为互连神经元微缩组织制造磁平台
Summary
这项工作提出了一个自下而上的方法，以工程的局部磁力控制神经元组织。装有磁性纳米粒子（MNPs）的神经元状细胞被镀在上面，由垂直磁化的微模式平台控制。还描述了磁定性、MNP细胞吸收、细胞生存能力和统计分析。
Abstract
将神经元引导到有组织的神经网络的能力对再生医学、组织工程和生物面具有重大影响。许多研究旨在利用化学和地形线索引导神经元。然而，关于大面积的微米规模组织控制的报告很少。在这里，描述了一种有效的方法，用于将神经元放置在预设位点，并利用嵌入微模式磁性元素的磁平台，以微米级分辨率引导神经元生长。已经证明，用磁性纳米粒子（MNPs）加载神经元可以将其转换成受磁梯度影响的敏感磁性单位。按照这种方法，制作了一个独特的磁性平台，在该平台上，PC12细胞，一个常见的神经元样模型，被镀层并装载了超参数纳米粒子。沉积了具有稳定垂直磁化的铁磁（FM）多层薄膜，为磁态模式提供有效的吸引力。这些MNP加载的PC12单元，镀层和分化在磁平台上，优先附着在磁性模式上，中性生长与图案形状完全一致，形成定向网络。介绍了磁性、蜂窝MNP吸收、细胞生存能力和结果统计分析的定量表征方法。这种方法能够控制神经网络的形成，并通过磁力的操纵改善神经元到电极的接口，它可以成为网络体外研究的有效工具，并可能提供新的治疗生物接触方向。
Introduction
神经元的微观模式对组织再生1、2、3、4、5和神经电子设备6、7、8的发展具有巨大的潜力。然而，与生物组织一样，微米比例定位神经元在高空间分辨率下构成重大挑战。在这种尺度上形成预先设计的结构需要通过局部控制索马活力和轴向外生长来指导神经细胞过程。先前的研究表明，使用化学和物理线索9，10，11，12来指导神经元生长。在这里，一种新的方法侧重于通过磁场梯度13、14、15、16、17控制细胞定位，将装有MNP的细胞转变为磁敏单元，可以远程操作。
Kunze等人用磁芯片和MNP加载的细胞来描述诱导细胞反应所需的力，他们证明早期轴向拉长可以通过细胞内部的机械张力触发。Tay等人证实，具有增强磁场梯度的微结构基板允许使用钙指示染料19对与MNPs一起使用的神经回路进行无线刺激。此外，曾等人将纳米粒子凝聚在细胞内，导致局部纳米粒子介质力接近细胞张力20。这导致了微磁基板的定义模式的制造，帮助研究细胞对机械力的反应。通过在细胞20内凝聚纳米粒子，实现了因应用局部纳米粒子介导力而产生的细胞张力。Lee等人开发了一种互补的金属氧化物半导体（CMOS）-微流体混合系统，他们在CMOS芯片中嵌入了一系列微电磁铁，以控制标有磁珠21的单个细胞的运动。
阿隆等人利用微尺度、预编程、磁垫作为磁性”热点”定位细胞22。也可以利用微模式磁阵列在细胞内刺激特定活动，使纳米粒子在特定的亚细胞位置23。细胞MNP的吸收已经成功地证明在水痘，大鼠和小鼠原神经元24，25，26。在这里，这已经证明在老鼠PC12 pheo色细胞细胞系，这之前有报道说，显示高吸收MNPs27。近年来，国会议员的医疗应用多种多方面，包括药物输送和治疗癌症治疗28、29、30、31。具体来说，研究涉及MNP和神经元网络32，33，34，35的应用。然而，在单细胞水平上使用MNP的神经元的磁组织值得进一步研究。
在这项工作中，描述了一种自下而上的方法，通过预先设计的平台设计本地磁力，用于控制神经元排列。介绍了调频多层机型微米尺度模式的制造。这种独特的调频多层结构创造了稳定的垂直磁化，从而对所有磁模式产生有效的吸引力。通过孵化，MNP 被加载到 PC12 单元中，将其转换为磁性敏感单元。在磁平台上镀层和分化的MNP加载单元优先附着在磁性模式上，中性生长与模式形状完全一致，形成定向网络。介绍了几种方法来描述调频多层和MNPs的磁性特性，并介绍了细胞MNP吸收和细胞生存性检测技术。此外，还详细介绍了神经元生长的形态参数和结果的统计分析。
Protocol
注意：在生物安全柜中执行所有生物反应。 1. 磁平台制造 光刻 使用抄写笔将玻璃滑入 2 x 2 厘米2。 用丙酮清洁玻璃滑梯，然后在超声波浴缸中分别清洁 5 分钟。用超高纯度 （UHP） 氮干燥。 在 60s 的 60s 中使用旋转涂层，将玻璃涂上光层，达到 1.5μm 厚度，在 60 年代以 100 °C 烘烤。使用光面罩或无面膜光刻技术，使用合适的波长，使用所需的图?…
Representative Results
制造了具有不同几何形状的磁平台（图1A）。磁模式通过溅射沉积：14个多层的Co80Fe20和Pd，分别为0.2纳米和1纳米。电子显微镜显示磁模式的总高度为~18纳米（图1B）。这种独特的调频多层沉积创造了一个稳定的平台，与基板平面相对于基板平面的垂直磁化同位素（PMA），使MNP加载的细胞对整个磁模式的吸引力，?…
Discussion
具有代表性的结果表明，在微米尺度上控制和组织神经元网络形成的方法的有效性。MNP 加载的 PC12 电池仍然可行，并被转换为磁敏单位，这些单元被从调频电极到特定部位的磁力所吸引。这在图 5C中表现得最好，其中细胞优先粘附在六边形较大的顶点上，而不是细线。此外，细胞的分支也随着磁模式的发展而发展。所有的控制实验都明确表明，磁力引导细胞体和生长的本地…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究得到了以色列科学和技术部以及以色列科学基金会（569/16）的支持。
Materials
| 16% Paraformaldehyde (formaldehyde) aqueous solution | ELECTRON MICROSCOPY SCIENCES | 15710 | |
| 6-well cell culture plate | FALCON | 353846 | |
| 96-well cell culture plate | SPL life sciences | 30096 | |
| Amphotericin B solution | Biological Industries | 03-028-1B | |
| AZ 1514H photoresist | MicroChemicals GmbH | ||
| AZ 351 B developer | MicroChemicals GmbH | ||
| Bovine serum albumin (BSA) | Biological Industries | 03-010-1B | |
| Cell and Tissue cultur flask | Biofil | TCF002250 | 75.0 cm^2 250 mL Vent cap, Non-treated |
