通过侧向柔光照明促进脑类器官培养
1Department of Obstetrics and Gynecology, Guangdong Provincial Key Laboratory of Major Obstetric Diseases,The Third Affiliated Hospital of Guangzhou Medical University, 2Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes, 3School of Biotechnology and Health Sciences,Wuyi University
Summary
脑类器官为研究器官发育和人类疾病病理学提供了前所未有的机会。虽然脑类器官培养系统取得了巨大成功，但在应用这项技术方面仍然存在操作困难。本方案描述了一种促进介质改变和类器官转移的脑类器官程序。
Abstract
目前，脑类器官培养技术操作仍复杂，难以大规模应用。有必要找到一个简单而实用的解决方案。因此，本研究提出了一种更可行的脑类器官方案。为了解决早期介质变化和类器官转移中不可避免的不便，目前的研究通过应用工程原理优化了操作技术。采用柔和的光灯横向照亮胚体（EB）样品，通过增强的漫反射效果，肉眼可以看到EB。利用旋转产生的二次流动原理，类器官向孔中心聚集，这有利于介质变化或类器官转移的操作。与分散的细胞相比，胚状体在移液器中沉降得更快。利用这种现象，可以以简单的方式有效地去除大多数游离细胞和死细胞碎片，防止EB因离心而受到损害。本研究促进脑类器官培养的操作，并有助于促进脑类器官的应用。
Introduction
与二维（2D）培养系统相比，三维（3D）培养系统具有几个优点，包括某些器官的复杂结构的真实复制和有效复制1。因此，脑类器官是人脑发育与疾病2、药物筛选、细胞治疗等研究领域的重要辅助方法之一。
通过旋转悬浮法培养脑类器官有利于它们的发育和成熟3.虽然大脑类器官培养系统取得了巨大的成功，但它们仍然面临着限制其应用的关键挑战。例如，手工栽培涉及复杂的操作步骤，并给实现大规模应用带来了障碍。此外，在脑类器官培养的每个发育阶段，需要改变不同的培养基和细胞因子4.然而，在早期阶段，类器官或EB具有微小的尺寸（约200μm至300μm），并且在没有适当的设备的情况下几乎无法在视觉上进入。不可避免地，当介质发生变化时，一定量的珍贵类器官样品被冲走。已经探索了许多技术来克服其他类型的类器官培养物中的这一点，一些例子包括将整个类器官芯片浸入培养基中3天而不进行干预5;在旧介质被吸收后，使用吸水纸5通过盖玻片添加新鲜介质;或应用复杂的微流体管道进行流体交换6，7，8。在类器官培养的早期阶段遇到的另一个障碍是难以用肉眼进行直接观察，这可能导致操作不良，导致类器官移植步骤中的类器官损伤和丢失。因此，有必要建立一个更可行的方案，促进介质变化和类器官转移以产生类器官。
为了克服这些问题，提出了一种基于工程原理的相应优化操作，这显着且方便地促进了许多类器官手术。在自然界中，当太阳透过窗户缝隙照射到房屋中时，肉眼可以看到光束中舞动的灰尘。由于太阳光对尘埃的漫反射，一些光线被折射到眼球中以产生视觉图像。受9，10现象原理的启发，本研究制作了柔和的光灯并横向照亮了EB。研究发现，EB可以在不影响观察范围的情况下在视觉上清晰可见。由于涡流11，通过旋转培养板在液体中产生指向中心的二次流。原本分散的EB积聚在板的中心。基于此，针对类器官沉降速度快于细胞的现象，提出一种无需离心即可进行介质更换和类器官转移的简便操作方法。通过这种转移操作，培养基中的类器官可以有效地与游离细胞和死细胞碎片分离。
这里提出了一种易于操作的方案，从人多能干细胞中产生脑类器官。通过应用工程原理优化操作技术，使3D文化中的操作与2D文化中的操作一样简单可行。改进的液体交换方法和类器官转移操作也有助于其他类型的类器官培养和自动培养机的设计。
Protocol
该议定书是在《赫尔辛基宣言》之后进行的。经广州医科大学第三附属医院伦理委员会批准（医德伦理审查[2021]022号）。在实验之前，每种培养基都是根据Juergen A. Knoblich的配方12 （补充表1-4）制备的，或者使用市售的脑类器官试剂盒（见 材料表）。本研究中使用的iPSC先前由我们的实验室建立，并已获得知情豁免。SCA3-iPSCs由一名31岁的女性脊髓小脑共济…
Representative Results
本研究诱导iPSCs（图2B）进入脑类器官（图2C）。早期培养的EB表示OCT4标记（图2A），表明良好的多能性。在后期阶段，EBs发育成成熟的脑类器官（图2D）。该研究将iPSC从正常健康个体和SCA3患者培养成脑类器官（图3A）。SCA3，也称为马查多- 约瑟夫病（MJD），是由ATXN3基因25<…
Discussion
大脑类器官为医学研究开辟了新的途径。该技术的许多有用应用才刚刚开始探索28.本研究发现，遗传性疾病性脑类器官与正常脑类器官的转录组测序结果可以反映疾病与健康之间的差异。例如，RNA-seq数据分析结果（图3B）与许多报道的SCA3疾病29，30，31，32的研?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究由广东省自然科学基金（批准号：2020A0505100062）、广州市科技重点课题项目（第201904020025号）、国家自然科学基金项目（31872800号、32070582、82101937）和广州市博士后科研资助项目（陈邦珠）资助。
Materials
| 0.2 μm Filter | NEST Biotechnology, China | 331001 | |
| 1000 μL wide-bore pipette tip | Thermo Fisher Scientific, USA | 9405163 | |
| 200 μL wide-bore pipette tip | Thermo Fisher Scientific, USA | 9405020 | |
| 2-Mercaptoethanol | Merck, Germany | 8057400005 | |
| 4% Paraformaldehyde | Servicebio, China | G1101 | |
| 6-well low adhesion plate | NEST Biotechnology, China | 703011 | It is used for EBs suspension cultures |
| Aaccute cell detachment solution | STEMCELL Technologies, Canada | 07920 | It is used to digest iPSC into single cells. |
| AggreWell800 24-well | STEMCELL Technologies, Canada | 34811 | Microporous culture plate for EBs preparation. |
| Anti-Adherence Rinsing Solution | STEMCELL Technologies, Canada | 07010 | Rinsing solution for cultureware to prevent cell adhesion |
| B27-vit. A supplement | Thermo Fisher Scientific, USA | 12587010 | |
| bFGF | Peprotech, USA | GMP100-18B | |
| BSA | Beyotime Biotechnology, China | ST025 | |
| Centrifuge | Eppendorf, Germany | 5810 R | It can be used for centrifugation of various types of centrifuge tubes, reagent bottles and working plates. |
| Cover glass | Shitai Laboratory Equipment, China | 10212020C | |
| DAPI | Beyotime Biotechnology, China | C1002 | Used for nuclear staining. After DAPI was combined with double stranded DNA, the maximum excitation wavelength was 364nm and the maximum emission wavelength was 454nm. |
