Name: increasing durability of dissociated neural cell cultures using biologically active coralline matrix
Uploaded: 2020-06-03T13:30:00.000+00:00
Duration: 9 min 22 s
Description: Watch this Scientific Journal Video about Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix at JoVE.com

这项工作由以色列贸易和劳工部的KAMIN计划和Qrons Inc.资助，地址为777 Brickell Avenue Miami，FL 33131，US。

该协议中动物的使用得到了国家动物护理和使用委员会的批准。
注意:碳酸钙珊瑚骨架应以文石的结晶形式使用。到目前为止，用于神经培养物的珊瑚类型是 Porites Lutea，Stylophora pistillata和 Trachyphyllia Geoffroyi。 骨架可以全部购买或磨碎。
1. 清洁珊瑚骨架碎片
注意:以下步骤应在室温下的化学罩中进行，因为下面描述的溶液是危险的，可能会导致灼伤和刺激。
<ol><li>用锤子打破珊瑚骨架并将其分成0.5-2厘米的碎片。为了溶解有机和非有机残留物，将珊瑚骨架碎片浸泡在10%次氯酸钠溶液中10分钟，然后用双蒸水（DDW）洗涤一次。</li>
	<li>为了去除残留的有机残留物，将碎片浸泡在1M NaOH溶液中5分钟，然后用DDW洗涤一次。通过将片段浸泡在30%H2O2 溶液中10分钟，继续去除有机沉积物，然后用DDW洗涤片段3倍。</li>
	<li>尽可能多地去除多余的DDW，并将珊瑚碎片留在引擎盖中干燥（1-8小时）。</li>
</ol>2. 清洁玻璃盖玻片
<ol><li>将玻璃盖玻片转移到100毫米玻璃培养皿中。加入 10 mL 95% 乙醇 15 分钟。</li>
	<li>取出乙醇并用DDW 3x洗涤，每次洗涤之间等待10分钟。将培养皿放在80°C预热的加热板上，直到DDW蒸发。干燥时轻轻搅拌盘内的盖玻片几次，以防止它们相互粘连。</li>
	<li>高压灭菌盖玻片。</li>
</ol>3.珊瑚骨架颗粒的制备
<ol><li>使用研钵和研杵研磨珊瑚骨架碎片（手动研磨），直到完全分解。结果是尺寸范围为20μm-200μm的颗粒混合物。</li>
	<li>或者，使用电动研磨机以 1，000 rpm 的速度研磨珊瑚骨架碎片 30 秒（刀片长度 = 6 厘米;宽度 = 0.5 厘米–1.0 厘米）。得到的晶粒尺寸与手工研磨产生的粒度范围相似。</li>
</ol>4. 特定尺寸范围的谷物的纯化
注意:如果需要控制基质中颗粒的大小，请使用以下基于过滤的颗粒纯化程序。
<ol><li>将颗粒转移到手动或电动40μm过滤网过滤器上。</li>
	<li>通过过滤器筛分谷物，将谷物分为两个特定范围（如果使用电动过滤器，建议使用以下条件:振荡= 600振幅/分钟，弹跳= 6 / s）。该过程产生两组晶粒尺寸，一组<40μm，第二组>40μm。 注意:这两种尺寸可以通过使用不同网格的过滤器来确定。如果需要更有限的范围，则通过具有不同网格的过滤器重新筛选步骤4.2中的每个组。</li>
	<li>高压灭菌谷物。</li>
</ol>5. 准备珊瑚粒涂层的餐具或盖玻片
<ol><li>用于包衣烧瓶、平板或培养皿 注意:以下步骤应在无菌条件下进行。<ol><li>将珊瑚骨架颗粒加入溶解在汉克斯溶液中的 20 μg/mL 聚-D-赖氨酸 （PDL） 溶液中。推荐浓度为每 1 mL PDL 溶液 5 mg/10 mg 谷物。</li>
		<li>将溶液倒入烧瓶和培养皿（约2mL / 25cm2）中，并在4°C下孵育过夜。 颗粒下沉并附着在底部。</li>
		<li>第二天，用无菌DDW清洗烧瓶和盘子一次。让烧瓶和培养皿在引擎盖中干燥。 注意:最好使用新鲜涂层的烧瓶和培养皿。如果在4°C下保存，包被的烧瓶和培养皿可以在包衣后使用长达一周。 但是，矩阵的有效性可能会受到影响。</li>
	</ol></li>
	<li>镀膜玻璃盖玻片（直径 12 毫米，厚度 0.17 毫米）<ol><li>将珊瑚骨架颗粒以任何所需的浓度添加到DDW中。用于神经细胞的常见密度为 5 mg/mL 和 10 mg/mL。将 40 μL 谷物溶液倒在盖玻片的中心（图 4）。</li>
