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Obtaining Multi-electrode Array Recordings from Human Induced Pluripotent Stem Cell–Derived Neurons
从人诱导性多能干细胞神经元获得多电极阵列记录   

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Stem Cell Reports
Mar 2017

Abstract

Neuronal electrical properties are often aberrant in neurological disorders. Human induced pluripotent stem cells (hiPSCs)-derived neurons represent a useful platform for neurological disease modeling, drug discovery and toxicity screening in vitro. Multi-electrode array (MEA) systems offer a non-invasive and label-free platform to record neuronal evoked-responses concurrently from multiple electrodes. To better detect the neural network changes, we used the Axion Maestro MEA platform to assess neuronal activity and bursting behaviors in hiPSC-derived neuronal cultures. Here we describe the detailed protocol for neuronal culture preparation, MEA recording, and data analysis, which we hope will benefit other researchers in the field.

Keywords: Multi-electrode array (多电极阵列), Human induced pluripotent stem cells (人诱导性多能干细胞), Neurological diseases (神经系统疾病), Disease modeling (疾病建模)

Background

Human induced pluripotent stem cell (hiPSC) technology is currently being used to model neurological and psychiatric diseases in vitro. Recent studies have demonstrated that certain cellular phenotypes associated with particular disorders can be recapitulated in the dish. Neural electrical activity is at the essence of nervous system function, representing a key form of communication whose normal functioning is essential for emotion, memory, sensory modalities, and behavior in vivo. In disease conditions, the electrical properties can be affected, so it is important to understand neuronal circuit connectivity, physiology, and pathology in hiPSC-based models of neurological disease.

Patch-clamp and multi-electrode array (MEA) technology are the prevailing techniques used to assess electrophysiological activity, and thus neuronal function. While patch-clamp is a powerful intracellular method to investigate the activity and function of a single cell (Neher et al., 1978), an MEA plate has the ability to record extracellular action potentials (or spikes) and local field potentials simultaneously from thousands of different cells in the same plate over time, thus offering a better understanding of neuronal activity at a network level (Hutzler et al., 2006; Obien et al., 2014). MEAs are grids of tightly spaced electrodes that are capable of directly sensing changes in extracellular membrane potential in excitable cells and produce real-time trace of neuronal activity. The transparent CytoView 12-well MEA plate contains 64 recording electrodes per well, whereas the 48-well MEA plate contains 12 electrodes per well. The MEA system then parses these voltage traces for action potential waveforms, time-stamps the waveforms, and calculates action potential firing rates on-line for each electrode. This on-line firing rate is represented by color-coded heat-maps that depict real-time and simultaneous indications of network activity, and experimental effects (Figure 1).


Figure 1. Example plate activity heat map, and spike detector. Top: Heat map of MEA reading visualized using Axion BioSystems Integrated Studio (AxIS). Each box represents one well. Active channels represented by bright light blue dots on the map. Bottom: Offline spiking activity illustrated on AxIS data display.

Materials and Reagents

  1. 6-well cell culture plate (Corning, Costar®, catalog number: 3516 )
  2. Plastic pipette tips (Corning, Axygen®, catalog number: T-1000-B )
  3. Sterile 1.5 ml centrifuge tubes (Corning, Axygen®, catalog number: MCT-175-C )
  4. 15 ml and 50 ml centrifuge tubes (Corning, Falcon®, catalog numbers: 352096 and 352070 )
  5. Petri dish (Corning, Falcon®, catalog number: 351029 )
  6. Cell lifter (Corning, catalog number: 3008 )
  7. 12-well MEA plate (Axion BioSystem, catalog number: M768-GL1-30Pt200 )
  8. 0.22 µm filter (Corning, catalog number: 431097 )
  9. Kimwipes (KCWW, Kimberly-Clark, catalog number: 34155 )
  10. Sterile water (Thermo Fisher Scientific, GibcoTM, catalog number: 15230162 )
  11. Dispase (STEMCELL Technologies, catalog number: 07923 )
  12. Y-27632 (STEMCELL Technologies, catalog number: 72302 )
  13. Laminin (Thermo Fisher Scientific, GibcoTM, catalog number: 23017015 )
  14. LDN-193819 (Stemgent, catalog number: 04-0074 )
  15. SB431542 (Sigma-Aldrich, catalog number: S4317 )
  16. XAV939 (Stemgent, catalog number: 04-0046 )
  17. Recombinant Human Sonic Hedgehog (SHH) (R&D Systems, catalog number: 464-SH-025 )
  18. Brain-derived neurotrophic factor (BDNF) (R&D Systems, catalog number: 248-BD-025 )
  19. Glial-derived neurotrophic factor (GDNF) (R&D Systems, catalog number: 212-GD-050 )
  20. L-Ascorbic acid (Sigma-Aldrich, catalog number: A4403 )
  21. N6,2’-O-Dibutyryladenosine 3’,5’-cyclic monophosphate sodium salt (dbcAMP) (Sigma-Aldrich, catalog number: D0260 )
  22. 50% poly(ethyleneimine) (PEI) solution (Sigma-Aldrich, catalog number: P3143 )
  23. Accutase (STEMCELL Technologies, catalog number: 07920 )
  24. Tetrodotoxin (TTX) (Alomone Labs, catalog number: T-500 )
  25. N2 (Thermo Fisher Scientific, GibcoTM, catalog number: 17502048 )
  26. B27 (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
  27. Modified Eagle’s Medium-Non Essential Amino Acid (MEM-NEAA) (Thermo Fisher Scientific, GibcoTM, catalog number: 11140050 )
  28. L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  29. Penicillin-streptomycin (Pen-Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  30. Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (DMEM/F12) (Thermo Fisher Scientific, GibcoTM, catalog number: 11320082 )
  31. Neurobasal medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21103049 )
  32. N2 Supplement-A (STEMCELL Technologies, catalog number: 07152 )
  33. NeuroCultTM SM1 (STEMCELL Technologies, catalog number: 05711 )
  34. BrainPhysTM neuronal medium (STEMCELL Technologies, catalog number: 05790 )
  35. Boric acid (Sigma-Aldrich, catalog number: B6768 )
  36. Sodium tetraborate (Sigma-Aldrich, catalog number: 221732 )
  37. Hydrochloric acid fuming 37% (Merck, catalog number: 100317 )
  38. N2B27 medium (see Recipe 1)
  39. BrainPhys medium (see Recipe 2)
  40. Borate buffer (see Recipe 3)

