胶质都会增殖 (尽管在我们的经验中, AWESAM 的培养中没有小胶质)。每周交换一半培养基, 可防止任何可能残留的小胶质细胞的富集8, 单一保持健康至少三周。</li>
</ol>

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作者没有什么可透露的。

四步在协议之内是重要的: 1) 准备 HBEGF 浓度精确地 5 ng/毫升, 因为小增量在集中可能导致不成熟的文化,例如, 10 ng/ml HBEGF 将 de-differentiate 星形胶质细胞4;2) 在含有组织或细胞的溶液中避免气泡, 这会改变 pH 值, 阻碍星形胶质星的健康;3) pre-equilibrating 培养基, 达到生长健康的星形胶质细胞的最佳 pH 值和温度;4) 每周只交换一半培养基, 因为任何残留的小胶质细胞在存在足够的养分时不太可能增殖, 而培养基则定期交换8 (尽管到目前为止, 在 AWESAM 培养中没有发现小胶质)。对于这最后一点, 重要的是要注意, 在文化中的星形胶质细胞衍生的因素不应该完全消除, 因为它们可能支持星的生存, 分化和增殖。
重要的是要使用 DIV14 或更古老的文化作为成熟的星形胶质细胞源 (但对于转导或转染步骤, 早期的文化可以使用), 因为比 DIV14 还要年轻, 但仍有神经干细胞电位9。对于病毒转导, 神经元和星形胶质细胞转的混合培养 DIV7 (使用腺相关病毒) 有效表达病毒转 GFP10。对于质粒转染, DIV10-DIV24 星形胶质细胞经常使用和荧光标记的蛋白质成像2-5 天后,11。在我们的经验, 病毒转导和质粒转染工作最好的, 如果星形胶质细胞是转之间的 DIV9 和 DIV14。转染和转导效率依赖于结构和方法, 但我们发现转染效率为 20%, 星形胶质细胞单一采用体转染, 腺相关病毒 (AAV) 转导效率为 90% (有关更多详细信息, 请参见我们以前的工作4 ,例如, 使用构造)。当换星形胶质细胞与 AAV 粒子和染的体, 我们允许至少5和2天, 分别为表达的建设。在转导或转染之前, 星形胶质细胞也应允许在传代后至少一天恢复。
此外, 在准备不同时间点的 RNA/蛋白表达的培养基时, 可以在不同的密度下进行培养, 以获得类似数量的材料,例如, 在 DIV7 和 DIV21 单一的星形胶质细胞可以镀层密度分别为 1 x 106和每10厘米的50万细胞。例如, 整个蛋白质的裂解产量将约0.5 毫克/毫升的 DIV14 时收获后, 最初电镀 50万 AWESAM 星形胶质细胞在一个10厘米的菜。
AWESAM 协议代表一种快速、简单和廉价的方法来培养孤立于神经元的星形胶质细胞, 但与在体内一样, RNA 和蛋白质表达、形态学和 Ca2 +信令4的特征, 这是迄今为止没有其他协议提供。AWESAM 星形胶质细胞可以用于分析, 这是典型的单元培养研究, 包括免疫化学、免疫、RNA 表达分析、TIRF 显微镜和 Ca2 +成像4。特别是, 最近的进展, 在基因编码的 ca2 +指标允许可视化精细星过程更详细的比经典的 ca2 +染料14。Ca2 +信号, 水泡摄取和胞在厚和薄的星过程是生理学相关的, 因为这些过程可以影响大脑功能的营养支持, 血流, 突触信号和可塑性,细胞间通讯。AWESAM 协议允许研究与脑生理学相关的精细星过程15和疾病16,17, 没有神经元干扰, 并与伟大的可访问性,在体外文化用品。