		<li>将盖玻片放在预热至80°C的加热板上，等待完全蒸发（通常为15分钟）。在这些条件下，颗粒粘附在盖玻片上。高压灭菌涂层盖玻片。储存在无菌条件下。</li>
		<li>培养前一天，将盖玻片放在无菌24孔板的盖子上。向每个盖玻片中加入 100 μL 20 μg/mL PDL 溶液。使用尖端确保液体覆盖整个颗粒区域和盖玻片表面的其余部分。</li>
		<li>用盘子底部盖住盖子。用石蜡膜包裹板的侧面。在4°C孵育过夜。</li>
		<li>第二天，用DDW清洗一次，然后在引擎盖中晾干。 注意:最好使用新涂覆的盖玻片。</li>
	</ol></li>
</ol>6. 珊瑚骨架颗粒涂层玻璃盖玻片上海马解离细胞的培养
注意:海马解离细胞培养的方法是从先前发表的程序24，27修改而来的。描述了四只大鼠幼崽的培养物的制备。每个海马体的预期产量为1-1.5 x 106 个细胞。
<ol><li>设置<ol><li>准备一个空的60毫米培养皿。将 2 mL 最小必需培养基 （MEM） 倒入 60 mm 培养皿中，放在冰上。</li>
		<li>将 2 mL MEM 倒入 35 mm 培养皿中，并将其放入 37 °C、5–10% CO2 的培养箱中。将 2 mL MEM 倒入 15 mL 管中，并保持在 4 °C。</li>
		<li>将 1 mL 首日培养基倒入 15 mL 管中，并将其放入 37 °C、5-10% CO2 的培养箱中。 注意:首日培养基的成分在 材料表中描述。</li>
		<li>解冻200μL的2.5%胰蛋白酶溶液并在37°C下孵育。</li>
		<li>使用火焰准备三个直径为 0.5 mm、0.75 mm 和 1.0 mm 的玻璃巴斯德移液器。</li>
		<li>准备足够的手术工具:一把大剪刀、两把小剪刀、一把大镊子、四把小镊子和一把手术刀。</li>
		<li>在引擎盖中准备一个体视显微镜。</li>
	</ol></li>
	<li>使用一把大剪刀，将0-3天大的Sprague Dawley大鼠幼崽从身体上分离到一个空的60毫米培养皿中（步骤6.1.1）。处理尸体。</li>
	<li>用大镊子从嘴里拿起头。用小剪刀剪开覆盖头骨的皮肤。</li>
	<li>用干净的剪刀进入颅骨下方（小脑一侧），将头骨向右和向左切割以使其被移除。用小镊子，从大脑上剥下头骨。</li>
	<li>用干净的镊子将大脑与头部分开，并将其放入含有2mL冷MEM的60毫米培养皿中（见步骤6.1.2）。</li>
	<li>使用两个小镊子，在体视显微镜下解剖海马体。将海马体转移到含有MEM的制备的35毫米培养皿（来自步骤6.1.2）中。 注意:应为每个小狗重复步骤 6.3–6.6。</li>
	<li>用手术刀将海马切成约1毫米的切片。加入 200 μL 胰蛋白酶溶液（稀释至终浓度为 0.25%）。轻轻混合并在37°C，5–10%CO2 下孵育30分钟。</li>
	<li>孵育后，向培养皿中加入 2 mL 冷 MEM（步骤 6.1.2）以使胰蛋白酶失活。使用最大直径的玻璃巴斯德移液管（步骤6.1.5）将胰蛋白酶消化组织转移到含有预热至37°C的15 mL首日培养基的15 mL管中（步骤6.1.3）。 注意:用于进一步处理组织的最佳培养基与组织 （v:v） 比率为 1 mL/8 海马体。尽可能避免将培养基与组织一起转移。</li>
	<li>通过最大直径的玻璃移液管10-15次来研磨组织。然后，使用中等直径的玻璃移液管重复此过程。继续使用最小直径的移液器研磨。避免冒泡以减少细胞死亡。</li>
	<li>让剩余的组织碎片下沉2-5分钟，然后将上清液转移到另一个15mL管中。使用血细胞计数器计数细胞。 注意:优选的细胞密度为 200，000–400，000 个细胞/mL。使用首日培养基以470 x g 稀释或离心以浓缩细胞。</li>
	<li>在每个玻璃盖玻片上接种 100 μL 细胞。接种时，确保用细胞覆盖整个盖玻片。在37°C，5–10%CO2下孵育。</li>
	<li>第二天，向孔中加入 500 μL 神经元生长培养基。 注意:神经元生长培养基的组成在 材料表中描述。</li>
	<li>使用镊子轻轻地将每个盖玻片转移到适当的孔中，同时通过倾斜盖玻片去除第一天培养基。从盖子上吸出剩余的介质。</li>
	<li>在37°C，5–10%CO 2下孵育。 避免在整个孵育过程中更换培养基。 在这些条件下，培养物可以维持长达 1 个月。主要问题是湿度。因此，请确保保持培养箱的最大加湿。</li>
</ol>