Equipment

  1. Pipettes
    P10 (Gilson, catalog number: F144802 )
    P200 (Gilson, catalog number: F123601 )
    P1000 (Gilson, catalog number: F123602 )
  2. Tissue culture hood (Gelman, model: BH Class II Type A2 series )
  3. 37 °C water bath (Thermo Fisher Scientific, Thermo ScientificTM, model: Labline 183 , catalog number: 2835)
  4. Cell culture incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM I50i CO2 , catalog number: 50116047)
  5. Centrifuge (Eppendorf, model: 5810 , catalog number: 5810000424)
  6. Cell counter Countess II (Thermo Fisher Scientific, model: CountessTM II, catalog number: AMQAX1000 )
  7. Phase contrast microscope (Leica DMIL LED Inverted Fluorescence microscope) (Leica Microsystems, model: Leica DM IL LED )
  8. Maestro MEA system (Axion Biosystem)

Software

  1. Axion Biosystems Integrated Studio (AxIS, Axion Biosystem)
  2. Neural Metric tool (Axion Biosystem)
  3. Neuroexplorer (NEX, Plexon)
  4. MATLAB (R2016a)
  5. Excel (Microsoft Office)

Procedure

  1. Preparation of hiPSC-derived neurons
    Forebrain neurons were differentiated from hiPSCs as described previously (Xu et al., 2017).
    1. Aspirate old medium from wells of hiPSCs maintained in a 6-well plate.
    2. Add 1 ml dispase (1 mg/ml) into wells and incubate for 5 min at 37 °C.
    3. Add 2 ml DMEM/F12 into wells and transfer the lifted cell clumps into a 15 ml centrifuge tube using a 5 ml plastic pipette.
    4. Spin down cells at 160 x g for 3 min at room temperature.
    5. Remove and discard the supernatant.
    6. Re-suspend and rinse the cell clumps twice with 10 ml of DMEM/F12 to remove the remaining dispase.
      Note: It is important to completely remove dispase via multiple washes. Failure to remove dispase will affect cell reattachment when plated.
    7. Re-suspend cell clumps using 10 ml N2B27 medium (see Recipe 1) supplemented with 10 μM Y-27632, and culture them on uncoated 10 cm Petri dishes for 8 h at 37 °C.
    8. Collect cell aggregates and plate them on dishes pre-coated with 10 μg/ml poly-L-ornithine and 10 μg/ml laminin in N2B27 medium supplemented with 100 nM LDN193189, 10 μM SB431542, and 2 µM XAV939.
    9. From Day 5, 200 ng/ml SHH is added to the differentiated cells.
    10. Change medium every other day.
    11. On Day 15, passage cells with a cell lifter at a split ratio of 1:1.

    Note: These cells will be referred to as neural progenitor cells (NPCs).

    1. Expand the NPCs in N2B27 medium supplemented with 20 ng/ml BDNF, 2 µM XAV939, and 200 ng/ml SHH for five days.
    2. For neuronal differentiation, the NPCs are cultured in N2B27 medium supplemented with 20 ng/ml BDNF and 20 ng/ml GDNF, 0.2 mM ascorbic acid, and 0.5 mM dbcAMP.
    3. Exchange 50% medium every three days.
      Note: Neurons at days of 46 to 50 were dissociated and re-plated on MEA plate.

  2. MEA plate coating
    Coat MEA plate with PEI solution just before the day of seeding neurons according to Axion’s cell culture protocol on MEA plate.
    (https://www.axionbiosystems.com/sites/default/files/resources/icell_neuron_culture_protocol.pdf).
    1. Prepare 0.1% PEI solution by diluting 50% PEI solution in borate buffer (see Recipe 3).
    2. Filter the 0.1% PEI solution through a 0.22 µm filter.
      Note: 0.1% PEI solution can be stored at 4 °C for up to 1 month.
    3. Add 200 µl of 0.1% PEI solution to each well of the MEA plate and incubate plate at room temperature for 1 h.
    4. Aspirate the PEI solution from the MEA plate.
    5. Rinse each well 4 times with 500 μl sterile water.
    6. Air-dry the MEA plate with the lid off in tissue culture hood overnight.
      Note: It is essential to allow the MEA plate to air-dry overnight to achieve optimal cell adhesion.