| DMEM-F12 | Thermo Fisher Scientific, USA | 11330032 | |
| ES-quality FBS | Thermo Fisher Scientific, USA | 10270106 | |
| Ficoll Paque | General Electric Company, USA | 17-5442-02 | Isolate the peripheral blood mononuclear cells according to Ficoll-Paque method. |
| Gelatin | Sangon Bioteach, China | A609764 | |
| Glutamax supplement | Thermo Fisher Scientific, USA | 35050061 | |
| Glutamax supplement | Thermo Fisher Scientific, USA | 17504044 | |
| Goat anti-Chicken IgY secondary antibody | Abcam, UK | ab150171 | Goat anti-Chicken IgG. Conjugation: Alexa Fluor 647. Ex: 652 nm, Em: 668 nm. Use at 1:500 dilution. |
| Goat anti-Mouse IgG secondary antibody | Abcam, UK | ab150120 | Goat anti-Mouse IgG. Conjugation: Alexa Fluor 594. Ex: 590 nm, Em: 617 nm. Use at 1:500 dilution. |
| Goat anti-Rabbit IgG secondary antibody | Abcam, UK | ab150077 | Goat Anti-Rabbit IgG. Conjugation: Alexa Fluor 488. Ex: 495 nm, Em: 519 nm. Use at 1:500 dilution. |
| Heparin | Merck, Germany | H3149 | |
| Horizontal shaker | Servicebio, China | DS-H200 | Relative centrifugal force (RCF) of 0.11808 x g is more appropriate, according to the manufacturer INFORS HT (Switzerland). |
| Insulin | Merck, Germany | I9278-5ML | |
| KOSR | Thermo Fisher Scientific, USA | 10828028 | |
| Matrigel | Corning, USA | 354277 | Matrigel will solidify in the environment higher than 4 °C, so it should be sub packed at low temperature. |
| MEM-NEAA | Thermo Fisher Scientific, USA | 11140050 | |
| mTeSR1 | STEMCELL Technologies, Canada | 85850 | iPSC culture medium |
| N2 supplement | Thermo Fisher Scientific, USA | 17502048 | |
| Neurobasal | Thermo Fisher Scientific, USA | 21103049 | |
| OCT4 primary antibody | Abcam, UK | ab19857 | Host: Rabbit. Dissolve with 500 μL PBS. Use at 1:200 dilution. |
| Pathological frozen slicer | Leica, Germany | Leica CM1860 | |
| PAX6 primary antibody | Abcam, UK | ab78545 | Host: Mouse. Use at 1:100 dilution. |
| PBS | STEMCELL Technologies, Canada | 37350 | |
| Penicillin-Streptomycin | Thermo Fisher Scientific, USA | 15140122 | |
| PSC dissociation solution | Beijing Saibei Biotechnology, China | CA3001500 | Enzyme free dissociation solution can be used for iPSC digestion and passage. |
| Sendai Reprogramming Kit | Thermo Fisher Scientific, USA | A16518 | Establish iPSC according to the protocol of Sendai Reprogramming Kit. |
| Slide Glass | Shitai Laboratory Equipment, China | 188105W | |
| Soft light lamp | NUT | NUT | A simple self made device, refer to supplementary figure 2 for preparation. |
| STEMdiff Cerebral Organoid Kit | STEMCELL Technologies, Canada | 8570 | Contain: 1. EB Formation Medium; 2. Induction Medium; 3. Expansion Medium; 4. Maturation Medium. |
| STEMdiff Cerebral Organoid Maturation Kit | STEMCELL Technologies, Canada | 8571 | Maturation Medium |
| Sucrose | Sangon Bioteach, China | A502792 | |
| Triton X-100 | Merck, Germany | X100 | |
| TUJ1 primary antibody | Abcam, UK | ab41489 | Host: Chicken. Use at 1:1000 dilution. |
| Vaseline | Sangon Bioteach, China | A510146 | |
| Y-27632 | STEMCELL Technologies, Canada | 72302 | Prepare a 5 mM stock solution in PBS, resuspend 1 mg in 624 µL of PBS. |
| Weblink | |||
| Raw sequencing data | Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences | GSA-Human: HRA002430 | https://ngdc.cncb.ac.cn/gsa-human/ |
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Cite This Article
Chen, B., Lin, Q., Liu, N., Chen, D., Zhang, Y., Sun, X. Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination. J. Vis. Exp. (184), e63989, doi:10.3791/63989 (2022).