该协议旨在将背腹融合组合稳定地接种在多电极阵列上,用于体外模拟癫痫。
人脑类器官是源自人类多能干细胞 (hPSC) 的三维 (3D) 结构,概括了胎儿大脑发育的各个方面。背侧与腹侧区域指定的脑类器官 在体外融合产生 组合体,这些组合体具有与兴奋性和抑制性神经元的功能集成微回路。由于其结构复杂性和神经元群的多样性,组装体已成为研究异常网络 活动的有用体外 工具。多电极阵列 (MEA) 记录是一种从神经元群中捕获电场电位、尖峰和纵向网络动力学的方法,而不会影响细胞膜的完整性。然而,由于电极尺寸大且与电极的接触表面积有限,因此将组件粘附到电极上以进行长期记录可能具有挑战性。在这里,我们展示了一种将组装体接种到 MEA 板上以记录 2 个月内电生理活性的方法。尽管目前的协议利用人类皮质类器官,但它可以广泛适应分化为模拟其他大脑区域的类器官。该协议建立了一种稳健的纵向电生理测定法,用于研究神经元网络的发展,该平台有可能用于癫痫治疗开发的药物筛选。
人类多能干细胞 (hPSC) 衍生的脑类器官是空间自组织的 3D 结构,反映了体内的组织结构和发育轨迹。它们由多种细胞类型组成,包括祖细胞(神经上皮细胞、放射状神经胶质细胞、神经元祖细胞、神经胶质祖细胞)、神经元(皮质样兴奋性神经元和抑制性中间神经元)和神经胶质细胞(星形胶质细胞和少突胶质细胞)1,2。组装体代表了下一代脑类器官,能够在 3D 培养物中整合多个大脑区域和/或细胞谱系。它们为模拟类似于体内对应物的各种大脑区域之间的连接、捕获神经元和星形胶质细胞之间的相互作用以更好地模拟成熟和复杂的神经网络以及研究神经回路的组装提供了一个有用的工具。因此,组合体正在成为一种广泛使用的工具,用于概括癫痫病理生理学的特征,其中功能措施是必要的,以询问可能是疾病原因基础的异常神经网络 3,4,5,6。
为了模拟皮质谷氨酸能神经元和 GABA 能中间神经元之间的相互作用,几个小组开发了类似于背侧和腹侧大脑的单独类器官,然后将它们融合在一起形成一个多区域组合体 7,8,9,10。在这里,应用了先前描述的具有区域特异性神经亚型的组装体培养方案9。然而,一个重大障碍是缺乏可重复的功能测定来监测神经发育过程中的神经网络活动。类器官中网络的许多功能测试产生的结果在分化批次和细胞系之间具有高度可变性。涉及切片或解离类器官的技术通过切断突触连接来改变其固有网络8。
多电极阵列 (MEA) 以高时间分辨率提供网络活动随时间变化的大规模视图,以表征类器官的电生理特性,而不会破坏培养条件或细胞膜完整性11。与膜片钳电生理学相比,MEA 能够基于大量神经元而不是单个细胞进行高通量数据采集。MEA 平台的电极密度各不相同,可满足脑类器官研究的不同需求12。如本协议所示,广泛使用的系统记录每孔 8 到 64 个电极 13,14,15。每孔多达 26,400 个电极的高密度 MEA 可以提高空间和时间分辨率,量化动作电位传播速度,并与光遗传学刺激相结合 14,16,17。因此,MEA 是体外模拟癫痫的有力工具和抗癫痫药物筛选的转化范式。
一个主要挑战是将大型组合体稳定在疏水金属表面上以进行长期记录。该协议概述了在 MEA 板上铺板完整组装体以进行长期纵向记录以及药理学测定的详细方法。该方案的独特优势包括将组装体稳定地附着在电极表面而不会失去电活性,使用市售的神经生理学基础培养基来加速电镀后的功能网络成熟,进行下游功能测定(如药物治疗)的可行性,以及广泛应用于使用其他区域特异性方案生成的类器官。
目标是提供一种具有高时间分辨率的功能测定,以研究网络活动、检查疾病特异性变化并测试具有癫痫治疗潜力的药物。为该方案中最具挑战性的步骤提供了视频说明,展示了在 MEA 板上铺板组装体的技术,以及来自这些培养物的代表性记录。
基于 MEA 的 iPSC 衍生组合体中网络活动的电生理记录方法已用于癫痫的体外建模22,23。这个整合兴奋性和抑制性突触连接的平台有可能解决神经元过度兴奋的机制和皮层中间神经元在癫痫发生过程中的作用。此外,该平台允许在体外稳定组装体和收集纵向电生理数据约 50 天,即使在存在药理学扰动的情况下也是如?…
The authors have nothing to disclose.