  3. Seeding neurons
    1. Aspirate medium from neurons, and add 1 ml DMEM/F12 to wash neurons once at room temperature.
    2. Aspirate DMEM/F12, add 0.5 ml accutase into neurons, and incubate for 5 min in a 37 °C incubator.
    3. Add 2 ml DMEM/F12 into cells, and gently pipette up and down several times.
    4. Transfer cell suspension into a 15 ml centrifuge tube, and wash plate once using 2 ml DMEM/F12 to transfer all cells into 15 ml centrifuge tube.
    5. Re-suspend cells and take 10 µl for viable cell counting using a cell counter.
      Note: Gentle pipetting is essential to increase cell viability.
    6. Spin cells at 201 x g for 4 min at room temperature.
    7. Aspirate the supernatant above the cell pellet without disturbing the pellet.
    8. Dilute the laminin solution in complete N2B27 medium to a final concentration of 10 µg/ml.
      Note: Thaw laminin stock at 4 °C and don’t vortex laminin solution.
    9. Re-suspend the cell pellet using 10 µg/ml laminin solution to a final concentration of 28,000 viable cells/µl.
    10. Seed a 5 µl droplet of the suspension (140,000 cells) directly on the center of each PEI coated well over the recording electrode area.
      Note: Do not let the tips to touch the MEA plate bottom.
    11. Add sterile water to the area surrounding the wells of MEA plate to prevent substrate evaporation (Figure 2A).
      Note: MEA reservoir water is no longer required following the media addition in step C13.
    12. Incubate the MEA plate with the seeded neurons at a 37 °C incubator for 1 h.
      Note: Do not allow the cell suspension droplets to dry before adding additional complete N2B27 medium.
    13. Gently add 100 µl complete N2B27 medium down the side of each well of the MEA plate (Figure 2B).
    14. Repeat step C13 four more times to reach a final volume of 500 µl/well.
    15. Incubate MEA plate at a 37 °C incubator with 5% CO2.
    16. Next day, aspirate spent medium and add 1 ml fresh N2B27 medium or Brainphys medium (see Recipe 2) supplemented with 20 ng/ml BDNF, 20 ng/ml GDNF, 0.5 mM dbcAMP, and 0.2 mM ascorbic acid.
      Note: The neurons can be viewed using a phase contrast microscope (Figures 2D-2E).
    17. Exchange 50% medium every 2-3 days by aspirating 500 µl of spent medium from each well and adding 500 µl of fresh complete medium.
      Note: To avoid any influence of immediate medium change on neuronal activity, start recording not earlier than 1 h after the last media change.


      Figure 2. Representative images of MEA plate handling. MEA plate with sterile water surrounding the wells to prevent the evaporation (A). Recommended method of pipetting medium into MEA wells to avoid damaging the cells (B). MEA plate placed in the plate cavity (C). hiPSC-derived neurons plated around recording electrodes (D). A zoomed-in view of the marked white square in panel (D), showing neurons surrounding one of the recording electrodes (E). Scale bars = 100 μm.

  4. MEA recordings and data acquisition
    1. Turn on the Maestro MEA system using the power switch on the back of the Middleman.
    2. Power on AxIS software and click on the temperature icon to 37 °C.
    3. Set up experiment in AxIS (Figure 3):
      1. Adjust Maestro acquisition settings by selecting neural mode from the configuration.
      2. Set analog settings, choose ‘Neural: spikes (1,200 x Gain, 200-5,000 Hz).
      3. Use ‘Median’ Referencing to reduce noise in neural mode.
      4. Select data streams for recording.
      5. Choose recording duration.
        Note: Neuronal activity is very variable and typically needs at least 5 min for adequate statistics.
      6. Add experiment descriptions, notes, and plate maps.
    4. Place the MEA plate in the plate cavity (Figure 2C).
      Note: Do not touch the metal bottom part of MEA plate.
    5. Equilibrate the plate on the Maestro for 5-10 min.
    6. Click ‘play’ to start the voltage offset and wait until it is complete.
    7. Press ‘Start Schedule’ to record data.
    8. Data will be automatically saved once the recording is done.


      Figure 3. A snapshot of experiment setup in AxIS

  5. TTX treatment
    Tetrodotoxin (TTX), a potent neurotoxin, inhibits the firing of action potentials in neurons by binding to the voltage-gated sodium channels in nerve cell membranes and blocking the passage of sodium ions into the neuron. Here, we test whether hiPSC-derived neurons can respond to TTX treatment (Figures 4 and 5).
    1. Dissolve TTX at a pH of 7.4 in sodium acetate buffer to get 1 mM stock solution.
    2. Record baseline for MEA plate before adding TTX.
    3. Add 0.5 µl TTX into MEA wells to get a final concentration of 1 µM TTX.
    4. Incubate cells for 5 min.
    5. Record data after TTX treatment.
    6. Wash out TTX using BrainPhys medium and incubate plate overnight.
    7. Record neuronal activity after TTX washout.

  6. Data filtering and data extraction
    Since raw data was sampled at 12.5 kHz, it can be analyzed offline both in the low-frequency band (10-300 Hz), representative of local field potential (LFP) signal (Buzsáki et al., 2012), as well as high-frequency band, representative of action potential (AP) (Quian Quiroga and Panzeri, 2009). For our current study, we chose to focus on the extracellular AP quantification and high-pass filter the data. Filtering of data and spike detection (as timestamps) was performed using AxIS, according to the following steps:
    1. Recorded raw data files (.csv) were opened in AxIS. Data were band-pass filtered in the range 200 Hz-3 kHz using the AxIS digital filter to yield multi-unit data. Neuronal spikes were detected using a threshold set to 6 times standard deviation (6SD) above the mean noise level (Pouzat et al., 2002; Obien et al., 2014). A new file (.spk) was obtained for each recording day containing information such as spike timestamps and waveforms, as well as electrode (channel) and well indices.
    2. Multiunit spike data were subsequently imported into NEX, which contains a toolbox to identify AxIS .spk data formats. From NEX, spike data was exported into MATLAB as timestamps, and grouped per channel per well.
    3. In MATLAB, each individual recording was saved as .mat file, and later loaded for analysis.

