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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E., Vago, R., Baranes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superior+survival+and+durability+of+neurons+and+astrocytes+on+3-dimensional+aragonite+biomatrices.">Superior survival and durability of neurons and astrocytes on 3-dimensional aragonite biomatrices.</a> Tissue Engineering. 13 (3), 461-472 (2007).</li><li>Shany, B., Vago, R., Baranes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+primary+hippocampal+neuronal+tissue+on+an+aragonite+crystalline+biomatrix.">Growth of primary hippocampal neuronal tissue on an aragonite crystalline biomatrix.</a> Tissue Engineering. 11 (3-4), 585-596 (2005).</li><li>Baranes, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interconnected+network+of+ganglion-like+neural+cell+spheres+formed+on+hydrozoan+skeleton.">Interconnected network of ganglion-like neural cell spheres formed on hydrozoan skeleton.</a> Tissue Engineering. 13 (3), 473-482 (2007).</li><li>Morad, T. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gliosis+of+astrocytes+cultivated+on+coral+skeleton+is+regulated+by+the+matrix+surface+topography.">Gliosis of astrocytes cultivated on coral skeleton is regulated by the matrix surface topography.</a> Biomedical Materials. 14 (4), 045005 (2019).</li><li>Green, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Therapeutic+Potential+for+Marine+Skeletal+Proteins+in+Bone+Regeneration.">A Therapeutic Potential for Marine Skeletal Proteins in Bone Regeneration.</a> Marine Drugs. 11 (4), 1203-1220 (2013).</li><li>Neto, A. S., Ferreira, J. M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+and+Marine-Derived+Porous+Scaffolds+for+Bone+Tissue+Engineering.">Synthetic and Marine-Derived Porous Scaffolds for Bone Tissue Engineering.</a> Materials. 11 (9), (2018).</li><li>Ahmad Khalili, A., Ahmad, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review+of+Cell+Adhesion+Studies+for+Biomedical+and+Biological+Applications.">A Review of Cell Adhesion Studies for Biomedical and Biological Applications.</a> International Journal of Molecular Sciences. 16 (8), 18149-18184 (2015).</li><li>. Visualization of the ultrastructural interface of cells with the outer and inner-surface of coral skeletons Available from: <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/19218486">https://www.ncbi.nlm.nih.gov/pubmed/19218486</a> (2019)</li><li>Drake, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+skeletal+organic+matrix+from+the+stony+coral+Stylophora+pistillata.">Proteomic analysis of skeletal organic matrix from the stony coral Stylophora pistillata.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (10), 3788-3793 (2013).</li><li>Ramos-Silva, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+skeletal+proteome+of+the+coral+Acropora+millepora:+the+evolution+of+calcification+by+co-option+and+domain+shuffling.">The skeletal proteome of the coral Acropora millepora: the evolution of calcification by co-option and domain shuffling.</a> Molecular Biology and Evolution. 30 (9), 2099-2112 (2013).</li><li>Peretz, H., Blinder, P., Baranes, D., Vago, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aragonite+crystalline+matrix+as+an+instructive+microenvironment+for+neural+development.">Aragonite crystalline matrix as an instructive microenvironment for neural development.</a> Journal of Tissue Engineering and Regenerative Medicine. 2 (8), 463-471 (2008).</li><li>Morad, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CaCO3 Matrix Dictates Astrocytes Transition to Astrogliosis. , (2019).</li><li>Prada, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocean+warming+and+acidification+synergistically+increase+coral+mortality.">Ocean warming and acidification synergistically increase coral mortality.</a> Scientific Reports. 7, (2017).</li></ol>

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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?...

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 &#8211; glucose</td>
			<td>Sigma</td>
			<td>#G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;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&#8217;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>H2O2</td>
			<td>Romical</td>
			<td>#007130-72-19</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mixture</td>
			<td>Sigma</td>
			<td>#N4888</td>
			<td></td>
		</tr>
		<tr>
			<td>HANK&#39;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&#181;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&#39;s modified eagle&#39;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 &#8211; 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?...

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

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

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

心肌组织工程生物衍生的注射材料的制备

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

科研

生物工程

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

形状记忆聚合物主动细胞培养

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50pg total RNA, or 0.01-2.5pg mRNA, per cell 1) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell 2. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used 3,4. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields 5-8. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: (1) greater numbers of genes to be analyzed per cell; (2) more quantitative analysis of gene expression; and (3) better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming 9-12, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature 13. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile 13, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.

增加从单细胞的数量标准实验室逆转录反应的mRNA的cDNA产量，使用声Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Shock Wave Application to Cell Cultures

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to target cells or tissue without any subsequent damage. Therefore, in vitro experiments are of increasing interest. Various methods of applying shock waves onto cell cultures have been described. In general, all existing models focus on how to best apply shock waves onto cells.
However, this question remains: What happens to the waves after passing the cell culture? The difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected! We therefore developed a model that mainly consists of a Plexiglas built container that allows the waves to propagate in water after passing the cell culture. This avoids cavitation effects as well as reflection of the waves that would otherwise disturb upcoming ones. With this model we are able to mimic in vivo conditions and thereby gain more and more knowledge about how the physical stimulus of shock waves gets translated into a biological cell signal (&#8220;mechanotransduction&#34;).

Shock waves nowadays are well known for their regenerative effects. Therefore in vitro experiments are of increasing interest. We therefore developed a model for in vitro shock wave trials (IVSWT) that enables us to mimic in vivo conditions thereby avoiding distracting physical effects.

冲击波应用细胞培养

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

在一个三维胶原蛋白矩阵分析细胞迁移

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

改良的3D 水凝胶培养的原发胶质细胞的体外Neuroinflammation 模型的建立

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

一种新型的腱绦缝合技术，采用组织工程胶原蛋白移植物修复大肌腱缺损

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

肾皮质细胞外基质衍生水凝胶的制备

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

牺牲纤维膜细胞培养生产细胞外基质纤维

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Methods Article

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