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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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…
Representative Results
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?…
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 24-well plates | Greiner | #60-662160 | |
| B-27 | Gibco | #17504-044 | |
| Bovine Serum Albumin (BSA) | Sigma | #A4503 | |
| D – glucose | Sigma | #G8769 | |
| Dulbecco's Minimal Essential Eagle (DMEM) | Sigma | #D5796 | |
| Electrical sieve | Ari Levy | #3700 | |
| Fetal Bovine Serun (FBS) | Biological Industries | #04-007-1A | |
| First Day Medium | 85.1% Minimum Essential Eagle’s medium (MEM), 11.5% heat-inactivated fetal bovine serum, 1.2% L-Glutamine and 2.2% D-Glucose. | ||
| Flasks | Greiner | #60-690160 | 25cm^2, Tissue culture treated |
| Fluoro-deoxy-uridine | Sigma | #F0503 | |
| Glass Coverslips | Menzel-Glaser | #BNCB00120RA1 | |
| H2O2 | Romical | #007130-72-19 | Hazardous |
| Ham's F-12 Nutrient Mixture | Sigma | #N4888 | |
| HANK'S solution | Sigma | #H6648 | |
| Kynurenic acid | Sigma | #K3375 | |
| L – glutamine | Sigma | #G7513 | |
| Manual strainer (40µm) | VWR | #10199-654 | |
| Minimun Essential Eagle (MEM) | Sigma | #M2279 | |
| Mortar and pestle | De-Groot | 4-P090 | |
| NaClO (Sodium Hypochlorite) | Sigma | #425044 | Hazardous |
| NaOH | Sigma | #S8045 | Hazardous |
| Neuronal Growth Medium | 45% MEM, 40% Dulbecco's modified eagle'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. | ||
| Petri dish | Greiner | #60-628160, #60-627160 | 60mm, 35mm, respectively. |
| Poly D – Lysine | Sigma | #P7280 | |
| Smart Dentin Grinder | KometaBio | #GR101 | |
| Trypsin | Gibco | #15-090-046 | |
| Uridine | Sigma | #U3750 |
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Citar este artigo
Weiss, O. E., Hendler, R. M., Baranes, D. Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix. J. Vis. Exp. (160), e60443, doi:10.3791/60443 (2020).