星形胶质细胞通过营养支持、血流、突触信号和可塑性以及细胞间通讯来影响大脑功能, 所有这些都是围绕着薄星过程的机制。这里描述的 AWESAM 协议允许对这些过程的研究, 不受神经元和其他神经胶质的干扰, 这是有用的例如, 因为许多蛋白质的表达, 甚至 Ca2 +信号重叠在不同的脑细胞类型.此外, 此方法克服了以前可用技术的局限性。特别是, 我们的协议提供了在体内样的形态学 (星形胶质细胞与薄的过程) 和基因表达快速, 方便, 便宜的方式。
细胞形态、基因表达以及许多其他的调控过程都受到环境的直接影响。在培养皿中, 这指的是周围细胞释放的因子, 也是细胞生长的媒介。对于星形胶质细胞, HBEGF 以前报告诱导一种星状形态学1 , 但也发现 de-differentiate 星形胶质细胞2。然而, 较低浓度的 HBEGF 后来用于产生更多的在体内样星形胶质细胞通过 RNA 测序在两个不同的协议3,4。此外, 星形胶质细胞的形态随培养基成分的变化而改变, 不管是什么协议4: 低浓度 HBEGF 的 Neurobasal 培养基都是生长中的正晶状胶质星, 而其他培养基成分 (如如,含血清的 DMEM) 产生多边形细胞 (即使是在 immunopanning 新分离的星形胶质)。
AWESAM 协议克服了以前技术的缺点生长星形胶质细胞单一4。以前的技术为生长星形胶质细胞单一有以下缺点: 多边形形态学, 这是不典型的在体状胶质星型,在体内(MD 方法)5;协议的长度和成本 (从诱导多能干细胞制备星形胶质细胞需要三月, 需要许多试剂, 包括昂贵的生长因子)6;低量的材料 (immunopanning 方法)3;星形胶质细胞的 de-differentiation 和3D 矩阵的要求 (Puschmann et al)1,2. 相比之下, AWESAM 协议提供:在体内样的形态学 (星形胶质细胞与薄的过程);快速、简便、廉价的方法;大量的材料;与 immunopanned 和 MD 星形胶质细胞相比, 最体内样的基因表达。此外, 2D 文化允许研究的 Ca2 +信号和囊泡回收在薄的过程中, 并在事件接近膜 (例如, TIRF 显微镜是不可能在3D 文化)。
MD 方法自 1980年5发布以来已被广泛使用, 为多边形星形胶质细胞单一提供了一种简单快捷的技术。简言之, MD 方法需要在胎儿小牛血清 (FCS) 含有 DMEM 的混合脑细胞, 其次是为星形胶质细胞提供丰富的震动步骤 (就像所有其他的脑单元类型从碟中分离出来) 和在同一培养基中的进一步培养。DMEM 补充与 FCS 是用于许多其他细胞类型, 从成纤维细胞和脂肪细胞到不同的癌细胞系, 所有这些共享的多边形形态学显示由 MD 星形胶质细胞在文化。直到早期的 2000s7, 几乎没有考虑到适合于星形胶质细胞的介质, 特别是有利于其典型的星状形态学发现的体内。2011年发布的一项协议确实实现了这种星状形态学: 称为 immunopanning 方法, 它采用了最优化的无血清培养基培养星形胶质细胞3。使用这种方法, 新的孤立的脑细胞暴露在一系列的菜肴的抗体, 靶向细胞类型特定的细胞表面蛋白, 以丰富的星形胶质。尽管 immunopanned 星形胶质细胞的形态学和表达谱有更多的在体内, 但大多数的体外研究仍依赖于 MD 型胶质蛋白的方法。MD 方法是简单和快速的, 而 immunopanning 方法包括更复杂和耗时的步骤在第一天的文化 (如较长的酶消化周期, 仔细分层的解决方案的不同密度, immunopanning 本身,几个离心步骤-所有之前电镀)。但是, 通过使用更专门的介质, AWESAM 方法提供了 MD 方法的速度和 immunopanning 协议中的在体内样貌。
总的来说, AWESAM 协议是有用的研究更多的在体内状的星形胶质细胞在2D 单一从神经元和其他神经胶质分离 (如以前的特点4由 immunostainings, immunoblots, 和 RNA 测序)。它允许研究薄的星过程, 并为可视化自发的 Ca2 +信号和接近膜的事件提供了很大的可访问性 (如, 由 TIRF 显微镜成像)。

