胚胎和肝脏斑马鱼细胞系球状体的3D培养方法
Summary
在这里，我们提出了一种有效，简单和快速的3D培养方案，用于形成两种斑马鱼（Danio rerio）细胞系的球状体：ZEM2S（胚胎）和ZFL（正常肝细胞）。
Abstract
鱼细胞系是有希望的 体外 生态毒性评估模型;然而，传统的单层培养系统（2D培养）具有众所周知的局限性（例如，培养寿命和维持某些 体内 细胞功能）。因此，已经提出了3D培养物，例如球状体，因为这些模型可以复制组织样结构，更好地重新捕获 体内 条件。本文介绍了一种有效、简单且快速的 3D 培养方案，用于用两种斑马鱼 （Danio rerio） 细胞系形成球状体：ZEM2S（胚胎）和 ZFL（正常肝细胞）。该方案包括将细胞接种在圆底、超低附着的 96 孔板中。在轨道振荡（70rpm）下5天后，每孔形成单个球体。形成的球体呈现稳定的尺寸和形状，该方法避免了在井中形成多个球体;因此，没有必要手工挑选相似大小的球体。这种球状体方法的简便性、速度和重现性使其可用于高通量 体外 测试。
Introduction
球状体是在3D培养中以紧密的细胞间接触培养细胞时形成的小细胞球体。球状体模拟体内组织环境的能力已经在各种细胞系和原代细胞中进行了研究1，2。然而，尽管用于哺乳动物毒性研究的球状体已经发展得很好，但用于非哺乳动物脊椎动物（例如鱼类）毒性研究的球体的开发仍在进行中3。对于鱼细胞系，球状体已经通过各种不同的方法开发，例如使用不同类型的孔板3，4，5，6，7的轨道振荡（OS）和使用磁性纳米颗粒8的磁悬浮方法。然而，其中一些球状体培养方法可能比其他方法具有更多的缺点。
例如，大微孔板（24孔板）中的回转方法可能会产生大量大小和形状不同的球体;事实上，已经证明了多球体结构的形成7。这需要付出巨大的努力来手工挑选具有相似大小和形状的球体进行实验。悬挂液滴3D培养方法通常用于生成哺乳动物细胞系1，2，9，10，11的球状体，从而每滴可以生成单个球状体，避免了上述问题。然而，尽管改进的悬挂液滴法（悬滴+轨道振荡）能够使用廉价的方法生成ZFL球体，但它有其缺点12。形成的细胞聚集体不能在液滴中长时间维持;因此，它们需要转移到孔板中。该过程需要密集的处理和在层流罩中长时间的工作，因为它是使用微量移液器12滴加进行的。此外，该方法需要10天才能完全形成ZFL球体（悬挂液滴5天+OS5天）12。这些缺点可能会限制3D鱼球体在毒性测试中的应用，特别是考虑到化学优先级和产品可持续性的潜在应用。
因此，本文描述了一种 3D 培养方案，该方案能够基于结合使用 96 孔、超低附着板（ULA 板）和轨道振荡器（22 mm 旋转直径）生成 ZFL（D. rerio 正常肝细胞）和 ZEM2S（D. rerio 胚泡期胚胎）细胞系的单个球状体。所应用的方法简单且可重复，并且可以在短时间内（5天）生成大量大小和形状相似的微球。该方法的优点可以支持鱼类三维模型在工业界和学术界的水生毒性研究中的应用，以及实施生态毒性测试替代方法的进展。
Protocol
在圆底96孔板中生成ZFL和ZEM2S细胞系的3D球状体的关键步骤如图 1所示。 注意：有关本协议中使用的所有材料的详细信息，请参阅 材料 表，有关本协议中使用的溶液和培养基，请参阅 表1 。 1. 细胞培养基和单层培养 在28°C的培养箱中以单层形式生长两种细胞系（ZFL，ZEM2S）没有CO2…
Representative Results
通过该方法形成每孔具有稳定尺寸和形状的单个球体。图2说明了ZFL和ZEM2S细胞的单个球体在轨道振荡（70rpm）下的ULA板孔中的形成过程。ZFL和ZEM2S细胞系在3D培养中具有不同的行为。ZEM2S细胞系具有自眼眶振荡第一天起就易于形成球状体形状的能力的特征（图2E），而ZFL细胞系需要5天才能达到所需的球状体形状（图2A-C<s…
Discussion
这是一种简单、简单且快速的生成斑马鱼肝脏和胚胎细胞系球状体的方法。该方法由该小组基于对现有3D球体方法的修改而开发，以克服与微球形成相关的科学研究中报告的问题，以及3D球体测定的数据准确性的不确定性。例如，报告的问题在于处理困难，生成微球的耗时性，选择相似尺寸和形状的微球进行测定的必要性，以及在悬挂液滴法3，7，</s…
Disclosures
The authors have nothing to disclose.
Acknowledgements
为了纪念这部作品的合著者Márcio Lorencini博士，他是化妆品领域的优秀研究人员，致力于促进巴西的化妆品研究。作者感谢生理学系（UFPR）的多用户实验室提供设备，并感谢高等教育人员改进协调组织（CAPES，巴西）（财务代码001）和Grupo Boticário的财政支持。
Materials
| 96-well Clear Round Bottom Ultra-Low Attachment Microplate, Individually Wrapped, with Lid, Sterile | Corning | 7007 | |
| DMEM, powder, high glucose, pyruvate | Gibco | 12800-017 | |
| Ham's F-12 Nutrient Mix, powder | Gibco | 21700026 | |
| HEPES (1M) | Gibco | 15630080 | |
| Image Processing and analysis in Java (ImageJ) 1.52p software | National Institutes of Health, USA |
Available at: https://imagej.nih.gov/ij/index.html | |
| Leibovitz's L-15 Medium, powder | Gibco | 41300021 | |
| Orbital shaker | Warmnest | KLD-350-BI | 22 mm rotation diameter |
| Dulbeccos PBS (10x) with calcium and magnesium | Invitrogen | 14080055 | |
| Penicillin-Streptomycin (10,000 U/mL) | Gibco | 15140122 | |
| RPMI 1640 Medium | Gibco | 31800-014 | |
| FBS – Fetal Bovine Serum, qualified, USDA-approved regions | Gibco | 12657-029 | |
| Sodium bicarbonate, powder, bioreagent for molecular biology | Sigma-Aldrich | S5761 | |
| Trypan blue stain (0,4%) | Gibco | 15250-061 | |
| Trypsin-EDTA (0.5%), no phenol red | Gibco | 15400054 | |
| ZEM2S cell line | ATCC | CRL-2147 | This cell line was kindly donated by Professor Dr. Michael J. Carvan (University of Wisconsin, Milwaukee, USA) |
| ZFL cell line | BCRJ | 256 |
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Cite This Article
de Souza, I. R., Micali Canavez, A. D. P., Schuck, D. C., Costa Gagosian, V. S., de Souza, I. R., de Albuquerque Vita, N., da Silva Trindade, E., Cestari, M. M., Lorencini, M., Leme, D. M. A 3D Culture Method of Spheroids of Embryonic and Liver Zebrafish Cell Lines. J. Vis. Exp. (191), e64859, doi:10.3791/64859 (2023).