在自制迷你生物反应器中从诱导多能干细胞中生成脑类器官
1Federal Research and Clinical Center of Physical-Chemical Medicine,Federal Medical Biological Agency, 2Faculty of Bioengineering and Bioinformatics,Lomonosov Moscow State University, 3Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Federal Research and Clinical Center of Physical-Chemical Medicine,Federal Medical Biological Agency, 4Department of Pathological Anatomy,North-Western State Medical University, 5Department of Biology,Lomonosov State University
Summary
在这里，我们描述了一种从人类诱导多能干细胞（iPSCs）产生脑类器官的方案。为了获得大量和高质量的脑类器官，我们使用自制的迷你生物反应器。
Abstract
iPSC衍生的脑类器官是一种有前途的技术，用于 体外 建模神经系统的病理学和药物筛选。这项技术最近才出现。它仍处于起步阶段，并且有一些限制尚未解决。目前的方案不允许获得足够一致的类器官用于药物发现和临床前研究。类器官的成熟可能需要长达一年的时间，这促使研究人员同时启动多个分化过程。这在空间和设备方面给实验室带来了额外的成本。此外，脑类器官通常在中心有一个坏死区，患有营养和缺氧。因此，大多数现行方案使用循环系统作为培养基以改善营养。
同时，没有用于类器官培养的廉价动态系统或生物反应器。本文描述了一种在紧凑且廉价的自制小型生物反应器中生产脑类器官的方案。该协议允许大量获得高质量的类器官。
Introduction
人类iPSC衍生的模型广泛用于神经发育和神经退行性疾病的研究1。在过去的十年中，3D脑组织模型，即所谓的脑类器官，基本上补充了传统的2D神经元培养物2。类器官在一定程度上概括了胚胎大脑的3D结构，并允许更精确的建模。许多方案已经发表，用于产生代表不同大脑区域的类器官：大脑皮层3，4，5，小脑6，中脑，前脑，下丘脑7，8，9和海马体10。已经有多个使用类器官研究人类神经系统疾病的例子11。此外，类器官在药物发现12中实施，并用于传染病研究，包括SARS-Cov-213，14。
大脑类器官的直径可达几毫米。因此，类器官的内区可能患有缺氧或营养不良，最终变得坏死。因此，许多方案包括特殊的生物反应器8、振荡器或微流体系统15。这些设备可能需要大量昂贵的细胞培养基。此外，这种设备的成本通常很高。一些生物反应器由许多机械部件组成，使其难以灭菌以重复使用。
大多数方案都受到”批处理效应“16的影响，这在从相同的iPSC获得的类器官之间产生了显着的变异性。这种变异性阻碍了需要一致性的药物测试或临床前研究。类器官的高产量足以选择大小均匀的类器官，可以部分解决这个问题。
时间因素也是一个重大问题。Matsui等人（2018）表明，脑类器官至少需要六个月才能达到成熟17。Trujillo等人（2019）还证明，只有在培养六个月后，类器官中才会发生电生理活性18。由于类器官成熟时间长，研究人员经常在完成前一个分化之前启动新的分化。多个并行的差异化过程需要额外的费用、设备和实验室空间。
我们最近开发了一种迷你生物反应器，主要解决了上述问题19。这款自制生物反应器由超低附着力或未经处理的培养皿组成，中间有一个塑料旋钮。这种塑料旋钮可防止类器官拥挤及其在培养皿中心的凝结，这是由振荡器的旋转引起的。本文描述了这种廉价而简单的自制迷你生物反应器如何能够大量产生高质量的脑类器官。
Protocol
注意：在整个方案中使用无菌技术，不包括步骤1.2和1.3。在应用于细胞或类器官之前，将所有培养基和溶液加热至37°C。在37°C的CO2 培养箱中培养细胞，在80%湿度下在5%CO2 中培养细胞。协议方案如图 1所示。 1. 将培养皿转变为微型生物反应器 将无菌的15 mL离心管切割成7-8毫米高的环;高压灭菌环。 将低附着力、…
Representative Results
协议方案如图1所示。该协议包括五种培养基，其中iPSCs在至少一个月内分化为脑类器官。开始分化，然后iPSCs达到75-90%的汇合度（图2A，B）。在培养基A中iPSC培养的第10-11天观察到向神经元分化的最初迹象，当时细胞开始聚集成”玫瑰花”（图2C）。在第14-15天，iPSCs分化为神经上皮祖细胞。99%的细胞在神经上皮标志物SOX1?…
Discussion
所描述的方案有两个关键步骤，允许产生大小均匀的高质量类器官。首先，类器官从在细胞数量和细胞成熟度上几乎相同的球体生长。其次，自制的生物反应器为每个类器官提供了一个统一的环境，在这个环境中，类器官不会聚集或粘在一起。