<table><tbody><tr><td>0.05% trypsin-EDTA</td><td>Gibco</td><td>25300054</td><td>warm in 37 &amp;ordm;C waterbath before use</td></tr><tr><td>0.25% trypsin-EDTA</td><td>Gibco</td><td>25200-056</td><td>warm in 37 &amp;ordm;C waterbath before use</td></tr><tr><td>B-27 supplement</td><td>Gibco</td><td>17504-044</td><td>50X stock</td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050-061</td><td>100X stock</td></tr><tr><td>Penicillin / streptomycin</td><td>Gibco</td><td>15070-063</td><td>50 stock; penicillin: 5000 U/ml; streptomycin: 5000 &amp;micro;g/ml</td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103049</td><td>without L-glutamine</td></tr><tr><td>DMEM</td><td>Gibco</td><td>41966029</td><td></td></tr><tr><td>HBEGF</td><td>Sigma</td><td>4643</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630080</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14170112</td><td></td></tr><tr><td>FCS</td><td>Gibco</td><td>10437028</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010049</td><td>1X</td></tr><tr><td>100 &amp;mu;m nylon cell strainer</td><td>BD</td><td>352360</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>T8154&amp;nbsp;</td><td></td></tr><tr><td>Cell culture incubator</td><td>Thermo Fisher Scientific</td><td>Hera Cell 240i cell culture incubator</td><td></td></tr><tr><td>Laboratory shaker</td><td>Heidolph</td><td>Rotamax 120&amp;nbsp;</td><td>use inside cell culture incubator</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810 R&amp;nbsp;</td><td></td></tr></tbody></table>

culturing in vivo-like murine astrocytes using the fast, simple, and inexpensive awesam protocol

AWESAM (a low-成本easy stellate strocyte m方法) 协议需要一种快速、简单且廉价的方法来生成大量的在体内像老鼠和大鼠星形胶质细胞单一: 脑细胞可以从不同的脑区分离出来, 经过一周的细胞培养, non-astrocytic 细胞通过将培养皿放在孵化器中6小时的振动器上而被动摇。其余的星形胶质细胞被传代成新的板块, 并有一个特定的特殊介质 (称为 NB + H)。含低浓度肝素结合 EGF 样生长因子 (HBEGF), 用于代替血清在培养基中。AWESAM 星形胶质细胞生长于 NB + H 后, 具有星状形态, 并具有优良的工艺特点。此外, 这些星形胶质细胞比以前发表的方法产生的星形胶质细胞有更多的体内样的基因表达。Ca2 +成像, 囊泡动力学, 和其他事件接近的膜可以因此研究的精细星过程体外,例如, 使用活细胞共聚焦或 TIRF 显微镜。值得注意的是, AWESAM 星形胶质细胞也表现出类似于星形胶质细胞体内的自发 Ca2 +信号。

