第一次混合胶质细胞培养的成年小鼠脊髓组织准备
Summary
神经性疼痛的发展涉及脊髓神经胶质细胞的病理变化。从成年脊髓组织的，设计用于在体外研究这些细胞中的胶质可靠的培养体系缺乏。因此，我们在这里展示了如何建立从成年小鼠的脊髓组织的主要胶质细胞混合培养。
Abstract
它已被广为接受的脊髓神经胶质反应显著到神经性疼痛的发展作出贡献。已通过体外细胞培养系统中获得关于在细胞和分子水平胶质活动巨大的信息。所用的体外系统，主要包括从新生儿脑皮质组织来源的原神经胶质细胞和永生化细胞系。然而，这些系统可能无法反映体内脊髓神经胶质细胞的特性。为了使用培养体系，更好地反映体内状况进一步调查脊髓神经胶质细胞在周围神经损伤引起的神经性疼痛发展中的作用，我们的实验室已经发展到建立原发性脊髓混合胶质细胞培养从方法成年小鼠。简单地说，脊髓从成年小鼠收集，并通过木瓜蛋白酶消化后髓鞘去除与加工窝点性梯度网上平台。单细胞悬浮液是在完整的Dulbecco补充有2-巯基乙醇（2-ME）改良的Eagle培养基（cDMEM）培养在35.9℃。这些培养条件为混合胶质细胞的生长进行了优化。在这些条件下，细胞准备用于12之间实验 – 14天（细胞通常是在这段时间对数期）建立培养物（D 0）之后，并且可以被保持在培养条件达到D 21。该培养系统可以用于研究脊髓神经胶质细胞的刺激时与各种物质和试剂的反应。除了神经性疼痛，这种系统可用于研究在涉及脊髓神经胶质细胞的病理改变其他疾病胶质反应。
Introduction
慢性疼痛是一种严重的健康问题，影响到在美国的约100万成年人，具有高达635 $ 1十亿预计每年成本。越来越多的证据表明脊髓神经胶质细胞的神经性疼痛，最具破坏性的种慢性疼痛2中的一个发展的显著贡献。一个适当的体外培养体系将在细胞和分子水平深入研究神经胶质细胞的作用显著帮助。
目前，研究人员使用的体外神经胶质培养系统包括从新生小鼠或大鼠大脑皮质组织来源主要是神经胶质细胞和永生化从新生小鼠衍生的神经胶质细胞系或大鼠原胶质细胞。新生细胞含有未分化细胞显著数字，可以进一步分化成神经胶质细胞（主要为星形胶质细胞和小胶质细胞），从而提供一bundant细胞实验使用3。然而，我们以前的研究已经表明，新生儿脑胶质细胞从成人脊髓神经胶质细胞在脂多糖（LPS）刺激的反应显著不同。例如，白细胞介素（IL）-4显示的增强的LPS诱导的一氧化氮的抑制作用（NO）产生的成年大鼠脊髓神经胶质相比新生儿大脑胶质4。此外，趋化因子产生刺激后的由神经肽的型材，降钙素基因相关肽（CGRP），是新生小鼠脑部的神经胶质细胞之间无可争议不同（在参考文献中描述的方法。5）和成年小鼠脊髓神经胶质细胞（ 图1）。建立的细胞系是易于使用和维护，可以在很短的时间内提供大量的细胞。在一般情况下，永生化细胞系中产生或者使用病毒介导的永生化系统（如广泛使用的小胶质细胞系BV2）6,7或以下第自发变换电子识别（如星形胶质细胞系的C8-D1A和小胶质细胞系的C8-B4）8,9的细胞系具有优异的学习星形细胞和小胶质细胞单独的分子特征。然而，从细胞系所获得的结果总是需要在原代细胞或在体内条件进一步验证。还应当指出的是，没有发生过一个从啮齿动物的脊髓神经胶质衍生的神经胶质细胞系的报告。
以帮助调查脊髓神经胶质细胞中的神经性疼痛，是从成年小鼠脊髓衍生已通过适应用来产生成年大鼠混合胶质细胞培养4先前报道的方法开发了一种培养系统，5的发展中的作用该小鼠脊髓神经胶质文化进一步细化最近10和更详细的这篇文章中介绍。成人脊髓混合胶质细胞培养Ç一个与来自适合的特定的研究的需要选出的菌株的小鼠建立的，并且细胞能够在培养物中维持长达21天后的培养开始。这种方法可以在神经性疼痛的研究中使用，以及在涉及脊髓内的病理变化，如肌萎缩性侧索硬化症（ALS），人免疫缺陷病毒（HIV）相关的感觉神经病，和多个各种神经疾病的调查硬化症（MS）。
Protocol
Representative Results
Discussion
它是执行所有步骤，包括媒体，以获得实验之间重复性的结果改变时间表-以一致的方式非常关键。下面的步骤是获得优良的品质成人混合胶质尤为关键。
酶消化是该协议的一个重要方面。至关重要的是，该组织片是为了获得单细胞悬浮液充分消化。然而，过度消化将导致显著较少的细胞。每个实验需要执行导频测试，以确定基于所述类型用于孵化小瓶的确切消化时间，每次?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Adult spinal cord mixed cultures were first developed using rats with grant support from an NIH/NIDA R01 award (PI De Leo) and an NIH T32 training grant (PI Green). These methods were further adapted for mouse spinal cords with support from the following grants: NIH/NIDA 5K01DA023503 (PI Cao), NIH/NINDS 5R21NS066130 (PI Cao), and NIH/NIGMS P20GM103643 (PI Meng). The authors would also like to thank Dr. Alejandro M. S. Mayer, Department of Pharmacology, Chicago College of Osteopathic Medicine, for his technical help in obtaining microglia-rich mixed glial cells.
Materials
| Dulbecco’s modification of Eagle’s media (DMEM) with 4.5g/L glucose | Lonza | 12-709F | The cDMEM media is a standard, widely used culture media. Individual researcher can decide where to purchase the DMEM and all other component used to make cDMEM media. |