Data analysis

Inbuilt and custom written MATLAB functions were employed to generate raster plots and illustrate spike activity for the investigated conditions.
Raster plots across experiments were used to visually examine wells whose overall spiking activity was different from control wells over time. Any unusual activity, or pattern, was further investigated using inter-stimulus interval, burst analysis or spike frequency analysis (see below). Each well was illustrated in a separate raster plot, and labeled with its corresponding condition name. For every individual channel, the timestamp of each spike detected was graphically plotted. In the raster plot, each column (y-axis) represents the channel number corresponding to each electrode, whereas the column (x-axis) represents the time each spike was detected (Figure 4).
Spike activity analysis included calculating the total number of spikes, the mean and maximum spike frequency, and the total number of unresponsive channels (or channels with no spiking activity) for each experimental condition. In this case, each condition was represented by three wells.


Figure 4. Representative raster plots of individual channel spiking activity from baseline, TTX treatment, and TTX washout. Raster plots showing high spiking activity during baseline (A), which is completely abolished by application of 1 µM TTX (B), and is recovered after washout of TTX (C); x-axis corresponds to time, y-axis corresponds to channel.

  1. Several parameters were initialized, including date of experiment, experimental conditions (i.e., control, treatment, media etc.), and well labels.
  2. As recording time varied slightly across experiments, data were truncated to the first 300 sec of recordings, and only relevant timestamps included for further analysis.
  3. Wells or channels with no spiking activity, or where only one spike for the whole recording was detected, were excluded from frequency analysis.
  4. The total number of channels with no spiking activity was counted for each well. The average number of channels per experimental condition was averaged.
  5. The total number of occurring spikes was counted for each channel within each well.
  6. Spike frequency was calculated for each channel as the total number of spikes divided by the recording duration.
  7. Wells containing the same cell type in the same experimental condition were pooled together for descriptive statistics analysis.
  8. Outliers were removed using a custom-written function by Brett Shoelson in 2009 based on an iterative implementation of the Grubbs Test (Grubbs, 1950).
  9. Descriptive statistics for the spike frequency, such as mean, maximum and standard error of the mean (SEM), were calculated for each well (or wells, if one condition included more than one wells).
  10. Data were plotted in a bar plot format to illustrate the differences in mean and maximum spiking frequency between conditions (Figures 5A and 5B), as well as the number of channels with no spiking activity (Figure 5C).


    Figure 5. TTX effects on hiPSC-derived neurons. Mean spike frequency (A), maximum spike frequency (B), and the average number of channels without spiking activity (C) for representative hiPSC-derived neurons. Bar graphs show mean ± SEM.

Recipes

  1. N2B27 medium (500 ml)
    5 ml N2
    10 ml B27
    5 ml MEM-NEAA
    2.5 ml L-glutamine
    5 ml Pen-Strep
    236.25 ml DMEM/F12
    236.25 ml neurobasal medium
  2. BrainPhys medium (500 ml)
    10 ml NeuroCultTM SM1 neuronal supplement
    5 ml N2 Supplement-A
    485 ml BrainPhysTM neuronal medium
  3. Borate buffer (500 ml)
    1.55 g of boric acid
    2.375 g of sodium tetraborate
    500 ml distilled water
    Adjust to a final pH of 8.4

Acknowledgments

The described methods were previously published in Xu et al. (2017). The work was partly funded by a Strategic Positioning Fund for Genetic Orphan Diseases (SPF2012/005) and a Joint Council Office Project grant (1431AFG122) from the Agency for Science Technology and Research (Singapore), and a Tier 1 grant R-172-000-297-112 from the Ministry of Education (Singapore) to M.A.P. C.R. is supported by the A*STAR Research Attachment Programme (ARAP). The authors declare that there are no conflicts of interests or competing interests.

References

  1. Buzsáki, G., Anastassiou, C. A. and Koch, C. (2012). The origin of extracellular fields and currents--EEG, ECoG, LFP and spikes. Nat Rev Neurosci 13(6): 407-420.
  2. Grubbs, F. E. (1950). Sample criteria for testing outlying observations. The Annals of Mathematical Statistics 21(1): 27-58.
  3. Hutzler, M., Lambacher, A., Eversmann, B., Jenkner, M., Thewes, R. and Fromherz, P. (2006). High-resolution multitransistor array recording of electrical field potentials in cultured brain slices. J Neurophysiol 96(3): 1638-1645.
  4. Neher, E., Sakmann, B. and Steinbach, J. H. (1978). The extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes. Pflugers Arch 375(2): 219-228.
  5. Obien, M. E., Deligkaris, K., Bullmann, T., Bakkum, D. J. and Frey, U. (2014). Revealing neuronal function through microelectrode array recordings. Front Neurosci 8: 423.
  6. Pouzat, C., Mazor, O. and Laurent, G. (2002). Using noise signature to optimize spike-sorting and to assess neuronal classification quality. J Neurosci Methods 122(1): 43-57.
  7. Quian Quiroga, R. and Panzeri, S. (2009). Extracting information from neuronal populations: information theory and decoding approaches. Nat Rev Neurosci 10(3): 173-185.
  8. Xu, X., Tay, Y., Sim, B., Yoon, S. I., Huang, Y., Ooi, J., Utami, K. H., Ziaei, A., Ng, B., Radulescu, C., Low, D., Ng, A. Y., Loh, M., Venkatesh, B., Ginhoux, F., Augustine, G. J. and Pouladi, M. A. (2017). Reversal of phenotypic abnormalities by CRISPR/Cas9-mediated gene correction in Huntington disease patient-derived induced pluripotent stem cells. Stem Cell Reports 8(3): 619-633.