星形胶质细胞与神经元共, 生长在 NB + 培养基上, 在2周的培养后呈星状 (图 2)。除了铌 + 培养基, 共星形胶质细胞还暴露在未知的神经元衍生因素, 可能有助于他们的生存和形态学。相比之下, MD 星形胶质细胞生长在 DMEM + 作为单一 (在摆脱其他细胞类型之前, 通过) 出现多边形后2周。AWESAM 星形胶质细胞在震动后生长, 在2周的培养后出现星状与许多精细的过程。
我们先前表明, 与 MD 和 immunopanned 星形胶质细胞4相比, AWESAM 星形胶质细胞的 RNA 表达谱更接近于星形胶质细胞在体内。在星形胶质细胞特异性蛋白表达方面, 皮质 ALDH1L1 在体内表现出高水平的高浓度和低浓度的胶质,在活体中12。这也适用于 AWESAM 星形胶质细胞, 和免疫的 ALDH1L1, 作为一个细胞质酶, 揭示更细的细节比骨架蛋白胶质纤维 (图 2B)。
除了 RNA 和蛋白表达, 星形胶质细胞在体内显示自发的 ca2 +信号 (即, 没有外部刺激), 但多边形 MD 星形胶质细胞在体外经常需要刺激来引出 ca2 +信号,例如, 通过添加 ATP 或谷氨酸。AWESAM 星形胶质细胞展示自发的 Ca2 +信号在体内, 在细胞体和精细的过程, 这可以可视化的病毒换细胞与膜靶 Lck-GCaMP3 (图 3)。在相邻的星形胶质细胞中也会出现 Ca2 +的自发波 (图 3A)。星 Ca2 +信号 (图 3B) 在精细过程和微可以使用特定于星形胶质细胞13的免费软件进行分析。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56092/56092fig1.jpg"> 图 1: 协议时间线.显示 AWESAM 协议的主要步骤的时间线, 包括在 DIV0 (剥离日)、DIV7 (通过日期) 和以后的时间点上执行这些步骤所需的时间。绿盒说明了当细胞可以被捕获为星状胶质细胞时, 相对较暗的色调与更复杂的星状形态和成熟度分别表现得更少。<a href="//cloudflare.jove.com/files/ftp_upload/56092/56092fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56092/56092fig2v3.jpg"> 图 2: 中等决定形态学.(A) 示意图显示星形胶质细胞的形态学变化时, 共与神经元 (如先前所描述的4) 与成长为单一, 并视媒介。星形胶质细胞特异性染色, 概述了共和 AWESAM 星状胶质蛋白和在 MD 星形胶质细胞的多边形形态学。(B) ALDH1L1 星形胶质细胞的标签比 ALDH1L1 更强烈, 而非胶质细胞是 AWESAM 星形胶质蛋白的精细过程。两种星形胶质细胞都没有表现出 MAP2 的神经元标记。<a href="//cloudflare.jove.com/files/ftp_upload/56092/56092fig2v3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56092/56092fig3.jpg"> 图 3: 自发 Ca2 +在星形胶质细胞中发出信号.(A) GCaMP-转 AWESAM 星形胶质细胞在包括薄的过程 (箭头) 中的整个胶质组织网络中呈现自发的 Ca2 +事件。一个 ca2 +波从从左到右的几个星形胶质细胞从不同的时间点的图像中传播, 其中浅色描述高 Ca2 +浓度的区域, 而黑色区域代表胞外区域。使用活体细胞共聚焦显微镜获取图像。(B) ca2 +浓度随着时间的推移在 color-coded 区域发生变化, 显示为 GCaMP 荧光强度的痕迹, 说明了 ca2 +信号波在几个星形胶质细胞之间的传播。<a href="//cloudflare.jove.com/files/ftp_upload/56092/56092fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol at JoVE.com

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

这里描述的 AWESAM 协议是最理想的培养小鼠星形胶质细胞与其他脑神经干细胞的快速, 简单, 便宜的方式。AWESAM 星形胶质细胞展示自发 Ca2 +信号, 形态学和基因表达谱类似于星形胶质细胞在体内。

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

culturing-vivo-like-murine-astrocytes-using-fast-simple-inexpensive

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

8.4K 次观看.  University of Oxford. 这里描述的 AWESAM 协议是最理想的培养小鼠星形胶质细胞与其他脑神经干细胞的快速, 简单, 便宜的方式。AWESAM 星形胶质细胞展示自发 Ca2 +信号, 形态学和基因表达谱类似于星形胶质细胞在体内。

视频: Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

小鼠脑皮层星形胶质细胞的分离和培养

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

科研

神经科学

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.

建模星形细胞瘤发病机制<em&gt;在体外</em&gt;和<em&gt;体内</em&gt;使用星形胶质细胞或条件，遗传工程小鼠神经干细胞

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

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

在缺血样条件下监测星形胶质细胞的反应性和增殖体外

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells.
The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

大鼠星形胶质细胞和小胶质细胞的原代培养及其在肌萎缩侧索硬化症研究中的应用

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro&#160;experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

小鼠星形胶质细胞的分离和直接神经元重编程

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through ...

