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>

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 &#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/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/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/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/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/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

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 ...

科研

生物工程

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 ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

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 ...

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A general protocol is described for the preparation of V. natriegens crude cell extracts using common laboratory equipment. This high yielding protocol has been specifically optimized for user accessibility and reduced cost. Cell-free protein synthesis (CFPS) can be carried out in small scale 10 &#956;L batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of &#62; 260 &#956;g/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1&#8722;2 full days by a single user. This protocol can be easily integrated into existing protein synthesis pipelines to facilitate advances in bio-production and synthetic biology applications.

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template.

快速生长的纳氏弧菌的无细胞蛋白表达

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A ...