该手稿得到了 R01NS127829 NIH/NINDS (LTD) 的支持。 图 1 是使用 biorender.com 生成的。
10 cm Corning Non TC-treated culture dishes | Corning | 08-772-32 | For suspension culture on the shaker |
100 mL Beaker | Fisher Scientific | FB100100 | |
100% Ethanol | Fisher Scientific | BP28184-4L | |
2-Mercaptoethanol (β-ME) | Thermo Fisher | 21985023 | Working concentration 100 μM |
48-well cell culture plate | Fisher Scientific | 50-202-140 | |
6-well cell culture plate | Fisher Scientific | 07-200-83 | |
Aggrewell 800 | Fisher Scientific | 501974754 | |
Allegra X-14R Refrigerated Centrifuge | Beckman Coulter | BE-AX14R | |
Allopregnanolone | Cayman | 16930 | Suspended 5mg into DMSO to get 1 mM stock solution. Aliquot and freeze at −80 °C. Dilute at 1:10,000 for use. Working concentration 100 nM. |
Automated cell counter | Thermo Fisher | AMQAX2000 | |
Axion CytoView MEA 6-well plates | Axion Biosystems | M384-tMEA-6B | |
Axion Maestro MEA platform | Axion Biosystems | Maestro | With temp environmental control |
B-27 supplement (regular, with Vitamin A) | Thermo Fisher | 21985023 | |
B-27 supplement (without Vitamin A) | Thermo Fisher | 12587010 | |
Basement membrane matrix- Geltrex | Thermo Fisher | A1569601 | |
Bead bath | Fisher Scientific | 10-876-001 | Isotemp |
Benchtop inverted microscope | Olympus | CKX53 | Kept in laminar flow clean bench |
Bicuculline | Sigma-Aldrich | 14340 | Working concentration 10 μM |
Bleach | CLOROX | 67619-26 | |
Borate buffer 20x | Thermo Fisher | 28341 | Working concentration at 1x |
BrainPhys media | StemCell Technologies | 5790 | |
Cell dissociation reagent (StemPro Accutase) | Thermo Fisher | A1110501 | |
Celltron orbital shaker | HT-Infors | I69222 | |
Detergent/enzyme (Terg-A-Zyme) | Sigma-Aldrich | Z273287 | Working concentration 1% m/v |
DMEM/F12 + HEPES/L-Glutamine | Thermo Fisher | 113300 | |
DMSO | Sigma-Aldrich | 67685 | |
Dorsomorphin | Sigma-Aldrich | P5499 | Dissolve 5mg into DMSO to get 10 mM stock solution. Aliquot and freeze at −20 °C. Dilute at 1:2000 for use. (working concentration 5 μM) |
D-PBS w/o calcium or magnesium | Thermo Fisher | 14190144 | |
Glial cell line-derived neurotrophic (GDNF) | Peprotech | 450-10 | Dissolve 100 μg in 1mL of PBS to 100 μg/mL. Aliquot and freeze at −20 °C. Dilute at 1:5000 for use. (working concentration 20 ng/mL). |
GlutaMAX supplement | Thermo Fisher | 35050061 | |
Hemacytometer | Election Microscopy Sciences | 63510-20 | |
HEPES | Thermo Fisher | 15630080 | |
Heraguard ECO Clean Bench | Thermo Fisher | 51029692 | |
Humidity controlled cell culture incubator | Thermo Fisher | 370 | set to 37 °C, 5% CO2 |
