Author Produced

丰富的少突胶质细胞培养和Myelinating产后小鼠组织联合培养的少突胶质细胞/神经元的推导

Neuroscience
 

Summary

本文介绍的方法,从中获得丰富的人口在原代​​培养的小鼠少突胶质前体细胞(OPCS),它分化为成熟的少突胶质细胞(OLS)。此外,本报告介绍的技术生产小鼠myelinating播种到小鼠背根神经节(DRGNs)突起床鼠标OPCs的合作文化。

Cite this Article

Copy Citation | Download Citations | Reprints and Permissions

O'Meara, R. W., Ryan, S. D., Colognato, H., Kothary, R. Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues. J. Vis. Exp. (54), e3324, doi:10.3791/3324 (2011).

Please note that all translations are automatically generated.

Click here for the english version. For other languages click here.

Abstract

确定基本的OL发展的分子机制,推动我们的OL生物学知识不仅是关键,但也有了解脱髓鞘疾病,如多发性硬化症(MS)的发病机制的影响。细胞发育通常与原代细胞培养模型研究。原代细胞培养有利于对一个给定的细胞类型的评价提供了一个受控环境中,体内多余的变量。虽然从大鼠的职等文化提供入职等生物大量的洞察力,类似的努力,建立从小鼠OL的文化遭到了主要障碍。发展文化小鼠的主要OLS方法是必须以利用现有的转基因小鼠线的优势。

已被描述为啮齿类动物组织中提取OPCs的多种方法,从神经球的推导,差速贴壁纯化和immunopurification 1-3范围。虽然许多方法提供了成功,最需要丰富的文化时间和/或昂贵的设备/试剂。为了解决这个问题,净化适应最初是由麦卡锡和DE Vellis 2描述的方法,从小鼠组织的OPCs的首选。此方法涉及物理分离OPCs的来自新生儿灭鼠皮质的神经胶质混合文化。结果是可以分化成OL丰富文化的纯化OPC人口。这种做法是有吸引力,因为其文化相对短的时间和不必要的生长因子或immunopanning抗体要求。

虽然在纯化文化探索的OL发展的机制是知识性的,它不提供有关生理环境评估髓鞘的形成。与神经元共培养OLS将给予调节OL介导的神经轴突髓鞘的分子基础的洞察力。对于很多OL /神经元共培养研究,背根神经节(DRGNs)已被证明是神经类型的选择。他们是由于其易于提取,少量污染的细胞,并形成致密突起的病床与OLS的合作文化的理想选择。 4-6,虽然研究使用鼠/鼠标myelinating xenocultures已经发表,这样的推导方法OL / DRGN myelinating产后小鼠组织中的合作文化并没有被描述。在这里,我们目前对如何有效地产生这样的文化,以及预期结果的例子详细的方法。这些方法有助于解决OL开发/ myelinating功能的相关问题,并在神经科学领域的有用工具。

Protocol

伦理声明

根据加拿大动物保健理事会(廉政公署)的指导方针,在这项工作中所使用的小鼠照料。获得批准的,从渥太华动物保健委员会根据协议号OGH - 119大学进行实验的伦理。

1。夹层 - 为OPC提取新生小鼠皮层

  1. 根据机构指引牺牲P0 - P2的鼠标。
  2. 解剖一个Petri菜含有冰冷MEM(不含抗生素)的大脑和地方。
  3. 盘转移到解剖镜下。
  4. 用脑背侧使用手术刀,使浅切口弧矢沿每个皮层(图1A)最内侧的边缘。这个切口应该只通过脑膜层,以促进其清除。
  5. 用细尖的镊子,以横向的方式脑膜剥离。如果做得仔细,这一层可以删除在一块。在这一步,去除嗅球。
  6. 随着大脑的腹侧方的,作出了深刻的矢状切口皮层与间脑的腹侧区(图1b)。
  7. 随着脑的背侧,撬在内侧横向时尚(图1C,C')的组织分开的脑皮层。在这一步中删除任何剩余的脑膜。
  8. 骰子成每个约4件,轻轻地转移到15 mL锥形管内含有350μL的MEM每鼠脑皮层。管置于冰上,直到所有的老鼠都被处理。
  9. 余下的小鼠重复步骤1.1-1.8。

2。解离新生儿皮质和胶质混合文化的维护

注意:应避免在以下所有步骤的气泡成细胞悬液。

  1. 添加15毫升的锥形管中,37℃水浴3分钟的新鲜解剖大脑。
  2. 大脑转移到无菌组织培养罩。
  3. 泡皮层轻轻地穿过一个P1000的枪头,以产生更小的片段。一旦有大到足以破坏一个畅通暂停通过枪头无脑片停止吹打。
  4. 添加到锥形管75μLOPC木瓜每大脑的解决方案。 OPC木瓜蛋白酶溶液必须预先加热37 ° C为前20分钟使用。
  5. 孵育在37℃水浴20分钟。大约每2分钟,轻轻颠倒管,以防止组织聚集。在此期间,每个聚- L -赖氨酸(PLL),涂(1毫克/毫升)的T25的瓶(小鼠大脑中的每一个瓶),并在37℃组织培养孵化器放置在添加5毫升混合胶质文化传媒8.5%的CO 2。
  6. 20分钟后,返回的组织悬液无菌罩和管加入2毫升每脑胶质混合培养基。让我们坐下来让灭活OPC木瓜蛋白酶溶液在室温为10分钟。
  7. 分装成5毫升塑料管的组织悬液。管的数量应匹配的解剖大脑,造成约2.5毫升每管。
  8. 使用无菌火焰抛光玻璃巴​​斯德吸管,轻轻磨碎每管组织。起初慢慢磨碎,并逐渐增加速度块游离。磨碎约10-15倍,但这个数字可能会有所不同的基础上消化的功效。