| L-Glutamine (100x) | Lonza | 17-605E | The L-glutamine is a standard, widely used component for various culture media. Individual researcher can decide where to purchase L-glutamine. |
| Antibiotic-Antimycotic Solution (100x) | Corning-Mediatech | 30-004-CI | This is a combination of penicillin, streptomycin and Amphotericin formulated to contain 10,000 units/ml penicillin G, 10 mg/ml streptomycin sulfate and 25 µg/ml amphotericin B. Individual researcher can decide where to purchase individual component. |
| 2-mercaptoethanol (2-ME) | Sigma-Aldrich | M3148 | A BioReagent, suitable for cell culture, molecular biology and electrophoresis. |
| Papain dissociation system | Worthington Biochemical Corporation | LK003150 | Individual components of this kit can be purchased separately. |
| Percoll | GE Health Care | 17-0891-01 | Percoll is sold as sterile solution. Undiluted Percoll can be re-autoclaved if needed. |
| Lab-line incubator/shaker | Barstead/Lab-line | MaxQ4000 | This is the incubator/shaker we have currently. Other types of shakers can be used instead. |
| Lipopolysaccharides, Salmonella Minnesota Re595 | Sigma-Aldrich | L-9764 | Other strains of LPS can also induce glial responses. The magnitude of the responses may vary. |
| Mouse IL-6 DuoSet ELISA | R&D Systems | DY406 | Each individual Researcher can select the ELISA kit he or she prefers. |
| Mouse TNF-alpha DuoSet ELISA | R&D Systems | DY410 | Each individual Researcher can select the ELISA kit he or she prefers. |
| Mouse IP-10 (CXCL10) DuoSet ELISA | R&D Systems | DY466 | Each individual Researcher can select the ELISA kit he or she prefers. |
| Mouse MCP-1 (CCL2) ELISA Opteia set | BD Biosciences | 555260 | Each individual Researcher can select the ELISA kit he or she prefers. |
| Mouse chemokine Q-Plex | Quansys Biosciences | 120251MS | This product was available for in-house assay at the time. Instead, we sent out samples to the manufacturer for analysis. |
| APC-anti-mouse-CD45 (clone 30-F11) | eBioscience | 17-0451 | Antibody of the same clone but from other vendors can be used. |
| FITC-anti-mouse-CD11b (clone M1/70) | eBioscience | 11-0112 | Antibody of the same clone but from other vendors can be used. |
| Accuri C6 flow cytometer | BD Biosciences | BD Accuri C6 | Each individual Researcher can use any flow cytometer he or she prefers. |
| FlowJo software | Tree Star, Inc. | FlowJo7.6.5 | Each individual Researcher can use any analysis software he or she prefers. |
References
- Gaskin, D. J., Richard, P. The economic costs of pain in the United States. J Pain. 13 (8), 715-724 (2012).