简介

神经元电特性在神经疾病中经常是异常的。 人诱导的多能干细胞(hiPSC)衍生的神经元代表神经疾病建模,药物发现和体外毒性筛选的有用平台。 多电极阵列(MEA)系统提供了一个非侵入性的无标记平台,可同时记录来自多个电极的神经元诱发反应。 为了更好地检测神经网络变化,我们使用了Axion Maestro MEA平台来评估hiPSC衍生的神经元培养物中的神经元活动和爆裂行为。 在这里,我们描述了神经元培养准备,MEA记录和数据分析的详细方案,我们希望这将有益于该领域的其他研究人员。
【背景】人类诱导多能干细胞(hiPSC)技术目前正用于体外模拟神经和精神疾病。最近的研究已经证明,与特定疾病有关的某些细胞表型可以在盘中重现。神经电活动是神经系统功能的本质,代表了正常功能对于情绪,记忆,感觉形态和体内行为至关重要的交流的关键形式。在疾病状况下,电特性可能受到影响,因此了解基于hiPSC的神经疾病模型中的神经元电路连接性,生理学和病理学非常重要。

膜片钳和多电极阵列(MEA)技术是用于评估电生理学活性并由此评估神经元功能的主要技术。尽管膜片钳是研究单个细胞的活性和功能的强大的细胞内方法(Neher等人,1978),MEA平板具有记录细胞外动作电位(或尖峰)的能力,和同一板中数千个不同细胞同时的局部场电位,从而更好地理解网络水平的神经元活动(Hutzler等人,2006; Obien等人。,2014)。 MEAs是紧密间隔电极的网格,能够直接感测可兴奋细胞中细胞外膜电位的变化,并产生神经元活动的实时踪迹。透明CytoView 12孔MEA板每孔含有64个记录电极,而48孔MEA板每孔含有12个电极。 MEA系统然后解析这些电压轨迹以获得动作电位波形,对波形进行时间标记,并且为每个电极在线计算动作电位点火率。这种在线点火速率是用彩色编码的热图来表示的,它们描绘了网络活动的实时和同时指示以及实验效果(图1)。

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图1. Plate活动热图示例和尖峰探测器顶部:使用Axion BioSystems Integrated Studio(AxIS)显示MEA读数的热图。每个盒子代表一个井。在地图上以明亮的浅蓝色点表示的活动通道。底部:在AxIS数据显示器上显示离线尖峰活动。

关键字:多电极阵列, 人诱导性多能干细胞, 神经系统疾病, 疾病建模

材料和试剂

  1. 6孔细胞培养板(Corning,Costar ,目录号:3516)
  2. 塑料移液器吸头(Corning,Axygen ®,目录号:T-1000-B)
  3. 无菌1.5ml离心管(Corning,Axygen,目录号:MCT-175-C)
  4. 15ml和50ml离心管(Corning,Falcon,产品目录号:352096和352070)。
  5. 培养皿(Corning,Falcon ,目录编号:351029)
  6. 细胞提升器(康宁,目录号:3008)
  7. 12孔MEA板(Axion BioSystem,目录号:M768-GL1-30Pt200)
  8. 0.22微米过滤器(Corning,目录号:431097)
  9. Kimwipes(KCWW,Kimberly-Clark,目录号:34155)
  10. 无菌水(Thermo Fisher Scientific,Gibco TM,目录号:15230162)
  11. Dispase(STEMCELL Technologies,目录号:07923)
  12. Y-27632(STEMCELL Technologies,目录号:72302)
  13. 层粘连蛋白(Thermo Fisher Scientific,Gibco TM,目录号:23017015)
  14. LDN-193819(Stemgent,目录号:04-0074)
  15. SB431542(Sigma-Aldrich,目录号:S4317)
  16. XAV939(Stemgent,目录号:04-0046)
  17. 重组人声波刺猬(SHH)(R& D Systems,目录号:464-SH-025)
  18. 脑源性神经营养因子(BDNF)(R& D Systems,目录号:248-BD-025)
  19. 胶质源性神经营养因子(GDNF)(R& D Systems,目录号:212-GD-050)
  20. L-抗坏血酸(Sigma-Aldrich,目录号:A4403)
  21. Ñ 6 ,2'-O-二丁3’ ,5'-环一磷酸钠盐(dbcAMP)(Sigma-Aldrich公司,目录号:D0260)
  22. 50%聚(乙烯亚胺)(PEI)溶液(Sigma-Aldrich,目录号:P3143)
  23. Accutase(STEMCELL Technologies,目录号:07920)
  24. 河豚毒素(TTX)(Alomone Labs,目录号:T-500)
  25. N2(Thermo Fisher Scientific,Gibco TM,目录号:17502048)
  26. B27(Thermo Fisher Scientific,Gibco TM,目录号:17504044)
  27. 改良伊格尔培养基 - 非必需氨基酸(MEM-NEAA)(Thermo Fisher Scientific,Gibco TM,目录号:11140050)
  28. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
  29. 青霉素 - 链霉素(Pen-Strep)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  30. Dulbecco改良的Eagle's培养基:营养混合物F-12(DMEM / F12)(Thermo Fisher Scientific,Gibco TM,目录号:11320082)
  31. 神经基础培养基(Thermo Fisher Scientific,Gibco TM,目录号:21103049)
  32. N2 Supplement-A(STEMCELL Technologies,目录号:07152)
  33. NeuroCult TM SM1(STEMCELL Technologies,目录号:05711)
  34. BrainPhys TM神经元培养基(STEMCELL Technologies,目录号:05790)
  35. 硼酸(Sigma-Aldrich,目录号:B6768)
  36. 四硼酸钠(Sigma-Aldrich,目录号:221732)
  37. 盐酸发烟37%(Merck,目录号:100317)
  38. N2B27培养基(见方法1)
  39. BrainPhys培养基(见方法2)
  40. 硼酸盐缓冲液(见方法3)