注:根据trituration将导致贫困的组织分解,而过trituration将产生不利影响细胞活力。重要的是不引入到解决方案中的泡沫,因为这将严重影响细胞活力。

  1. 一旦有不可见的组织团块,留在暂停,转移到50 mL锥形管中含有4毫升每脑胶质混合培养基(即4大脑= 16毫升混合胶质文化传媒)。
  2. 轻轻翻转50 ml锥形管和重复余下的5 mL管。
  3. 汇集的细胞悬液,分装成15毫升的锥形管(大约6.5毫升每15毫升管)。 15毫升管的数目应相匹配的解剖大脑。
  4. 离心管在1200转5分钟(约300克)。
  5. 小心吸出上清液,加入1毫升的温暖混合胶质文化传媒,每次15 mL锥形管。
  6. 慢慢地重新悬浮颗粒与P1000的枪头,小心,不要引入气泡。加入细胞悬液,每管预平衡PLL涂层的T25烧瓶,呈现总量的文化传媒至6 ml。
  7. 放置在3-4小时的组织培养孵化器的烧瓶中,让细胞附着到PLL基板。吹打媒体,并加入6毫升新鲜混合胶质细胞培养基的烧瓶执行一个全媒体的变化。这一步的碎片删除造成的trituration,并促进文化的生存能力。如果OL / DRGN联合培养的需要,是指本议​​定书第3条。
  8. 经过3天的文化,执行删除媒体4毫升,4毫升新鲜混合胶质培养基代替2 / 3的介质改变。在这一点上,星形胶质细胞的单层应在烧瓶底部形成。
  9. 第6天,进行2 / 3的媒体的变化和与终浓度为5μg/ mL的胰岛素补充的烧瓶。在这一点上,星形胶质细胞的单层应清晰可见,其中OPCs的将增殖的顶部。

3。 DRGN隔离

注意:要产生OL / DRGNs联合培养,DRGNs应建立后,胶质混合文化生成一天。这两种文化类型的种植独立,合并后9-10天。

  1. 牺牲P5 - P10鼠标根据机构指引。
  2. 提取脊柱,并转移到一个干净的培养皿。
  3. 修剪,尽可能的脊柱(图1D,D')尽可能多的肌肉和骨,因为这将缓解解剖背根神经节(病种付费)。
  4. 修剪脊柱转移到一个新的培养皿腹侧,。使用夹层剪刀,从尾端开始,穿过脊柱内侧在纵向的方式。
  5. 使用两个双钳,轻轻撬开脊柱脊髓暴露。
  6. 病种付费,可以发现下方和外侧脊髓。用细尖镊子,轻轻取出病种付费,同时避免损害神经节(图1E)。
  7. 将去除病种付费冰冷汉克的缓冲盐溶液(HBSS中,无抗生素)在一个新的培养皿。剥离的目标应提取40%小鼠背根节(图1F)。
  8. 已提取的病种付费后,修剪任何过长的根(图1G,G')的病种付费,以尽量减少污染细胞引入到文化(神经胶质细胞,成纤维细胞)。
  9. 病种付费转移到1.5 mL离心管中,含有500μL冰冷的HBSS。
  10. 离心机在1200转(约300克)5分钟,4 ° C至颗粒的病种付费。
  11. 离心管转移到无菌组织培养罩,从管道中删除的HBSS。
  12. 加入500μL预热(20分钟在37 ° C)DRG的木瓜蛋白酶的解决方案,并在37℃水浴10分钟中孵育管。反转管每隔2分钟,以防止组织聚集。
  13. 重复步骤3.10。
  14. 取出DRG的木瓜蛋白酶溶液,并添加500μL预热胶原酶一个解决方案,在37 ° C(20分)。在37℃水浴孵育10分钟,反相每2分钟。
  15. 重复步骤3.10。
  16. 去除上清液,加入1毫升DRGN媒体。反转管好几倍。
  17. 重复步骤3.10。
  18. 重复步骤3.16。
  19. 外套无菌火焰抛光玻璃巴​​斯德吸管吹打的HBSS几次0.25%BSA溶液与牛血清白蛋白(BSA)的。 BSA溶液的涂层将坚持的玻璃吸管的墙壁防止病种付费。
  20. 与BSA的涂层吸管轻轻磨碎的病种付费,在第一,和强度的增加,一旦团块开始游离。磨碎约10-15倍,但是这个数字是依赖于一定程度的消化,每管病种付费。
  21. 一旦实现了分解,通过一个50微米过滤到无菌的Petri菜含有7毫升DRGN媒体的暂停。过滤将消除碎片从细胞悬液,尽管这一步并不重要。
  22. 8.5%的CO 2孵育培养皿约1.25小时。
  23. 大衣几个12毫米盖玻片,用液氮在24孔盘(10微克/毫升的PBS)在此孵化时间。
  24. 一旦孵化完成后,观察培养皿中,在明亮的领域。被确定为大型健全,相黑暗的牢房DRGNs。摇动培养皿轻轻地抬起任何坚持DRGNs。