- Ji, R. R., Berta, T., Nedergaard, M. Glia and pain: Is chronic pain a gliopathy?. Pain. 154 (01), S10-S28 (2013).
- Ni, M., Aschner, M., Maines, M. D. Neonatal rat primary icroglia: isolation, culturing and selected applications. Curr Protoc Toxicol. , (2010).
- Cao, L., Fei, L., Chang, T. T., DeLeo, J. A. Induction of interleukin-1beta by interleukin-4 in lipopolysaccharide-treated mixed glial cultures: microglial-dependent effects. J Neurochem. 102 (2), 408-419 (2007).
- Malon, J. T., Maddula, S., Bell, H., Cao, L. Involvement of calcitonin gene-related peptide and CCL2 production in CD40-mediated behavioral hypersensitivity in a model of neuropathic pain. Neuron Glia Biol. 7 (2-4), 117-128 (2011).
- Blasi, E., Barluzzi, R., Bocchini, V., Mazzolla, R., Bistoni, F. Immortalization of murine microglial cells by a v-raf / v-myc carrying retrovirus. J Neuroimmunol. 27 (2), 229-237 (1990).
- Bocchini, V., et al. An immortalized cell line expresses properties of activated microglial cells. J Neurosci Res. 31 (4), 616-621 (1992).
- Alliot, F. O., Marty, M. C., Cambier, D., Pessac, B. A spontaneously immortalized mouse microglial cell line expressing CD4. Dev Brain Res. 95 (1), 140-143 (1996).
- Alliot, F. O., Pessac, B. Astrocytic cell clones derived from established cultures of 8-day postnatal mouse cerebella. Brain Res. 306 (1-2), 283-291 (1984).
- Malon, J. T., et al. Microglial content-dependent inhibitory effects of calcitonin gene-related peptide (CGRP) on murine retroviral infection of glial cells. J Neuroimmunol. 279, 64-70 (2015).
- Cao, L., DeLeo, J. A. CNS Infiltrating CD4(+) T lymphocytes Contribute to Murine Spinal Nerve Transection-Induced Neuropathic Pain. Eur J Immunol. 38 (2), 448-458 (2008).
- Mayer, A. M., et al. Effect of a short-term in vitro exposure to the marine toxin domoic acid on viability, tumor necrosis factor-alpha, matrix metalloproteinase-9 and superoxide anion release by rat neonatal microglia. BMC Pharmacol. 1, 7 (2001).
- Lawson, L. J., Perry, V. H., Dri, P., Gordon, S. Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain. Neuroscience. 39 (1), 151-170 (1990).
- Rock, R. B., et al. Role of Microglia in Central Nervous System Infections. Clin Microbiol Rev. 17 (4), 942-964 (2004).
- Yang, I., Han, S. J., Kaur, G., Crane, C., Parsa, A. T. The Role of Microglia in Central Nervous System Immunity and Glioma Immunology. J Clin Neurosci. 17 (1), 6-10 (2010).