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提交人声明，他们没有相互竞争的经济利益。

这里介绍的技术描述了一种改善培养中神经细胞的维持和功能的方法。这是通过将细胞粘附在由珊瑚骨架颗粒制成的基质上来实现的，该基质滋养细胞并促进其生长和活性。使用这种技术增加了神经培养模型模仿大脑中细胞环境的能力。
与经典神经细胞培养方法中使用的其他底物相比，引入基质作为培养底物具有几个优点。首先，它增加了细胞粘附性。珊瑚骨架和PDL的组合产生比玻璃和PDL更强的粘合性（未显示）。粘附性的这种增加已被证明对于培养物中贴壁非浮动细胞的存活至关重要32。其次，珊瑚骨架颗粒的碳酸钙晶体是钙离子的来源，实际上被细胞吸收。与珊瑚骨架基质上的存活水平相比，这很可能是增强持久性和增加密度（图5C，D）的原因26。
第三个优点在于，与玻璃盖玻片不同，珊瑚骨架矩阵实际上是非平面的。颗粒是3D结构的，具有复杂的曲面结构。当考虑到细胞面对不同形态、尺寸和密度的颗粒种群时，这些结构特性会有所不同。这种粗糙且非平面的培养物质比经典的平面盖玻片更好地模拟体内细胞的环境。事实上，我们已经证明珊瑚骨架的粗糙度、大小和形态会影响细胞分布、存活和活动8，26，29。
此外，珊瑚骨架基质作为微环境为细胞提供的3D配置与用于体外3D生长的传统水凝胶基质主要不同。当使用水凝胶，例如胶原蛋白和透明质酸时，细胞嵌入凝胶23中。凝胶在空间填充、刚性和其他属性方面类似于细胞外基质，但由于凝胶的粘度，这些培养物需要复杂的补料系统才能保持细胞良好的营养21，22。相比之下，珊瑚骨架3D颗粒使细胞能够占据其表面，其表面对培养基完全开放。此外，与水凝胶培养物相比，珊瑚骨架基质上的细胞很容易通过相位和荧光显微镜进行监测。
此技术有几个限制。尽管具有上述优点，但珊瑚骨架是一种生物材料，不能制造，而是从死珊瑚中收集，导致基质供应受到限制，尽管有些可以在商业上获得。此外，使用多种珊瑚物种的骨骼可能会引入培养特性的变化。另一方面，珊瑚骨架晶体都由一种物质文石制成，并且可以控制颗粒的大小，这一事实消除了基质之间的大部分化学和结构多样性。所有测试的生物材料的颗粒都足够大，可以迫使细胞三维生长。细胞爬上晶粒8，通过附着在晶体33 的尖端生长并在晶粒27之间交叉。或者，由化石沉积物和合成碳酸钙制成的地质文石是市售的，可用作基质。但是，它可能显示出对玻璃盖玻片的附着力较低。
鉴于使用多种珊瑚物种，重要的是要注意，一些研究表明珊瑚的生物矿化，这可能导致碳酸钙晶体中存在不同的有机沉积物。这些不能使用本协议33，34中描述的清洁过程去除。在确定用于生物应用的生物材料时，这一事实至关重要。在本研究的情况下，先前执行了几个步骤，以确保支架的均匀性尽可能高。首先，对三种类型的珊瑚进行了基质测试: Porites Lutea，Stylophora Pistillata和 Trachyphyllia Geoffroyi。这些珊瑚骨骼上神经细胞的生长和存活没有显着变化。这一结果可能表明，在这种情况下，骨架内的不同有机沉积物没有显着影响。其次，清洁珊瑚骨架的傅里叶变换红外光谱显示不存在有机残留物29。
协议中应考虑一些关键步骤。首先，在手动筛分谷物时，建议不时更换过滤器，因为过滤器容易堵塞。电过滤器在筛分时使用更大的力。因此，它往往不容易堵塞。其次，在将PDL-珊瑚颗粒混合物分散在培养皿和盖玻片上时，重要的是要经常移液混合物以避免颗粒下沉。这将确保溶液的均匀分散。重要的是要注意谷物与盖玻片的附着过程。颗粒附着在玻璃盖玻片上期间不正确的加热温度、振动和其他因素可能导致颗粒在细胞接种之前或之后脱落。建议通过仔细控制温度和尽量减少振动来解决此问题。此外，优选在涂覆烧瓶和培养皿后的设定时间进行实验。我们建议在培养前一天新鲜涂覆培养皿。用PDL谷物混合物长时间孵育培养皿会导致附着在培养皿上的谷物数量多样化。
根据培养细胞的需要，可以对上述技术进行一些修改。除了改善培养中神经细胞生长的能力外，珊瑚骨架基质还支持骨细胞30、31、肝细胞和心肌细胞的生长（未发表的数据）。为了提高其他细胞类型的培养耐久性，建议检查对不同珊瑚类型骨骼的最强细胞反应，以及优化颗粒的大小和密度。
也可以直接在谷物上生长神经细胞，而无需额外的涂层。然而，如该协议（步骤5）所述，用PDL预涂颗粒极大地提高了神经细胞的粘附和存活率，高于使用PDL镀膜玻璃获得的水平。也可以根据不同细胞类型的喜好使用其他涂层材料。
值得一提的是，基质中晶粒的大小是有限的。超过某个阈值，约200μm，很难将它们粘合到玻璃盖玻片上。因此，基体表面的粗糙度具有有限的范围。需要较大颗粒或骨架的实验不适合这种技术。人们可能会找到一种替代方法来附着较大的颗粒，也许使用胶水或凝胶，但这可能会掩盖细胞与颗粒的接触。另一种可能性是直接在骨骼上培养细胞，无需研磨。该方法需要修改细胞接种程序24，27。非地面珊瑚骨架的3D结构确实有助于神经细胞的存活，正如我们在26之前报道的那样。然而，如此复杂而密集的基质使显微镜工作变得困难。地面骨架的妥协设置保留了骨架的化学性质，使2D显微镜分析成为可能，并且由于颗粒的大尺寸，迫使细胞在3D8中生长。
此外，过量的珊瑚骨架颗粒可能是有毒的。如上所述，珊瑚骨架基质的钙成分被细胞35吸收。由高晶粒密度引起的钙过量可能对某些细胞类型造成负担甚至抑制，例如神经细胞36。因此，可能需要降低晶粒密度并针对每种细胞类型进行优化。此外，重要的是要记住，珊瑚骨架的可用性可能有限。珊瑚在水族馆和自然环境中生长缓慢。由于全球变暖和海洋酸化，它们的生长进一步受阻37.因此，珊瑚骨骼的丰度和可用性是有限的。
当与培养物中的珊瑚骨架颗粒接触时，解离神经细胞的加强为神经生物学研究开辟了新的途径。细胞生长，神经炎延伸和分支，以及突触发生和突触可塑性，可以通过增强的强度和延长的实验持续时间进行研究。
此外，珊瑚基质似乎会导致神经胶质细胞的某些细胞功能增加，例如生长、反应性和增殖。因此，这些条件也可能增加星形胶质细胞的能力，以利用分泌的营养因子富集解离的神经培养基，这可能适用于中枢神经系统的再生医学。