注:许多污染细胞会强烈坚持的培养皿中,从而丰富DRGNs细胞悬液。

  1. 细胞悬液转移到15 mL锥形管。轻轻冲洗4毫升DRGN媒体的菜收取任何残留DRGNs。将附加的4毫升的锥形管。
  2. 离心5分钟1200转(约300克)。
  3. 吸上清,沉淀重悬在500μL新鲜DRGN媒体。
  4. 计算取得DRGNs使用血球数量。请务必只DRGNs,而不是其他类型的细胞计数。 DRGNs可以识别他们的大球形细胞机构。
  5. 种子30,000-50,000 DRGNs在8.5%的CO 2一夜之间每1毫升DRGN媒体液氮涂盖玻片,在37℃组织培养箱内培养。
  6. 第二天早晨,富于变化的介质更换DRGN执行媒体与媒体与终浓度为1%笔/链球菌和10μmolFUDR的OL(减去,CNTF)。
  7. 第3和第5天,执行一个相同的媒体与步骤3.30 3 / 4媒体的变化。
  8. 第7天,进行全媒体与媒体(减号,CNTF,PEN /链球菌,FUDR)OL变化。
  9. 在第9天,DRGNs应该已经形成了一个广泛的突起床,现在已经准备好要与OPCs的共同培养​​。

4。 OL丰富的文化或OL / DRGN合作文化的建立混合胶质细胞培养纯化的OPCs

  1. 胶质混合文化的第9天,将烧瓶在5%的CO 2组织培养孵化器的轨道摇床。空T25烧瓶上放置的烧瓶,防止造成负面影响的混合胶质文化的轨道摇床所产生的任何热量。允许文化的平衡1小时,这个新的孵化器。
  2. 一旦烧瓶平衡,摇匀,在45分钟50转的烧瓶。这动摇的目的是消除任何松散附着的污染从单层细胞。
  3. 移动细胞到组织培养罩,从烧瓶中删除所有的媒体。更换4毫升新鲜混合胶质文化媒体与5μg/ mL的胰岛素补充。
  4. 将振动筛返回到烧瓶,并允许以平衡为大约3小时。
  5. 一旦烧瓶是平衡的,他们牢固地拧紧的轨道摇床,摇烧瓶,在220 RPM(过夜)约16小时。
  6. 第二天早晨,如果在DRGNs的情况下( OL丰富的文化),大衣几个无菌液氮(10微克/毫升的PBS)1小时12毫米盖玻片种植OLS。 24菜肴转移的盖玻片,用PBS冲洗,随后一个洗OL媒体。每孔加入1毫升的OL媒体和平衡在8.5%的CO 2。
  7. 在5%平衡30分钟10厘米的组织培养皿CO 2。每2烧瓶,将需要一盘。这些将用于附着力差的悬浮OPCs的富集。
  8. 一旦平衡时间已经过去了30分钟,动摇烧瓶转移媒体的菜肴。每一道菜,应该得到2烧瓶媒体,相当于每10 cm培养皿的细胞悬浮液约8毫升。
  9. 孵育5%CO 2 30分钟的菜肴,同时提供了在15分钟大关的温和的微调。这种微调,将有助于防止坚持10 cm培养皿OPCs的。
  10. 一旦孵化完成后,检查的菜肴,在明亮的领域。 OPCs的被认定为小细胞团块,通常3-5个细胞,但有时会形成类似神经球的大聚合。许多非OL系细胞,应坚决坚持的板块基地。轻轻旋流板分离任何松散坚持OPCs的,并从每个板块转移到15 mL锥形管的细胞悬液。
  11. 在1200转(约300克)离心5分钟。
  12. 1毫升的OL媒体与一个P1000的枪头,随后再悬浮与P200的枪头重悬沉淀。
  13. 使用血球计数细胞。
  14. 对于丰富的OL文化,种子25000 - 50000 OPCs的每12毫米液氮涂在终体积为1 mL的OL媒体盖玻片。
  15. 适合OL / DRGN联合培养,从第DRGNs执行一个完整的OL媒体(减去,CNTF)的变化[3],并轻轻地增加50,000从OPC富集细胞悬液细胞。请小心不要破坏DRGN突起床期间增加的OPCs。
  16. 广场文化,在37℃孵化器在8.5%的CO 2,避免删除,直到固定。小鼠OPCs的敏感,pH值的变化,从孵化器中删除,将改变的OL媒体的pH值。另外值得注意的,除了周围的细胞培养的空 ​​井DH 2 O将防止培养基蒸发,从而最大限度地减少浓度的溶质的波动范围内的OL媒体。这将提供一个更一致的OPCs的的环境。

5。处理免疫荧光显微镜文化

  1. 在-20 ° C,100%甲醇为10分钟,或3%多聚甲醛在室温为15分钟,修复文化。
  2. 通透盖玻片0.1%TRITON - X - 100的10分钟,冲洗1小时10%山羊血清磷酸盐缓冲液(PBS)和块。
  3. 原发性抗体孵育盖玻片在封闭液过夜稀释在4 ° C
  4. 洗净的盖玻片,用PBS 3倍,并培育与Alexa的福陆公司共轭二次稀释抗体阻断45分钟的解决方案(Invitrogen公司)。
  5. 4',6 - diamidino - 2 -苯基吲哚(DAPI)染液,用PBS冲洗盖玻片几次。
  6. DAKO公司荧光灯安装在盖玻片安装介质。
  7. 通过免疫荧光显微镜分析的幻灯片。在这个协议中,幻灯片一个ZEISS AXIOVERT 200M倒置荧光分析显微镜或蔡司LSM 510 META激光扫描共聚焦显微镜。