细胞质量和细胞成熟状态对于执行实验方案至关重要。在iPSC汇合75-90%处开始神经元分化至关重要。如果细胞密度太低，iPSC可以分化成…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了俄罗斯联邦科学和高等教育部（RT-PCR分析）的075-15-2019-1669赠款以及俄罗斯科学基金会第19-15-00425号赠款（用于所有其他工作）的支持。作者还感谢Pavel Belikov在视频编辑方面的帮助。手稿中的人物是用 BioRender.com 创作的。
Materials
| Advanced DMEM/F-12 | Gibco | 12634010 | DMEM/F-12 |
| AggreWell400 | STEMCELL Technologies Inc | 34425 | 24-well culture plate with microwells |
| B-27 Supplement | Gibco | 17504044 | Neuronal supplement B |
| GlutaMAX Supplement | Gibco | 35050061 | 200 mM L-alanyl-L-glutamine |
| Human BDNF | Miltenyi Biotec | 130-096-285 | |
| Human FGF-2 | Miltenyi Biotec | 130-093-839 | |
| Human GDNF | Miltenyi Biotec | 130-096-290 | |
| KnockOut Serum Replacement | Gibco | 10828028 | Serum replacement |
| mTESR1 | STEMCELL Technologies Inc | 85850 | Pliripotent stem cell medium |
| N2 Supplement | Gibco | 17502001 | |
| Neurobasal Medium | Gibco | 21103049 | Basal medium for neuronal cell maintenance |
| Penicillin-Streptomycin Solution | Gibco | 15140130 | |
| Plasmocin | InvivoGen | ant-mpt-1 | Antimicrobials |
| Purmorphamine | EMD Millipore | 540220 | |
| StemMACS Y27632 | Miltenyi Biotec | 130-106-538 | Y27632 |
| StemMACS Dorsomorphin | Miltenyi Biotec | 130-104-466 | Dorsomorphin |
| StemMACS LDN-193189 | Miltenyi Biotec | 130-106-540 | LDN-193189 |
| StemMACS SB431542 | Miltenyi Biotec | 130-106-543 | SB431542 |
| Trypan Blue Solution | Gibco | 15250061 | |
| Versen solution | Gibco | 15040066 | 0.48 mM EDTA in PBS |
| β-mercaptoethanol | Gibco | 31350010 |
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Tags
Keywords: Brain OrganoidInduced Pluripotent Stem CellsMini Bioreactors3D ModelCentral Nervous System PathologiesRegenerative MedicineDifferentiationShakerLow-adhesionBioreactorsNutrient MediumPlastic CrumbsChloroformLiquid PlasticPetri DishPluripotent Stem CellsDulbecco Modified Eagle MediumFisher-12 MediumSerum Replacement
Citar este artigo
Eremeev, A., Belikova, L., Ruchko, E., Volovikov, E., Zubkova, O., Emelin, A., Deev, R., Lebedeva, O., Bogomazova, A., Lagarkova, M. Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors. J. Vis. Exp. (178), e62987, doi:10.3791/62987 (2021).