解离神经细胞（在本例中为海马细胞）的培养物是通过提供高细胞分离和可及性来研究神经生长和功能的有价值的实验模型1，2，3。这种类型的培养物经常用于神经科学、药物开发和组织工程，因为可以收集大量信息，例如生长速率和活力、神经毒性、神经突生长和网络、突触连接和可塑性、形态修饰、神经突组织和布线等1，4，5，6，7。
尽管培养物很重要，但培养的细胞通常被迫在二维单层的玻璃盖玻片上生长。这些严格的环境修饰会随着时间的推移显着降低神经细胞的生存能力，因为玻璃盖玻片是具有低粘附强度的非培养基质，表现出较低的支持细胞生长的能力8，9，10，11。
由于培养的神经细胞被迫在具有挑战性的条件下生长，因此提高其生存能力的基本方法是尽可能模仿其自然环境12，13。这可以通过使用生物材料来实现，这些生物材料将充当基质并模仿细胞的细胞外基质，使它们能够形成组织样结构并协助其营养14。
使用生物材料是改善细胞培养物的一种有前途的方法，因为它们充当生物相容性支架，提供机械稳定性并增强各种细胞特性，包括粘附、存活、增殖、迁移、形态发生和分化15，16，17。几种类型的生物材料用于改善体外细胞的状况。其中包括生物聚合物，或生物成分，通常是细胞细胞外基质的一部分。这些生物材料大多用作聚合包衣剂或水凝胶18，19，20的形式。一方面，上面提到的基质为细胞提供了一个熟悉的3D环境来生长，鼓励它们粘附在培养皿上，并给予它们机械支撑21，22。另一方面，它们的聚合形式和细胞在水凝胶中的限制扰乱了细胞对生长培养基中存在的培养成分的访问，并且还使得通过显微镜方法对细胞的随访更加困难23。
珊瑚外骨骼是源自海洋的生物基质。它们由碳酸钙制成，具有机械稳定性，并且可生物降解。与玻璃盖玻片相比，先前使用珊瑚骨架作为培养中生长神经细胞的基质的研究显示出更大的粘附力24，25。此外，在珊瑚骨架上生长的神经细胞证明了它们摄入骨骼组成的钙的能力，这在营养匮乏的条件下保护神经细胞26。此外，珊瑚骨架是一种支持和培育基质，可增加神经细胞的存活，促进神经网络的形成，提高突触连接率，并能够形成组织样结构27，28。最近的研究还表明，珊瑚骨架基质的表面形貌在神经胶质细胞的分布和活化中起着至关重要的作用8，29。此外，珊瑚骨架可有效作为培养其他细胞类型的基质，例如培养中的骨细胞30，31，肝细胞和心肌细胞（未发表的数据）。
因此，珊瑚骨架是体外培养细胞的有前途的基质。因此，下面详述的方案描述了在珊瑚骨架上培养神经细胞的技术，以产生比现有方法更稳定和繁荣的神经培养物。该方案也可用于培养心肌细胞，肝细胞和其他细胞类型。