6。全细胞蛋白提取OL丰富的文化

  1. 从孵化器和冷却,冰浴3分钟,取出24以及文化。
  2. 小心取出媒体,并添加10-20μL裂解液(50毫米的Tris - HCl,150 mM氯化钠,0.1%SDS,0.5%去氧胆酸钠,1%TRITON - X - 100,用0.1%的胃酶抑素,抑肽酶,PMSF,亮肽素,钒酸钠),每孔(至少8%的样品水井的建议)。
  3. 使用大口径的P1000的吸头,刮井和裂解液转移到1.5 mL离心管。
  4. 穿过30轨半注射器裂解约15倍,上冰30分钟的寒意。
  5. 14,000 RPM(〜20,000 G)为15分钟在4 ° C离心管
  6. 传输上清至新的离心管中,并储存于-80 ° C。

7。 SDS - PAGE分析在丰富的OL文化蛋白质

  1. 经SDS - PAGE降低标准的12%聚丙烯酰胺凝胶的缓冲解决每个样本中的蛋白质30微克。
  2. 半干转移到PVDF膜凝胶。
  3. 1小时座膜在TBST在5%脱脂奶粉(10毫米的Tris - HCl pH值8.0,150 mM氯化钠,0.1%Tween - 20的)。
  4. 孵育膜与稀释的抗体阻断1小时的解决方案。
  5. 10分钟TBST洗膜3次。
  6. HRP标记的二抗封闭液为45分钟的孵育膜。
  7. 用TBST洗膜几次,Amersham公司的ECL Plus Western印迹法检测试剂(通用电气医疗集团)孵育5分钟。
  8. 检测标准的科学成像胶片蛋白条带。

8。代表性的成果:

在这个协议中,OPCs的是扩大单层星形胶质细胞内胶质混合文化。这是来自P0 - P2的新生小鼠皮层神经胶质混合文化。每日1 次,在体外(DIV1),胶质混合文化包含不同形态的细胞相衬显微镜(图2a)。在DIV3,星形胶质细胞的单层烧瓶的基础上开始形成,并在DIV8时,OPCs可以清楚地观察单层表面上。在DIV9,增殖的OPCs的已达到足够的密度,一夜之间高速轨道晃动纯化。一旦纯化过程已经完成,其结果是一个OPC丰富的细胞群。 DIV1后净化时,OPCs有简单的形态,延长几道工序(图2b)。在DIV3后纯化,细胞有延长一个复杂的过程小梁,让人联想到不成熟的OLS。在DIV6后的净化,纯化的OLS已夷为平地,预计单张般的膜结构。这种形态的发展是典型体外成熟OLS 。

OL系(图3a)表明纯化的细胞免疫荧光显微镜。种子OPCs的最初表达的硫酸软骨素蛋白多糖(NG2),并发展成髓鞘相关糖蛋白(MAG)积极不成熟OLS后三天内播种(图3B)。在DIV6,很多OLS表达髓鞘碱性蛋白(MBP),并具有典型的成熟OL形态。 %的OL系的细胞在不同时间点进行了量化,以确定纯度的OL丰富的文化(图3C)。在DIV1后的净化,文化为50 ± 14%NG2 +已经OPCs的,没有MAG + veMBP +已经OLS 。这表明OL系细胞的纯化是有区别的OLS可以忽略不计的数字,在播种时在易制毒化学阶段。在DIV3,许多OLS分化成MAG +已经细胞(24 ± 5.9%),而一些保留的易制毒化学表型,并保持NG2 +(13 ± 8.0%) 。在DIV3,MAG +已经细胞(3.2 1.2%)的比例很小,也表达的MBP。在DIV6,20 ± 5.9%OLS MAG +已经 12 ± 7.3%,而坚持NG2 +已经 OPCs的。此外,21 ± 9.3%的细胞内的文化是MBP + VE在这个时间点OLS 。 SDS - PAGE分析显示分级2'3' -环核苷酸的3' -磷酸二酯酶(CNP)在6天的文化时期的MBP表达,进一步体现了文化OPCs的的能力末期分化成成熟的OLS(图3D )。总的来说,这些数据建立了这种方法生产OL丰富文化系统适合从OPCs的OL成熟的研究手段。

该协议还介绍了建立OL / DRGN联合培养,使用只鼠的组织来源的方法。然而,以产生共同的文化,DRGNs必须先单独培养,以产生足够的突起网络。这些产后小鼠神经元的文化是生长在低血清媒体9天与10μM的FUDR补充,以防止污染成纤维细胞增殖和GL胶质细胞。在9天的体外过程中,孤立的DRGNs产生一个致密突起的床(图4a )。这突起的床是免疫阳性神经元的标志物神经丝蛋白200(NF)和Tuj1(图4b)。此时,纯化OPCs的可能是添加的突起病床,和一个额外的6天生产myelinating联合培养培养。