从成年小鼠脊髓中高效提取星形胶质细胞和小胶质细胞

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic cells exhibit rapid responses to environmental changes and display significant heterogeneity in terms of morphology, transcriptional profiles, and functions. While our understanding of the functions of glial cells in health and disease has advanced substantially, there remains a need for in vitro, cell-specific analyses conducted in the context of insults or injuries to comprehensively characterize distinct cell populations. Isolating cells from the adult mouse offers several advantages over cell lines or neonatal animals, as it allows for the analysis of cells under pathological conditions and at specific time points. Furthermore, focusing on spinal cord-specific isolation, excluding brain involvement, enables research into spinal cord pathologies, including experimental autoimmune encephalomyelitis, spinal cord injury, and amyotrophic lateral sclerosis. This protocol presents an efficient method for isolating astrocytes and microglia from the adult mouse spinal cord, facilitating immediate or future analysis with potential applications in functional, molecular, or proteomic downstream studies.

作者聚焦:从成年小鼠脊髓中分离神经胶质细胞 

This protocol outlines the isolation of purified astrocytes and microglia from the adult mouse spinal cord, facilitating subsequent applications such as RNA analysis and cell culture. It includes detailed cell dissociation methods and procedures designed to enhance both the quality and yield of isolated cells.

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于 体外 测定和转录组学研究

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...

Generating a Primary Culture of Astrocytes from a Rat Pup Brain

Source: Mili&#263;evi&#263;, K., et al., <a href="https://app.jove.com/v/63483">Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis.</a> J. Vis. Exp. (2022)
The video demonstrates a technique for culturing primary astrocytes from the rat cortex. The cortex is mechanically dissociated to obtain a homogeneous glial cell suspension, which is then plated on polypeptide-coated plates to promote selective proliferation of astrocytes. Subsequently, the astrocytes are enzymatically dissociated from the plates and transferred to fresh media, allowing the culture of pure astrocytes.

从大鼠幼犬大脑中产生星形胶质细胞的原代培养物

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Primary cell culture preparation

...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

A Simple Technique to Isolate and Culture Murine Astrocytes

Source: Wolfes, A. C., et al., <a href="https://app.jove.com/v/56092">Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol.</a> J. Vis. Exp. (2018)
The video demonstrates a simple method to culture murine astrocytes in isolation from other brain cells. During culturing, by using astrocyte-specific media and inhibiting the adherence of non-astrocytic cells, the method selectively cultures astrocytes. These cultured astrocytes exhibit stellate morphology with fine processes.

一种分离和培养小鼠星形胶质细胞的简单技术

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Rat or Mouse Brain Dissection
...

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

摘要

探索更多视频

此视频中的章节

小鼠脑皮层星形胶质细胞的分离和培养

建模星形细胞瘤发病机制

在缺血样条件下监测星形胶质细胞的反应性和增殖体外

大鼠星形胶质细胞和小胶质细胞的原代培养及其在肌萎缩侧索硬化症研究中的应用

小鼠星形胶质细胞的分离和直接神经元重编程

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于体外测定和转录组学研究

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于体外测定和转录组学研究

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于体外测定和转录组学研究

从大鼠幼犬大脑中产生星形胶质细胞的原代培养物

一种分离和培养小鼠星形胶质细胞的简单技术

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

摘要

探索更多视频

此视频中的章节

小鼠脑皮层星形胶质细胞的分离和培养

建模星形细胞瘤发病机制

在缺血样条件下监测星形胶质细胞的反应性和增殖体外

大鼠星形胶质细胞和小胶质细胞的原代培养及其在肌萎缩侧索硬化症研究中的应用

小鼠星形胶质细胞的分离和直接神经元重编程

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于 体外 测定和转录组学研究

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于 体外 测定和转录组学研究

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于 体外 测定和转录组学研究

从大鼠幼犬大脑中产生星形胶质细胞的原代培养物

一种分离和培养小鼠星形胶质细胞的简单技术

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于体外测定和转录组学研究

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于体外测定和转录组学研究

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于体外测定和转录组学研究