共培养实验的奥里戈丁细胞和奥里戈丁酸基质的生成
Summary
本文展示了一种用于纯化寡核苷酸物和生产寡核苷酸调节介质的有效方法，可用于共培养实验。
Abstract
在中枢神经系统中，寡核苷酸以其在轴向髓中的作用而闻名，通过盐性传导加速作用电位的传播。此外，越来越多的报告表明，寡核苷酸与神经元在骨髓外相互作用，特别是通过可溶性因子的分泌。在这里，我们提出了一个详细的协议，允许从胶质细胞培养物中也含有星形细胞和微胶质细胞的寡核体系细胞进行纯化。该方法依赖于在37°C处过夜摇动，这允许选择性地分离上覆的寡核瘤细胞和微胶质细胞，并通过差分粘附消除微胶质。然后，我们描述了寡核苷酸的培养物和寡核苷酸条件介质（OCM）的生产。我们还在共培养实验中提供OCM治疗或寡核苷酸的动力学，以及纯化海马神经元，研究寡核苷酸-神经元相互作用。
Introduction
寡核苷酸（OLs）是中枢神经系统（CNS）的胶质细胞，产生围绕轴xon的骨髓素。OLs源自寡核苷酸前体细胞（OPCs），在胚胎中枢神经系统的心室区增殖，然后迁移和分化成完全成熟的OLs（即骨髓形成细胞）1。OPCs在早期发育期间是丰富的，但也坚持在成人大脑中，在那里他们代表主要的增殖细胞群2。单个 OL 在非兴奋部分（即节点间）中产生多个阳极，并且每个骨髓环的边缘附着在构成参数域的斧子上，这对骨髓素1、3的绝缘特性至关重要。在副节点之间是称为Ranvier节点的未骨髓间隙。这些节点富含电压门控钠通道（Nav），允许通过盐性传导4再生和快速传播作用电位。这种紧密的相互作用也使得斧道能量支持通过神经元从OLs5，6的乳酸盐。
寡核苷酸系细胞的成熟和骨髓过程由它们与神经元的相互作用7严格调节。事实上，OLs和OPCs，也叫NG2细胞，表达神经递质的受体阵列，并能接收兴奋和抑制神经元的输入，使它们能够感知神经元活动，可以触发其增殖和/或分化到骨髓细胞2。反过来，OPCs/OP将微囊和蛋白质分泌到细胞外空间，单独或协同地介导神经调节和神经保护功能8，9，10，11，12。然而，控制寡核遗传细胞和神经元之间多种相互作用模式的分子机制尚未完全破译。
此外，在几个CNS病理条件下，OLs主要受到影响，从而干扰它们与神经元的相互作用。例如，在多发性硬化症 （MS） 中，神经功能障碍是由中枢神经系统中的焦脱髓引起，其次是 OLs 损失，可导致斧道损伤和相关残疾积累。重组可以发生，尽管在大多数情况下还不够。过去十年的进展，由于免疫疗法的发展，已经降低了复发率，但促进再骨髓化至今仍是一个未满足的需求。因此，更好地了解OLs的作用、功能和影响对于开发针对各种CNS条件的新疗法特别感兴趣。
在这里，我们描述了OLs纯化和培养的方法。这使得能够精确检查调节其发展和生物学的内在机制。此外，这种高度丰富的OLs培养物允许生产寡核苷酸条件介质（OCM），这种介质可以添加到纯化神经元培养物中，从而深入了解OLs分泌因子对神经元生理学和连通性的影响。此外，我们描述了如何实现一个体外共培养系统，其中纯化的寡核苷酸和神经元结合在一起，允许解决调节（再）髓化的机制。
Protocol
本实验中对大鼠的护理和使用符合机构政策和准则（UPMC、INSERM和欧洲共同体理事会指令86/609/EEC）。以下协议是为12只幼崽的标准垃圾建立的。 1. 烧瓶的准备（±5分钟） 注：在无菌条件下在层流罩中进行解剖的前一天执行以下步骤。 使用 5 mL 的聚乙烯胺（PEI，100 mg/L，参见补充文件 1中的协议）涂上 150 厘米2瓶 （T150） 与过滤?…
Representative Results
在此协议中，OL系系细胞通过摆脱星形细胞和微胶质从胶质培养中纯化。对OL培养物的纯度和质型检查可以通过用胶质标记物进行免疫染色来评估。对不同标记物表达的分析表明，OL培养物大多是O4+细胞的90%~4%，85%~7%NG2+细胞，4.7%~2.1%的PLP+细胞，7.2%~2.5%的细胞为GFAP+星细胞（均值=S.D.，n= 3; 图 2.此外，4….