在DIV6 OL / DRGN合作文化,很多的MBP +已经 OLS可以观察之间的NF +已经 DRGN突起(图5a)。经仔细检查,OLS证明,使众多DRGN突起的联系,经常鞘的MBP +已经膜(图5B,C) 。

图1
图1。特别是新生小鼠皮层和背根神经节隔离方面的解剖显微镜图像。 (一)背视图新鲜提取的新生小鼠大脑。虚线表示切口必须作出便于去除脑膜层的面积。(二 )脑腹面观,虚线表示皮层符合腹间脑的地方。深的切口,必须沿虚线援助隔离的皮质(C - C')的可视化描述如何撬从脑的其余部分的皮质(D)一个新鲜分离的P5 - P10鼠标脊柱之前修剪走多余的肌肉和骨骼(D)(E)在脊柱病种付费的位置。(F)(G)应该从一个鼠标隔离病种付费的大致数量。一个背根神经节,需要很长的根修剪前酶消化。虚线应修剪根部的区域。(G'),DRG的后根修剪。

图2
图2。 OPCs的是扩大内部胶质混合文化,纯化,并随后作为OL丰富的文化区别(一)第一阶段混合胶质文化对比度的图像,在不同的发展阶段。在DIV1,细胞出现几个扁平细胞轮。胶质混合文化的分层开始DIV3,星形胶质细胞形成一个统一的容量瓶中,赖以OPCs的增殖基地单层。许多OPCs的DIV8坚持的星形胶质细胞单层的表面(箭头),(b)一旦混合胶质文化纯化,DIV1 OPCs的扩展只有几道工序。在DIV3,细胞延长了许多过程,让人联想到中间阶段OLS。在DIV6,夷为平地的OLS(星号)显示有张膜(虚线)。比例尺,50微米。

图3
图3。 OL丰富的文化表征。 (一)的共聚焦显微镜图像的隔离OLS在不同的发展阶段。 NG2 +已经 OPCs的简单形态,而MAG +已经 OLS拥有多种乔木进程。 MBP +已经 OLS延长膜的髓鞘像张。 (二)比例尺,50微米。纯净的OL系细胞的来源称作OPCs,并分化成MAG + VE,MBP +已经OLS超过6格。在DIV1,所有OPCs的NG2 正极 ,而没有MAG + ve或MBP + VE。在DIV3,MAG + ve和少数的MBP +已经 OLS现在很明显的。 OLS的大部分DIV6,MAG和MBP +已经在所剩不多的NG2 +已经 OPCs的。 (三)比例尺,100微米。平均%OL细胞系在不同的发展阶段,超过6格值± SD 。在DIV1,所有的OL系细胞NG2 + VE,占50 ± 14%的总细胞内的文化。 DIV3和DIV6,OL系细胞分别占36 ± 6.8%和32 ± 8.4%的细胞总数,包括不同比例的NG2 +已经 MAG + ve和MBP的+已经 OLS。(D)SDS - PAGE电泳进行从丰富文化展示超过6 DIV文化时期分级OL标记CNP和MBP表达OL源性蛋白。

图4
图4。表征DRGN文化预OPC播种。 (a)第一阶段在9 DIV文化时期前OPC播种DRGNs对比度的图像。 DRGNs起源体型大细胞与几道工序,并产生一个日益复杂的突起网络。比例尺,100微米(二)固定在DIV9(预OPC播种)和神经元特异性标记Tuj1和NF200染色DRGN文化的共聚焦图像。 DRGNs已经产生了突起网络赖以OPCs可能是种子生产OL / DRGN myelinating联合培养。比例尺,50微米。

耳鼻喉科“> 图5
图5。 OLS共培养DRGNs OL介导DRGN突起的MBP +已经膜包装的结果(一)一个4场共聚焦图像蒙太奇一个DIV6 OL / DRGN共培养。许多的MBP +已经 OLS可以看出,与底层的DRGN突起床交互。比例尺,100微米(B)放大的共聚焦视图的MBP +已经 OLS包装多个DRGN突起。比例尺,50微米(C)(b)凡 DRGN突起正在OL膜包裹地区的“数字放大倍率表示。比例尺,25微米。

Discussion

这份报告介绍了隔离分化中的OL,丰富的文化或OL / DRGN联合培养的小鼠OPCs的一个方法。单独培养时,OPCs的分化成的MBP +已经 OLS,生产髓鞘膜张。 DRGN突起病床时,OLS enwrap与MBP的DRGN突起+已经膜。这种模式的好处OL介导的神经轴突ensheathment复杂的基础调查。

虽然建立这样的文化具有极大的价值,在技术上是具有挑战性的。特别是,要求方面,包括高效的组织消化/解离,维持平衡文化传媒pH值,并DRGN媒体的变化。重要的是要考虑消化的长度,组织量被消化和trituration金额影响疗效和最终结果的组织分解。这不是不寻常经验的研究人员获得从分离的神经系统组织细胞的产量低。此外,小鼠OPCs的倾向,特别是在碱性条件下,培养基的pH值的变化要敏感。文化维持在8.5%的CO 2,目的是为了防止这种情况,因为OPCs的出现,以更好地容忍超过基本微酸性条件。喂养DRGNs方面,媒体的变化,必须迅速执行,不会变干的神经元,但是,一定要轻柔,以不破坏发展中国家突起床。突然媒体的变化可能撞出从基板突起的床,并有可能导致在其完整的解离盖玻片。