设备

  1. 移液器
    P10(Gilson,目录号:F144802)
    P200(Gilson,目录号:F123601)
    P1000(Gilson,目录号:F123602)
  2. 组织培养罩(Gelman,型号:BH Class II Type A2系列)
  3. 37℃水浴(Thermo Fisher Scientific,Thermo Scientific TM,型号:Labline 183,目录号:2835)。
  4. 细胞培养培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heracell TM 150i CO 2,目录号:50116047)
  5. 离心机(Eppendorf,型号:5810,目录号:5810000424)
  6. 细胞计数器Countess II(Thermo Fisher Scientific,型号:Countess TM II,目录号:AMQAX1000)
  7. 相差显微镜(Leica DMIL LED倒置荧光显微镜)(Leica Microsystems,型号:Leica DM IL LED)
  8. Maestro MEA系统( Axion生物系统

软件

  1. Axion生物系统集成工作室(AxIS,Axion生物系统)
  2. 神经测量工具(Axion Biosystem)
  3. Neuroexplorer(NEX,Plexon)
  4. MATLAB(R2016a)
  5. Excel(Microsoft Office)

程序

  1. hiPSC衍生的神经元的制备
    如前所述,前脑神经元与hiPSC分化(Xu等人,2017)。
    1. 从保存在6孔板中的hiPSC的孔中吸取旧培养基。
    2. 向孔中加入1ml分散酶(1mg / ml),并在37℃孵育5分钟。
    3. 加入2毫升的DMEM / F12的井和提升的细胞团转移到15毫升离心管使用5毫升塑料移液器。

    4. 在室温下将细胞以160×g离心3分钟
    5. 去除并丢弃上清液。
    6. 重新悬浮并用10毫升DMEM / F12冲洗细胞团块两次以除去剩余的分散酶。
      注意:通过多次清洗完全去除分配是非常重要的。

      如果未能移除分配,电镀时会影响电池重新连接。
    7. 使用补充有10μMY-27632的10ml N2B27培养基重新悬浮细胞团(参见配方1),并将其在未涂覆的10cm培养皿中于37℃培养8小时。
    8. 收集细胞聚集物,并将它们在补充有100nMLDN193189,10μMSB431542和2μMXAV939的N2B27培养基中预涂有10μg/ ml聚-L-鸟氨酸和10μg/ ml层粘连蛋白的培养皿上培养。
    9. 从第5天起,将200ng / ml的SHH加入到分化细胞中。
    10. 每隔一天更换一次。
    11. 在第15天,用细胞提升器以1:1的分流比传代细胞。

    注:这些细胞将被称为神经祖细胞(NPC)。

    1. 在补充有20ng / ml BDNF,2μMXAV939和200ng / ml SHH的N2B27培养基中展开NPC 5天。
    2. 对于神经元分化,将NPCs在补充有20ng / ml BDNF和20ng / ml GDNF,0.2mM抗坏血酸和0.5mM dbcAMP的N2B27培养基中培养。
    3. 每三天交换50%中。
      注意:在46到50天的神经元被解离并重新涂在MEA板上。

  2. MEA板涂层
    根据MEA平板上的Axion细胞培养方案,在接种神经元之前,用PEI溶液包裹MEA平板。
    https://www.axionbiosystems.com/sites/default/files/资源/ icell_neuron_culture_protocol.pdf )。
    1. 通过在硼酸盐缓冲液中稀释50%PEI溶液来制备0.1%PEI溶液(参见方案3)。
    2. 通过0.22μm过滤器过滤0.1%PEI溶液。
      注:0.1%PEI溶液可以在4°C下保存长达1个月。
    3. 加入200μl的0.1%PEI溶液到MEA平板的每个孔中,并在室温下孵育平板1小时。
    4. 从MEA板上吸取PEI溶液。

    5. 用500μl无菌水冲洗每个孔4次
    6. 在组织培养罩中盖上隔夜的MEA板风干。
      注意:让MEA板过夜风干以达到最佳的细胞粘附是必不可少的。

  3. 播种神经元
    1. 从神经元中吸出培养基,并加入1ml DMEM / F12在室温下洗一次神经元。
    2. 吸出DMEM / F12,添加0.5毫升accutase到神经元,孵化5分钟,在37°C孵化器。
    3. 加入2毫升的DMEM / F12细胞,并轻轻吸管上下几次。
    4. 将细胞悬液转移到15ml离心管中,用2ml DMEM / F12洗板一次,将所有细胞转移到15ml离心管中。
    5. 重悬细胞,并使用细胞计数器取10μl用于活细胞计数。
      注意:温和的移液是增加细胞活力的必要条件。