Discussion
在这里，我们提供了一个详细的协议，以获得高度丰富的寡核苷酸细胞培养从混合胶质培养，改编自以前出版的方法16，并随后生产OL条件介质。这种摇动技术不贵，可重复三次，是获得高量纯化OL的最佳方法，因为在含有PDGF®的博滕斯坦-萨托（BS）培养的细胞中培养。胶质细胞是在P2时使用威斯塔大鼠的脑皮质制备的，此时绝大多数的寡核瘤系细胞是表达NG2和O415</su…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
作者要感谢雷米·朗扎诺在手稿编辑方面明智的建议。这项工作由ICM、INSERM、ARSEP基金会向NSF提供赠款和布韦特-拉布鲁耶尔价格资助。
Materials
| 5-fluorodeoxyuridine | Sigma | F0503 | |
| B27 supplement | ThermoFisher | 17504044 | |
| D-(+)-Glucose solution | Sigma | G8769 | |
| DNase (Deoxyribonuclease I) | Worthington | LS002139 | |
| Dulbecco's Modified Eagle Medium | ThermoFisher | 31966021 | |
| Ethanol 100% | Sigma | 32221-M | |
| Ethanol 70% | VWR Chemicals | 83801.360 | |
| Fetal Calf Serum | ThermoFisher | 10082147 | |
| L-cysteine | Sigma | C7352 | |
| Neurobasal | ThermoFisher | 21103049 | |
| Papain | Worthington | LS003126 | |
| Penicillin-Streptomycin | ThermoFisher | 15140122 | |
| Phosphate Buffered Saline without calcium and magnesium | ThermoFisher | A1285601 | |
| Polyethylenimine(PEI) | Sigma | P3143 | |
| Tetraborate decahydrate | Sigma | B9876 | |
| Trypsin | Sigma | Sigma | |
| Uridine | Sigma | U3750 | |
| Bottenstein-Sato (BS) media | |||
| apo-Transferrin human | Sigma | T1147 | |
| BSA (Bovine Serum Albumin) | Sigma | A4161 | |
| Dulbecco's Modified Eagle Medium | ThermoFisher | 31966021 | |
| Insulin | Sigma | I5500 | |
| PDGF | Peprotech | AF-100-13A | |
| Penicillin-Streptomycin | ThermoFisher | 15140122 | |
| Progesterone | Sigma | P8783 | |
| Putrescine dihydrochloride | Sigma | P5780 | |
| Sodium selenite | Sigma | S5261 | |
| T3 (3,3',5-Triiodo-L-thyronine sodium salt) | Sigma | T6397 | |
| T4 (L-Thyroxine) | Sigma | T1775 | |
| Co-culture media | |||
| apo-Transferrin human | Sigma | T1147 | |
| B27 supplement | ThermoFisher | 17504044 | |
| Biotin | Sigma | B4639 | |
| BSA (Bovine Serum Albumin) | Sigma | A4161 | |
| Ceruloplasmin | Sigma | 239799 | |
| Dulbecco's Modified Eagle Medium | ThermoFisher | 31966021 | |
| Hydrocortisone | Sigma | H4001 | |
| Insulin | Sigma | I5500 | |
| N-Acetyl-L-cysteine | Sigma | A8199 | |
| Neurobasal | ThermoFisher | 21103049 | |
| Penicillin-Streptomycin | ThermoFisher | 15140122 | |
| Progesterone | Sigma | P8783 | |
| Putrescin | Sigma | P5780 | |
| Recombinant Human CNTF | Sigma | 450-13 | |
| Sodium selenite | Sigma | S5261 | |
| T3 (3,3',5-Triiodo-L-thyronine sodium salt) | Sigma | T6397 | |
| Vitamin B12 | Sigma | V6629 | |
| Tools | |||
| 0.22 µm filter | Sartorius | 514-7010 | |
| 1 mL syringe | Terumo | 1611127 | |
| 100 mm Petri dish | Dutscher | 193100 | |
| 15 mL tube | Corning Life Science | 734-1867 | |
| 50 mL tube | Corning Life Science | 734-1869 | |
| 60 mm Petri dish | Dutscher | 067003 | |
| 70 µm filter | Miltenyi Biotec | 130-095-823 | |
| Binocular microscope | Olympus | SZX7 | |
| Curved forceps | Fine Science Tools | 11152-10 | |
| Fine forceps | Fine Science Tools | 91150-20 | |
| Large surgical scissors | Fine Science Tools | 14008-14 | |
| Scalpel | Swann-morton | 233-5528 | |
| Shaker | Infors HT | ||
| Small surgical scissors | Fine Science Tools | 91460-11 | |
| Small surgical spoon | Bar Naor Ltd | BN2706 | |
| T150 cm2 flask with filter cap | Dutscher | 190151 | |
| Animal | |||
| P2 Wistar rat | Janvier | RjHAn:WI |
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Citar este artículo
Mazuir, E., Dubessy, A., Wallon, L., Aigrot, M., Lubetzki, C., Sol-Foulon, N. Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments. J. Vis. Exp. (156), e60912, doi:10.3791/60912 (2020).