这个模型系统的潜在好处极大地掩盖其技术要求的性质。这种系统的优点之一是产后小鼠细胞培养推导的使用,规避需要牺牲养殖女性收获胚胎组织。另一个优点是生长因子的缺乏扩大的OPCs(GFS)的要求。混合胶质文化提供了一个环境,支持OPCs的,大概是由于星形胶质细胞源性营养因子的存在传播。其他方法,如通过神经球7,8推导,依靠政府飞行服务队的有丝分裂的属性,如碱性成纤维细胞生长因子(bFGF),表皮生长因子(EGF)和血小板衍生生长因子(PDGF)为OPC扩张。同样,产后(P5 - 10)DRGNs的小鼠,避免与神经生长因子(NGF),神经营养因子在体外胚胎DRGNs 9,10生存所需补充的文化传媒的要求。它的利益,以避免使用神经生长因子,因为它产生负面影响的OLS myelinating能力培养DRGNs 4时。避免使用GF -为辅的媒体也有经济利益,作为在大规模使用时,这些试剂成为昂贵的。

或许,这种文化模式最重要的好处是它从鼠标的唯一组织的推导,从而提供机会来自各种各样的转基因小鼠线OPCs的和DRGNs。这允许DRGN和/或OPC特定的属性,执政髓鞘的研究。这将是重要的,特别是阐明受体/配体的相互作用,调节OL介导的神经轴突髓鞘。总之,这是由于神经科学的研究及其应用朝着理解的基础髓鞘的分子线索方面很有价值的技术。

Disclosures

没有利益冲突的声明。

Acknowledgments

该项目被授予一个从加拿大多发性硬化症协会RKRWO的资助,是一个从加拿大多发性硬化症协会的助学金的获得者。特别提款权是一种多发性硬化症协会从加拿大和加拿大卫生研究院的博士后奖学金获得者。

Materials

Name Company Catalog Number Comments
Dulbecco's Modified Eagle Medium (DMEM) Multicell 319-005-CL
Hank's Balanced Salt Solution (HBSS) Invitrogen 14170-112
Minimum Essential Media (MEM) GIBCO, by Life Technologies 12360-038
Fetal Bovine Serum (FBS) GIBCO, by Life Technologies 10091-148
Penicillin-Streptomycin (Pen/Strep) GIBCO, by Life Technologies 15140-122
GlutaMAX Invitrogen 35050-061
Poly-l-lysine Sigma-Aldrich P2636
Bovine Serum Albumin (BSA) Sigma-Aldrich A4503
Human merosin purified protein (LN2) EMD Millipore CC085
Recombinant rat ciliary neurotrophic factor (CNTF) PeproTech Inc 450-50
L-thyroxine Biochemika 89430
Holo-transferrin Sigma-Aldrich T0665
B27 supplement GIBCO, by Life Technologies 0080085-SA
Bovine insulin Sigma-Aldrich I6634
3,3',5-Triiodo-L-thyronine Sigma-Aldrich I6634
Progesterone Sigma-Aldrich P8783
Putrescine Sigma-Aldrich P7505
Sodium Selenite Sigma-Aldrich S5261
5-Fluoro-2'-deoxyuridine (FuDR) Sigma-Aldrich F0503
Papain solution Worthington Biochemical LS003126
DNaseI Roche Group 1010159001
L-cysteine Sigma-Aldrich C7352
24-well tissue culture dishes Cellstar 662-160
T25-tissue culture flasks with vent cap Corning 430639
10 cm tissue culture dishes Corning 430167
10 cm petri dish Fisher Scientific 0875713
Collagenase A Roche Group 103578
CellTrics 50 μm filter (optional) PARTEC 04-004-2327
Myelin Basic Protein (MBP) Antibody AbD Serotec MCA409S
NG2 antibody EMD Millipore AB5320
Myelin-Associated Glycoprotein (MAG) antibody EMD Millipore MAB1567
2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP) antibody Covance SMI-91R-100
Actin pan Ab-5 antibody Fitzgerald 10R-A106AX
Neurofilament-200 (NF) antibody Sigma-Aldrich N4142
Tubulin beta-3 chain (Tuj1) antibody EMD Millipore MAB5544
Alexa Fluor 488 goat anti-rabbit IgG (H+L) secondary antibody Invitrogen A11008
Alexa Fluor 555 goat anti-mouse IgG (H+L) secondary antibody Invitrogen A21422
Alexa Fluor 647 goat anti-rat (IgG) (H+L) secondary antibody Invitrogen A21247
Goat anti-mouse IgG (H+L)-HRP conjugated secondary antibody Bio-Rad 170-6516
Goat anti-rat IgG (H+L)-HRP conjugated secondary antibody Santa Cruz Biotechnology, Inc. SC-2065
4',6-diamidino-2-phenylindole (DAPI) Sigma-Aldrich D9542

Media Recipes

100X OL-Supplement*

IngredientAmount to add
DMEM100 mL
BSA1.02 g
Progesterone0.6 mg
Putrescine161 mg
Sodium Selenite0.05 mg
3,3',5-Triiodo-L-thyronine4 mg