    6. 在室温下将201×g离心细胞4分钟

    7. 。将细胞沉淀上方的上清液吸出,不要搅动沉淀
    8. 将完全N2B27培养基中的层粘连蛋白溶液稀释至终浓度为10μg/ ml。
      注意:在4°C解冻层粘连蛋白库存,不要涡旋层粘连蛋白溶液。
    9. 使用10μg/ ml层粘连蛋白溶液重悬细胞沉淀至终浓度为28,000活细胞/μl。
    10. 将5μl悬浮液(140,000个细胞)直接在每个PEI涂覆的孔的中心上在记录电极区域上接种。
      注意:不要让提示碰到MEA板的底部。
    11. 将无菌水添加到MEA板的孔周围以防止基质蒸发(图2A)。
      注意:在步骤C13添加介质后,不再需要MEA水库水。
    12. 将带有种子的神经元的MEA平板在37℃培养箱中孵育1小时。
      注意:在添加额外的N2B27培养基之前,不要让细胞悬液液滴干燥。

    13. 轻轻加入100μl完整的N2B27培养基到MEA平板各孔的一侧(图2B)
    14. 重复步骤C13四次以达到500μl/孔的最终体积。
    15. 在含有5%CO 2的37℃培养箱中孵育MEA板。
    16. 第二天,抽出用过的培养基并加入1ml新鲜的N2B27培养基或补充有20ng / ml BDNF,20ng / ml GDNF,0.5mM dbcAMP和0.2mM抗坏血酸的Brainphys培养基(见方案2)。
      注意:使用相差显微镜可以观察神经元(图2D-2E)。
    17. 每隔2-3天更换50%培养基,每孔吸取500μl废培养基,加入500μl新鲜完全培养基。
      注意:为避免即时培养基变化对神经元活动的影响,请在最后一次培养基更换后1小时内开始记录。


      图2. MEA印版处理的代表性图像。用无菌水MEA板周围的井防止蒸发(一)。推荐将培养基移液到MEA孔中以避免破坏细胞的方法(B)。将MEA板置于板腔(C)中。 (D)周围的hiPSC衍生的神经元。 (D)中标记的白色正方形的放大视图,显示了围绕记录电极(E)之一的神经元。比例尺= 100微米。

  4. MEA记录和数据采集
    1. 打开Maestro MEA系统,使用Middleman背面的电源开关。
    2. 启动AxIS软件并点击温度图标到37°C。
    3. 在AxIS中设置实验(图3):
      1. 通过从配置中选择神经模式调整Maestro采集设置。
      2. 设置模拟设置,选择“神经:尖峰(1,200×增益,200-5,000赫兹)”。
      3. 使用“中位数”参考来减少神经模式中的噪音。
      4. 选择数据流进行录制。
      5. 选择录制时间。
        注意:神经元活动变化很大,通常需要至少5分钟才能获得足够的统计数据。
      6. 添加实验说明,笔记和版图。
    4. 将MEA板放入板腔(图2C)。
      注意:请勿触摸MEA板的金属底部。

    5. 在Maestro上平衡平板5-10分钟
    6. 点击“播放”开始电压偏移,然后等待完成。
    7. 按“开始时间表”记录数据。
    8. 一旦记录完成,数据将自动保存。


      图3. AxIS中实验设置的快照

  5. TTX治疗
    河豚毒素(TTX)是一种有效的神经毒素,通过与神经细胞膜上的电压门控钠通道结合,阻断钠离子进入神经元,抑制神经元动作电位的发放。在这里,我们测试hiPSC派生的神经元是否可以对TTX治疗产生反应(图4和5)。

    1. 在pH 7.4的乙酸钠缓冲液中溶解TTX,得到1 mM的储存液
    2. 在添加TTX之前记录MEA板的基线。
    3. 向MEA孔中加入0.5μlTTX,使终浓度达到1μMTTX。
    4. 孵育细胞5分钟。
    5. 记录TTX治疗后的数据。
    6. 用BrainPhys培养基冲洗TTX,孵育过夜。
    7. 记录TTX清除后的神经元活动。

  6. 数据过滤和数据提取
    由于原始数据采样频率为12.5 kHz,因此可以在低频段(10-300 Hz),代表局部场电位(LFP)信号(Buzskiki et al。, 2012),以及动作电位(AP)(Quian Quiroga和Panzeri,2009)的高频段。对于我们目前的研究,我们选择侧重于细胞外AP量化和高通滤波的数据。根据以下步骤使用AxIS进行数据和尖峰检测(作为时间戳)的过滤:
    1. 录制的原始数据文件( .csv )在AxIS中打开。使用AxIS数字滤波器在200Hz-3kHz范围内对数据进行带通滤波以产生多单元数据。使用设定为高于平均噪声水平6倍标准差(6SD)的阈值检测神经元峰值(Pouzat等, 。每个记录日获得一个新的文件(。 spk ),其中包含尖峰时间戳和波形以及电极(通道)和油井指数等信息。
    2. 随后将多单元峰值数据导入到NEX,其中包含一个工具箱来识别AxIS。 spk 数据格式。在NEX中,秒杀数据作为时间戳输出到MATLAB,并按每个通道分组
    3. 在MATLAB中,每个单独的记录都被保存为。 mat 文件,然后加载进行分析。