| Cell culture dish | Greiner Bio-One | 627-160 | 35 mm |
| Cell Proliferation Kit (XTT-based) | Biological Industries | 20-300-1000 | |
| Centrifuge tube | Biofil | CFT021500 | 50 mL |
| Co80Fe20 at% sputter target | ACI Alloys | 99.95% | |
| Collagen type I | Corning Inc. | 354236 | Rat Tail, concentration range 3-4 mg/mL |
| Confocal microscope | Leica | TCS SP5 | |
| Cy2-conjugated AffiniPure Donkey Anti-rabbit secondary antibody | Jackson ImmunoResearch Laboratories, Inc. | 711-165-152 | |
| DAPI fluoromount-G | SouthernBiotech | 0100-20 | |
| Disposable needle | KDL | 23 G | |
| Disposable syringe | Medispo | 1160227640 | 10 mL |
| Donor horse serum | Biological Industries | 04-124-1A | |
| ELISA reader | Merk Millipore | BioTek synergy 4 hybrid microplate reader | |
| Ethanol 70% | ROMICAL LTD | 19-009102-80 | |
| Ethanol absolute (Dehydrated) | Biolab-chemicals | 52505 | |
| Fetal bovine serum (FBS) | Biological Industries | 04-127-1A | |
| Fresh murine β-NGF | Peprotech | 450-34 | |
| GMW C-frame electromagnet . | Buckley systems LTD | 3470, 45 mm | |
| Hydrochloric acid 32% | DAEJUNG CHEMICAL & METALS | 4170-4100 | |
| ImageJ | US National Institutes of Health, Bethesda | NeuronJ plugin | |
| Inductively coupled plasma (ICP) | Ametek Spectro | SPECTRO ARCOS ICP-OES, FHX22 MultiView plasma | |
| Keithley source-measure | Keithley | 2400 | |
| Keithley switching system | Keithley | 3700 | |
| L-glutamine | Biological Industries | 03-020-1B | |
| Light microscope | Leica | DMIL LED | |
| Maskless photolithography | Heidelberg Inst. | MLA150 | |
| Microscope Slides | BAR-NAOR | BN1042000C | |
| Nitric acid 70% | Sigma-Aldrich | 438073 | |
| Normal donkey serum (NDS) | Sigma | D9663 | |
| PBS 10x | hylabs | BP507/1LD | |
| PC12 cell line | ATCC | CRL-1721 | |
| Pd sputter target | ACI Alloys | 99.95% | |
| Penicillin-streptomycin nystatin solution | Biological Industries | 03-032-1B | |
| PrestoBlue cell viability reagent | Molecular probes | A-13261 | resazurin-based |
| Rabbit antibody to α-tubulin | Santa Cruz Biotechnology, Inc. | ||
| RF magnetron sputtering system | Orion AJA Int. | Orion 8 | |
| RPMI 1640 with l-glutamine | Biological Industries | 01-100-1A | |
| Sonication bath | KUDOS | SK3210HP | Frequency: 53 kHz. Ultrasonic power: 135 W |
| SQUID magnetometer | Quantum Design, CA | ||
| Triton X-100 | CHEM-IMPEX INTERNATIONAL | 1279 | non-ionic surfactant |
| XTT cell viability reagent |
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Citer Cet Article
Indech, G., Plen, R., Levenberg, D., Vardi, N., Marcus, M., Smith, A., Margel, S., Shefi, O., Sharoni, A. Fabrication of Magnetic Platforms for Micron-Scale Organization of Interconnected Neurons. J. Vis. Exp. (173), e62013, doi:10.3791/62013 (2021).