*Store at -80 °C in 250 μL aliquots

OL media

IngredientAmount to add
DMEM23.75 mL
100X OL-Supplement250 μL
Bovine insulin (from 1 mg/mL stock)125 μL
GlutaMAX250 μL
Holo-transferrin (from 33 mg/mL stock)37.5 μL
B27 Supplement500 μL
FBS125 μL
CNTF (from 50 ng/μL stock)25 μL

Mixed glial culture media (made up in DMEM)

IngredientFinal concentration
FBS10%
Pen/Strep (0.33% from stock)33 units/mL Penicillin and 33 μg/mL Streptomycin
GlutaMAX1%

DRGN media (made up in DMEM)

IngredientFinal concentration
FBS10%
Pen/Strep (1% from stock)100 units/mL Penicillin and 100 μg/mL Streptomycin

Digestion Solution Recipes:

OPC papain solution (made up in MEM)

IngredientFinal concentration
Papain solution1.54 mg/mL
L-cysteine360 μg/mL
DNaseI60 μg/mL

DRG papain solution (made up in HBSS)

IngredientFinal concentration
Papain1.54 mg/mL
L-cysteine360 μg/mL

DRG Collagenase A solution (made up in HBSS)

IngredientFinal concentration
Collagenase A4 mg/mL

DOWNLOAD MATERIALS LIST

References

  1. Avellana-Adalid, V. Expansion of rat oligodendrocyte progenitors into proliferative "oligospheres" that retain differentiation potential. J Neurosci Res. 45, 558-570 (1996).
  2. McCarthy, K. D., de Vellis, J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol. 85, 890-902 (1980).
  3. Barres, B. A. Cell death and control of cell survival in the oligodendrocyte lineage. Cell. 70, 31-46 (1992).
  4. Chan, J. R. NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes. Neuron. 43, 183-1891 (2004).
  5. Camara, J. Integrin-mediated axoglial interactions initiate myelination in the central nervous system. J Cell Biol. 185, 699-712 (2009).
  6. Ishibashi, T. Astrocytes promote myelination in response to electrical impulses. Neuron. 49, 823-832 (2006).
  7. Chen, Y. Isolation and culture of rat and mouse oligodendrocyte precursor cells. Nat Protoc. 2, 1044-1051 (2007).
  8. Pedraza, C. E. Production, characterization, and efficient transfection of highly pure oligodendrocyte precursor cultures from mouse embryonic neural progenitors. Glia. 56, 1339-1352 (2008).
  9. Lewin, G. R., Ritter, A. M., Mendell, L. M. On the role of nerve growth factor in the development of myelinated nociceptors. J Neurosci. 12, 1896-1905 (1992).
  10. Greene, L. A. Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons. II. Sensory neurons. Dev Biol. 58, 106-113 (1977).

Comments

46 Comments

  1. very nice video and presentation....

    Reply
    Posted by: Anonymous
    December 28, 2011 - 12:44 PM
  2. Nice explanation. I have been carrying out this procedure for some time now and find that it works quite well.

    How many OPCs you generally obtain from a single mouse brain?

    Reply
    Posted by: Anonymous
    January 26, 2012 - 4:35 PM
  3. Hi - thanks for your comment, typically I yield about ²00,000 OPCs per mouse brain (on average).

    Reply
    Posted by: Anonymous
    February 2, 2012 - 1:04 AM
  4. Excellent. I am using B6 mice, usually P4-5 (because we also take cerebellar neurons from these same pups). I usually yield between 100-130,000 cells/brain. I see some differences in our protocols (I seed three brains/T75, use trypsin instead of papain, etc.). What steps have you found to dramatically increase your OPC yield post shake-off?

    Reply
    Posted by: Anonymous
    February 2, 2012 - 12:17 PM
  5. The largest factor in OPC yield is the efficiency of your digestion (ie. minimal cell death). In addition, the age of the mice from which you isolate the mixed glial cultures is a factor. I think if you go too far past P², many OPCs will be postmitotic, and therefore limit their proliferation capacity. I usually try to go as young as possible (P0) as this seems to work best. If you absolutely need to use P4-P5, maybe try to culture your monolayers longer than 9 days - try waiting ² weeks prior to shake off. Hope this helps - RO

    Reply
    Posted by: Anonymous
    February 2, 2012 - 1:06 PM
  6. Thanks for the great info. That is interesting, as many of the protocols for the "DeVellis-method" use different enzymatic preparations (trypsin +/- EDTA, +/- DNAase, papain, etc.). I had basically adapted my rat OPC protocol for mice, but am realizing that they are just not the same. I will trip your papain method and let you know how things pan out. Also, is there a reason you use T²5 flasks instead of combining brains into a T75?

    Reply
    Posted by: Anonymous
    February 2, 2012 - 4:55 PM
  7. No there is no specific reason for using T²5, simply this is what I have always been using. I would imagine combining 3 mice into a T75 flask would roughly be the same thing. Good Luck!

    Posted by: Anonymous
    February 7, 2012 - 12:00 PM
  8. Hi Ryan, nice video and explanation. Do you do lectin panning to further get rid of microglia or do you find that a 30 min incubation at 37 degrees is enough? Also, how do you know cells are ready to shake? are they always ready at day 9? what do you look for specifically?
    thank you

    Reply
    Posted by: luisa t.
    April 10, 2013 - 12:39 PM
  9. hello...I have gone through this video...very nicely presented...but I have one doubt that if I put the flasks on shaker which only maintains temp and speed but not CO² level, the pH will change...would it not allow the OPCs to survive at all??? and can I use HEPES in medium to solve this problem???
    M willingly waiting for your reply....