<table><tbody><tr><td>24-well plates</td><td>Greiner</td><td>#60-662160</td><td></td></tr><tr><td>B-27</td><td>Gibco</td><td>#17504-044</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>#A4503</td><td></td></tr><tr><td>D &amp;ndash; glucose</td><td>Sigma</td><td>#G8769</td><td></td></tr><tr><td>Dulbecco&#039;s Minimal Essential Eagle (DMEM)</td><td>Sigma</td><td>#D5796</td><td></td></tr><tr><td>Electrical sieve</td><td>Ari Levy</td><td>#3700</td><td></td></tr><tr><td>Fetal Bovine Serun (FBS)</td><td>Biological Industries</td><td>#04-007-1A</td><td></td></tr><tr><td>First Day Medium</td><td></td><td></td><td>85.1% Minimum Essential Eagle&amp;rsquo;s medium (MEM), 11.5% heat-inactivated fetal bovine serum, 1.2% L-Glutamine and 2.2% D-Glucose.</td></tr><tr><td>Flasks</td><td>Greiner</td><td>#60-690160</td><td>25cm^2, Tissue culture treated</td></tr><tr><td>Fluoro-deoxy-uridine</td><td>Sigma</td><td>#F0503</td><td></td></tr><tr><td>Glass Coverslips</td><td>Menzel-Glaser</td><td>#BNCB00120RA1</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Romical</td><td>#007130-72-19</td><td>Hazardous</td></tr><tr><td>Ham&#039;s F-12 Nutrient Mixture</td><td>Sigma</td><td>#N4888</td><td></td></tr><tr><td>HANK&#039;S solution</td><td>Sigma</td><td>#H6648</td><td></td></tr><tr><td>Kynurenic acid</td><td>Sigma</td><td>#K3375</td><td></td></tr><tr><td>L - glutamine</td><td>Sigma</td><td>#G7513</td><td></td></tr><tr><td>Manual strainer (40&amp;micro;m)</td><td>VWR</td><td>#10199-654</td><td></td></tr><tr><td>Minimun Essential Eagle (MEM)</td><td>Sigma</td><td>#M2279</td><td></td></tr><tr><td>Mortar and pestle</td><td>De-Groot</td><td>4-P090</td><td></td></tr><tr><td>NaClO (Sodium Hypochlorite)</td><td>Sigma</td><td>#425044</td><td>Hazardous</td></tr><tr><td>NaOH</td><td>Sigma</td><td>#S8045</td><td>Hazardous</td></tr><tr><td>Neuronal Growth Medium</td><td></td><td></td><td>45% MEM, 40% Dulbecco&#039;s modified eagle&#039;s medium (DMEM), 10% Nutrient mixture F-12 Ham, 0.25% (w/v) bovine serum albumin (BSA), 0.75% D-glucose, 0.25% L-Glutamine, 0.5% B-27 supplement, 0.1% kynurenic acid, 0.01% of 70 % uridine and 30% fluoro-deoxy-uridine.</td></tr><tr><td>Petri dish</td><td>Greiner</td><td>#60-628160, #60-627160</td><td>60mm, 35mm, respectively.</td></tr><tr><td>Poly D &amp;ndash; Lysine</td><td>Sigma</td><td>#P7280</td><td></td></tr><tr><td>Smart Dentin Grinder</td><td>KometaBio</td><td>#GR101</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>#15-090-046</td><td></td></tr><tr><td>Uridine</td><td>Sigma</td><td>#U3750</td><td></td></tr></tbody></table>