数据分析


内置和自定义的书面MATLAB函数被用来生成栅格图,并说明所调查条件的尖峰活动 通过实验的栅格图用于目视检查随时间推移其总体掺加活性与对照孔不同的孔。任何不寻常的活动,或模式,进一步调查使用刺激间期,突发分析或尖峰频率分析(见下文)。每个井都在一个单独的栅格图中进行了说明,并标有相应的条件名称。对于每个单独的频道,检测到的每个尖峰的时间戳都以图形方式绘制。在栅格图中,每列(y轴)代表与每个电极对应的通道编号,而列(x轴)代表检测到每个峰值的时间(图4)。
峰活性分析包括计算每个实验条件的峰值总数,平均峰值频率和最大峰值频率,以及无响应信道(或没有尖峰活动的信道)的总数。在这种情况下,每种情况都由三口井代表。


图4.代表从基线,TTX处理和TTX洗脱的单个通道峰电活动的代表性栅格图。在基线(A)期间显示高峰电活动的栅格图,其通过应用1μMTTX完全消除(B),TTX(C)冲洗后回收。 x轴对应于时间,y轴对应于通道。

  1. 几个参数被初始化,包括实验日期,实验条件(即em>,控制,处理,媒体等等)和well标签。
  2. 由于各个实验的记录时间略有不同,因此数据被截断至记录的前300秒,只有相关的时间戳才能进行进一步的分析。
  3. 没有加标活动的井或通道,或只检测到一个加标记的通道被排除在频率分析之外。
  4. 对每个孔计数没有加标活动的通道总数。每个实验条件的平均通道数被平均。

  5. 每个通道内的每个通道计数出现的峰值总数
  6. 每个通道的峰值频率计算为峰值总数除以记录时间。
  7. 将含有相同细胞类型的孔在相同的实验条件下合并在一起进行描述性统计分析。
  8. 根据Grubbs测试(Grubbs,1950)的迭代实施,Brett Shoelson在2009年使用定制函数删除了异常值。
  9. 针对每个孔(或者如果一个条件包括多于一个孔的孔)计算峰值频率的描述性统计,如平均值,最大值和平均值的标准误差(SEM)。
  10. 以条形图格式绘制数据以说明条件(图5A和5B)之间的平均和最大峰电频率的差异,以及没有尖峰活动的通道数量(图5C)。

    图5. TTX对hiPSC衍生的神经元的影响。平均尖峰频率(A),最大尖峰频率(B)和代表hiPSC衍生的神经元的无峰电流活动的平均数量(C)。条形图显示平均值±SEM。

食谱

  1. N2B27培养基(500毫升)
    5毫升N2
    10毫升B27
    5毫升MEM-NEAA
    2.5毫升L-谷氨酰胺
    5毫升Pen-Strep
    236.25毫升DMEM / F12

    236.25毫升神经基础培养基
  2. BrainPhys中等(500毫升)
    10毫升NeuroCult TM SM1神经元补充剂
    5毫升N2补充-A
    485毫升BrainPhys TM神经元培养基
  3. 硼酸盐缓冲液(500毫升)
    1.55克硼酸
    2.375克四硼酸钠
    500毫升蒸馏水
    调整到最终的pH值为8.4

致谢

所描述的方法先前在Xu等人发表。 (2017)。这项工作部分由科学技术与研究机构(新加坡)的遗传孤独病战略定位基金(SPF2012 / 005)和联合理事会办公室项目资助(1431AFG122)资助,一级资助R-172- 000-297-112从教育部(新加坡)到MAP C.R.得到A * STAR研究附件计划(ARAP)的支持。作者声明不存在利益冲突或利益冲突。

参考

  1. Buzsáki,G.,Anastassiou,C.A。和Koch,C。(2012)。 细胞外场和电流的起源 - 脑电,ECoG,LFP和尖峰 Nat Rev Neurosci 13(6):407-420。
  2. Grubbs,F.E。(1950)。 测试外围观测的样本标准 “数理统计年鉴” 21(1):27-58。
  3. Hutzler,M.,Lambacher,A.,Eversmann,B.,Jenkner,M.,Thewes,R.和Fromherz,P.(2006)。 高分辨率多晶体管阵列记录培养脑切片中的电场电位 J Neurophysiol 96(3):1638-1645。
  4. Neher,E.,Sakmann,B。和Steinbach,J.H。(1978)。 细胞外膜片钳:一种解决生物膜中单个开放通道电流的方法。 Pflugers Arch 375(2):219-228。
  5. Obien,M.E.,Deligkaris,K.,Bullmann,T.,Bakkum,D.J。和Frey,U。(2014)。 通过微电极阵列记录揭示神经元功能 Front Neurosci 8:423.
  6. Pouzat,C.,Mazor,O。和Laurent,G。(2002)。 使用噪音特征来优化穗分选和评估神经元分类质量 J Neurosci方法 122(1):43-57。
  7. Quian Quiroga,R.和Panzeri,S。(2009)。 从神经元种群中提取信息:信息理论和解码方法 Nat Rev Neurosci 10(3):173-185。
  8. Xu,X.,Tay,Y.,Sim,B.,Yoon,SI,Huang,Y.,Ooi,J.,Utami,KH,Ziaei,A.,Ng,B.,Radulescu,C.,Low, D.,Ng,AY,Loh,M.,Venkatesh,B.,Ginhoux,F.,Augustine,GJ和Pouladi,MA(2017)。 CRISPR / Cas9介导的基因校正在亨廷顿病患者衍生的诱导多能干中逆转表型异常细胞。干细胞报告 8(3):619-633。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Xu, X., Radulescu, C. I., Utami, K. H. and Pouladi, M. A. (2017). Obtaining Multi-electrode Array Recordings from Human Induced Pluripotent Stem Cell–Derived Neurons. Bio-protocol 7(22): e2609. DOI: 10.21769/BioProtoc.2609.
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