IWP-2 | Selleckchem | S7085 | Aliquot and freeze at −80 °C. It will precipitate if thawed at room temp. Frozen aliquots should be placed directly into 37 °C before use. |
Knockout serum replacement (KOSR) | Thermo Fisher | 10828010 | |
mTeSRplus (medium + supplements) | StemCell Technologies | 100-0276 | cGMP, stabilized feeder-free medium for human iPSC cells |
N2 supplement | Thermo Fisher | 17502048 | |
Neurobasal A | Thermo Fisher | 21103049 | |
Non-essential amino acids (NEAA) | Thermo Fisher | 11140050 | |
NT3 | Peprotech | 450-03 | Dissolve 100 μg in 1mL of PBS to 100 μg/mL. Aliquot and freeze at −20 °C. Dilute at 1:5000 for use. (working concentration 20 ng/mL). |
NuFF Human neonatal foreskin fibroblasts | MTI-GlobalStem | GSC-3002 | |
Parafilm | PARAFILM | P7793 | |
Penicilin/Streptomycin | Thermo Fisher | 15140122 | |
Pipette (P10, P200, P1000) | Eppendorf | EP4926000034 | Autoclaved cut P1000 tips for organoid collection |
Poly (Ethyleneimine) (PEI) | Sigma-Aldrich | P3143 | Dilute stock in sterile borate buffer. Working concentration 0.07%. See details in manuscript. |
Recombinant human epidermal growth factor (EGF) | R&D Systems | 236-EG-200 | Suspended in PBS. Aliquot and freeze at -20 °C. |
Recombinant Human fibroblast growth factor (FGF)-basic | Peprotech | 100-18B | Suspended in PBS. Aliquot and freeze at -20 °C. |
Recombinant human-brain-derived neurotrophic factor (BDNF) | Peprotech | 450-02 | Centrifuge briefly before reconstitution. Dissolve 100 μg in 1 mL of PBS to 100 μg/mL. Aliquot and freeze at −20 °C. Dilute at 1:5000 for use. (working concentration 20 ng/mL). |
Retinoic acid (RA) | Sigma-Aldrich | R2625 | Dissolve 100 mg into 3.3 mL of DMSO to get 100 mM stock solution. Aliquot the stock 100 μL/tube and freeze at −80 °C. Take 200 μL of 100 mM stock and dilute 10x (add 1.8 mL of DMSO) to make 10 mM stock. Aliquot 50 μL/tube and store at −80 °C. Dilute at 1:100,000 for use. (working concentration 100 nM). |
ROCK inhibitor Y-27632 | Tocris | 1254 | 1:200 from 10 mM stock |
SAG (smoothened agonist) | Selleckchem | S7779 | Aliquot and freeze at −80 °C. Stock concentration 1mM. Use at 1:10,000 dilution (working concentration 100 nM). |
SB-431542 | Tocris | 1614 | Dissolve 5mg into 1.3 mL of DMSO to get 10 mM stock solution. Aliquot and freeze at −80 °C. Dilute at 1:1000 for use. (working concentration 10 μM) |
Serological pipette filler | Fisher Scientific | 14-387-166 | |
Steriflip vacuum tube top filter | Sigma-Aldrich | SE1M179M6 | |
Sterile cell culture hoods | Baker Company | SG-600 | |
Trypan blue solution (0.4%) | Thermo Fisher | 15250061 | |
Trypsin-EDTA (0.25%) | Thermo Fisher | 25200056 | |
Zoom stereomicroscope | Olympus | SZ61/SZ51 | Kept in laminar flow clean bench |