increasing durability of dissociated neural cell cultures using biologically active coralline matrix

解离的海马神经元和神经胶质细胞的培养是通过提供高细胞分离和受控环境来研究神经生长和功能的有价值的实验模型。然而，体外海马细胞的存活受到损害:大多数细胞在培养的第一周死亡。因此，确定提高培养中神经细胞耐久性的方法非常重要。
来自珊瑚骨架的结晶文石形式的碳酸钙可用作神经培养的优质活性基质。通过培育、保护和激活神经胶质细胞，珊瑚骨架比其他基质更好地增强这些细胞在体外的存活和生长。
该协议描述了一种在珊瑚基质上培养海马细胞的方法。该基质是通过将珊瑚骨架颗粒附着在培养皿、烧瓶和玻璃盖玻片上而产生的。颗粒通过将细胞引入精细的三维（3D）环境以生长并形成组织样结构来帮助改善细胞的环境。珊瑚骨架引入的3D环境可以通过研磨针对细胞进行优化，从而可以控制颗粒的大小和密度（即基质粗糙度），这种特性已被发现会影响神经胶质细胞的活性。此外，谷物的使用使培养物的观察和分析变得更加容易，尤其是在使用光学显微镜时。因此，该协议包括生成和优化珊瑚基质的程序，作为改善体外神经细胞维持和功能的工具。

为了制备珊瑚骨架基质，使用锤子将整个珊瑚骨架（图1A）破碎成0.5-2厘米的碎片（图1B），并通过三个步骤（方案中的步骤1）彻底清除有机残留物使用10%次氯酸盐溶液，1M NaOH溶液和30%H 2 O2溶液（图1C）。当骨骼颜色从棕色（图1D）变为白色（图1E）时，珊瑚碎片被很好地清理干净。然后，使用研钵和研杵（图2B）或电动研磨机（图2C）研磨清洁的碎片（图2A）。两种程序产生了相似的结果:尺寸在20μm-200μm之间的颗粒混合物（图2D-G）。
与可溶性底物相比，晶粒基质的优点之一是可以改变其几种结构和物理性质，使其更具定量性。在这个珊瑚骨架晶粒基质中，操纵了两个晶粒混合物属性:晶粒尺寸和晶粒密度。为了减少上述研磨程序产生的20 μm–200 μm尺寸多样性，选择了使用40 μm过滤网的筛分方法。使用手动（图3 A-D）或电动（图3E）过滤器，生产了两种类型的谷物混合物:一种颗粒尺寸范围为40μm–200μm（图3 F，H），第二种尺寸为40μm或更小（图3G，I）。只需施用不同量的谷物并通过加热将它们附着在盖玻片上（图4C）或通过重力将它们附着到烧瓶和培养皿上（图4H）来确定覆盖玻片和培养皿的颗粒密度。通过这种方式，产生了低密度（图4 D，F，I）和高密度（图4E，G，J）的矩阵。
图5显示了在涂有<40μm（10mg / mL）珊瑚骨架颗粒的盖玻片上培养海马细胞的结果。相差显微照片显示，细胞在基质不存在（图5A）和存在（图5B）的情况下形成了复杂的神经网络。细胞核染色（图5C，D）表明接种后14天，基质上的细胞密度高于未包被的盖玻片。用微管相关蛋白2（MAP2）染色显示，与对照（图5E）相比，基质上形成了复杂的神经元和树突状网络（图5F）。此外，在使用神经胶质纤维酸性蛋白（GFAP）可视化的珊瑚骨架基质上生长的神经胶质细胞经历了显着的形态变化。与在对照基质上生长的神经胶质细胞相比，它们获得了更尖锐的外观和更长的过程，在那里它们看起来更圆更平坦（图5G，H）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60443/60443fig01.jpg"> 图 1:清洁珊瑚骨架。 （A）珊瑚的骨架 Stylophora pistillata。（B） 清洁前分解骨架碎片。（C）用次氯酸盐溶液，氢氧化钠和过氧化氢处理后的（B）碎片。（D） 扩大（B）所示未清理的碎片。（E） 扩大（C）所示的清洁碎片。刻度:A = 20 毫米;B，C = 10 毫米;D，E = 3 mm。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60443/60443fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60443/60443fig02.jpg"> 图 2:研磨珊瑚骨架。 （A） 清洗珊瑚骨架碎片。（B）用于手工研磨的研钵和研杵。红色箭头指向生成的颗粒。（三）磨床。红色箭头指向生成的颗粒。（四）手工研磨后的谷物。（五）机械研磨后的谷物。（f） 扩大（d）。（g） 扩大（e）。刻度:A = 15 毫米;深，东 = 3 毫米;F，G = 500 μm。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60443/60443fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60443/60443fig03.jpg"> 图 3:控制晶粒尺寸。 （公元至日）手动应变程序。（A） 使用40 μm过滤器。（B） 将未过滤的研磨颗粒放入 50 mL 管中的过滤器，并使用涡旋过滤。（C） 在 50 mL 管底部过滤 <40 μm 颗粒。（D）最小尺寸与网格相似的颗粒（>40μm，黄色箭头）。（E） 机械过滤器，用于从 20 μm–200 μm 的颗粒混合物中过滤出 <40 μm 的颗粒。（F） 在过滤前研磨后演示不同的粒度。（G） 展示应变颗粒<40微米。 （H） 扩大（F）。（一） 扩大（g）。刻度:F，G = 900 μm;H，I = 300 μm。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60443/60443fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60443/60443fig04.jpg"> 图 4:控制矩阵的密度。 （A） 一个 1.5 mL 离心管，将 <40 μm 颗粒稀释至 10 mg/mL，DDW 用于盖玻片，PDL 用于其他培养皿。（B） 装有 40 μL 10 mg/mL 溶液的玻璃盖玻片。（C） 将 40 μL 10 mg/mL 溶液的盖玻片在预热至 80 °C 的热板上干燥，使颗粒粘附在盖玻片上。（D） DDW 蒸发后晶粒浓度为 5 mg/mL 的涂层盖玻片。（E） DDW蒸发后颗粒浓度为10 mg/mL的涂层盖玻片。（f） 扩大（d）。（g） 扩大（e）。（H） T-25烧瓶中10 mg/mL溶液，60 mm板。（I） 装有浓度为5毫克/毫升的谷物的包被烧瓶。（J） 装有浓度为10毫克/毫升的谷物的包衣烧瓶。刻度:B，D，E，I，J = 3 毫米;F，G = 600 μm。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60443/60443fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60443/60443fig05.jpg"> 图5:珊瑚骨架基质上海马神经细胞的培养。 图像显示了从培养物中生长14天的0-3天大鼠幼崽海马中提取的细胞，并用GFAP（红色，神经胶质细胞），MAP2（绿色，神经元体细胞和树突），DAPI（蓝色，细胞核）染色。（A）没有基质的情况下细胞的相衬图像。（B）珊瑚骨架基质上生长的细胞的相差图像。（C）在没有用DAPI染色的基质的情况下生长的细胞。（D）在用DAPI染色的珊瑚骨架基质上生长的细胞。（E）在没有MAP2染色基质的情况下生长的细胞。（F）在珊瑚骨架基质上生长的细胞染色为MAP2。（G）生长的细胞没有对GFAP进行基质染色。（H）在珊瑚骨架基质上生长的细胞染色GFAP。（I） （C）、（E）、（G） 的合并图像。（J） （D）、（F）、（H） 的合并图像。刻度 = 40 μm。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60443/60443fig05large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix at JoVE.com

Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

解离海马细胞培养是神经科学中的关键实验工具。当珊瑚骨架用作基质时，由于其神经保护和神经调节作用，神经细胞在培养中的存活和功能得到增强。因此，在珊瑚基质上生长的神经细胞显示出更高的耐久性，因此更适合培养。

使用生物活性珊瑚基质提高解离神经细胞培养物的耐久性

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high ...

increasing-durability-dissociated-neural-cell-cultures-using

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

1.2K 次观看.  Ariel University. 解离海马细胞培养是神经科学中的关键实验工具。当珊瑚骨架用作基质时，由于其神经保护和神经调节作用，神经细胞在培养中的存活和功能得到增强。因此，在珊瑚基质上生长的神经细胞显示出更高的耐久性，因此更适合培养。

视频: Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

在三维自组装多肽水凝胶的人类神经祖细胞的培养

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

科研

生物工程

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.

二层共培养小学的大鼠皮层神经元和神经胶质

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 ...

神经科学

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing &#946;-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100&#946;/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

Marrow stromal cells (MSCs) with neural potential exist within the bone marrow. Our protocol enriches this population of cells via hypoxic preconditioning and thereafter directs them to become mature Schwann cells.

骨髓衍生祖细胞的低氧预处理作为成熟雪旺细胞产生的来源

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

发育生物学

Preparation of Neuronal Co-cultures with Single Cell Precision

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

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

神经元共培养与单细胞精度的制备

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

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

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

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

研究对牙髓寄生虫信号反应的神经质生长的共同培养方法

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

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

IDG-SW3 Cell Culture in a Three-Dimensional Extracellular Matrix

Osteocytes are considered to be nonproliferative cells that are terminally differentiated from osteoblasts. Osteoblasts embedded in the bone extracellular matrix (osteoid) express the Pdpn gene to form cellular dendrites and transform into preosteocytes. Later, preosteocytes express the Dmp1 gene to promote matrix mineralization and thereby transform into mature osteocytes.This process is called osteocytogenesis. IDG-SW3 is a well-known cell line for in vitro studies of osteocytogenesis. Many previous methods have used collagen I as the main or the only component of the culturing matrix. However, in addition to collagen I, the osteoid also contains a ground substance, which is an important component in promoting cellular growth, adhesion, and migration. In addition, the matrix substance is transparent, which increases the transparency of the collagen I-formed gel and, thus, aids the exploration of dendrite formation through imaging techniques. Thus, this paper details a protocol to establish a 3D gel using an extracellular matrix along with collagen I for IDG-SW3 survival. In this work, dendrite formation and gene expression were analyzed during osteocytogenesis. After 7 days of osteogenic culture, an extensive dendrite network was clearly observed under a fluorescence confocal microscope. Real-time PCR showed that the mRNA levels of Pdpn and Dmp1 continually increased for 3 weeks. At week 4, the stereomicroscope revealed an opaque gel filled with mineral particles, consistent with the X-ray fluorescence (XRF) assay. These results indicate that this culture matrix successfully facilitates the transition from osteoblasts to mature osteocytes.

Here, we present a protocol for culturing IDG-SW3 cells in a three-dimensional (3D) extracellular matrix.

三维细胞外基质中的 IDG-SW3 细胞培养

Osteocytes are considered to be nonproliferative cells that are terminally differentiated from osteoblasts. Osteoblasts embedded in the bone extracellular ...

Co-Culture of Schwann Cells with DRG Neurons on a Pre-Stretched Membrane

Source: Liu, C., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54085">An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons.</a> J. Vis. Exp. (2016).
The video demonstrates the co-culturing of dorsal root ganglion (DRG) neurons with Schwann cells. DRG neurons are first grown on a pre-stretched membrane for aligned axon growth. Schwann cells are then introduced, which attach to the axons, insulating them and promoting neural cell survival and function.

雪旺细胞与 DRG 神经元在预拉伸膜上的共培养

1. Culture of dorsal root ganglion (DRG) on Pre-stretched Surface

	Preparation of Pre-stretched Anisotropic Surface
	
		Mix a 10:1 solution of base and ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Co-culture and Interaction Studies

Generating a 3D Co-Culture Model of Human Corneal Fibroblasts and Neurons

Source: Sharif, R., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56308">Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea.</a> J. Vis. Exp. (2018)
This video demonstrates a technique to generate a three-dimensional co-culture model system of primary human corneal fibroblasts and differentiated neuronal cells. This co-culture system can be used to study the stromal-nerve interactions.

生成人角膜成纤维细胞和神经元的 3D 共培养模型

1. Primary HCFs (human corneal fibroblasts) Culture and 3D Constructs Assembly

	To assemble 3D constructs from explants previously grown in EMEM 10% FBS ...

Preparation of Collagen-Coated Compartmented Culture Dishes for Neuronal Cell Culture

Source: Zepecki, J. P., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/59744">Real-Time monitoring of human glioma cell migration on dorsal root ganglion axon-oligodendrocyte co-cultures.</a> J. Vis. Exp. (2019)
This video demonstrates constructing a three-compartment cell culture system using collagen and non-reactive grease. This system is used to observe interactions between glioma cells and axons in real-time.

用于神经元细胞培养的胶原包被区室培养皿的制备


	Compartmented culture of Rat Dorsal Root Ganglia (DRGs), Oligodendrocytes (OPCs), and hGCs (human glioma cells)

1. Preparation of compartmented culture ...