    Reply
    Posted by: Anonymous
    February 4, 2012 - 12:35 PM
  10. Thank you for your comment, you are correct in that it will not work without CO² buffering. HEPES buffer will likely not solve this problem either. I'm afraid you will absolutely need to have CO² buffering for this isolation to work.

    Reply
    Posted by: Anonymous
    February 7, 2012 - 11:56 AM
  11. Hi, very INFORMATIVE video, I zalak parikh working on glial cells. In your protocol you are not using any filter after papain digestion but I am facing one problem of Fibroblasts contamination. What could be the possible solution for this problem?
    thank you
    Zalak.

    Reply
    Posted by: Anonymous
    February 24, 2012 - 7:59 AM
  12. Hi Zalak, thanks for your comment. What glial cell type are you trying to culture? Oligodendrocytes, astrocytes or microglia? I have never experienced fibroblast contamination.. are you sure they are fibroblasts?

    Reply
    Posted by: Anonymous
    February 24, 2012 - 10:47 AM
  13. Hi there. Excellent video, very informative. I was just wondering if you can incubate the flasks in 5% CO² instead of 8% CO²?
    Thanks
    Matt

    Reply
    Posted by: Anonymous
    April 11, 2012 - 12:48 PM
  14. Hi Matt, Actually no, you need to have these at at least 8.5% CO² - otherwise you will have trouble establishing your mixed glial cultures - Good luck

    Reply
    Posted by: Anonymous
    April 11, 2012 - 10:22 PM
  15. Hi Ryan. Very nice video and presentation.
    I have to establish a DRG neuronal culture. Majority of all the papers I've read are using a embryonic source of DRG unlike you using post-natal mouse. What do you think will be the major difference between the cultures of the two??

    Reply
    Posted by: Lipi B.
    October 1, 2012 - 12:23 PM
  16. Hi Lipi,
    You're right most papers use embryonic DRGs. The benefit of using post-natal DRGs is that you don't have to sacrifice the mother, and if you use P5-P10 mice, there is no need to use nerve growth factor (in my hands). Embryonic DRG neurons require NGF supplementation to the media for their survival. In terms of the difference between cultures, embryonic dervied ones may have a bit more contaminating cells, since I find they divide pretty quickly. Otherwise I imagine they would be the same as post-natal derived cultures. Good luck!

    Reply
    Posted by: Ryan O.
    October 1, 2012 - 4:37 PM
  17. Thank you Ryan.

    Reply
    Posted by: Lipi B.
    October 3, 2012 - 2:39 AM
  18. Thank you Ryan.

    Reply
    Posted by: Lipi B.
    October 3, 2012 - 2:51 AM
  19. Great article Ryan. I had a question for you. Our lab generates mixed glial cell cultures by isolating NPCs from P1 mouse pup brains,expanding the NPCs via neurospheres in the presence of GFs, and then plating the spheres on matrigel for subsequent differentiation without GFs. My main goal in using the neurosphere protocol is to obtain OPCs, but I routinely get only ²0-30% O1+ OPCs following 8 days of differentiation on matrigel. GFAP+ astrocytes seem to dominate the whole culture, with OPCs growing on top of them. I wanted to try to incorporate your protocol into the neurosphere protocol by dissociating the spheres using TrypLE or Accutase, plating them on Poly-D-Lysine, letting the astrocytes grow to confluency while having OPCs proliferate on top, and then use the shaking technique to isolate OPCs. Do you think this is possible? When we perform in vitro studies with the NPCs, we usually plate the neurospheres on matrigel for a day and then trypsinize the cells for seeding into new dishes. Not only do I think the trypsin digest kills many OPCs, but I also remove astrocytes from the matrigel that then contaminate my cultures. Thanks for the help!

    Reply
    Posted by: Brett M.
    November 3, 2012 - 7:37 PM
  20. Hi Brett - To answer your question, yes I think it would be possible to do what you are suggesting. However, it seems to be a pretty roundabout approach. I think the more streamlined approach would be to simply seed your dissociated P1 neural cells onto PLL substrate to generate a monolayer. That way you would avoid having to generate neurospheres (which I imagine takes about 10 days) and then subsequently generating an astrocyte monolayer with the spheres (which would take another 10+ days). In my experience, OPCs don't like trypsin. If you wanted, you could try 0.05% Trypsin instead of 0.²5% and that seems to be more gentle. And if you don't incubate for too long, most astrocytes should remain on the plate. Hope this helps! -Ryan

    Reply
    Posted by: Ryan O.
    November 5, 2012 - 12:14 PM
  21. Ryan, thanks for the help! I tried your protocol and everything appeared to go well. However, one issue I ran into occurred during the overnight shaking step and subsequent differential adhesion assay to isolate a more pure population of OPCs. What I noticed was that after the overnight shaking I had many large oligospheres (maybe 10-²0 cells in size). These spheres did not stick down during the 30 minute plate incubation and I had difficulties trying to dissociate them as you described using the P1000 and P²00 tips. When I took the cells counts and plated the cells onto chamber slides, many smaller clumps remained and stuck down to the matrix. However, the clumps did not flatten out on the gel matrix very well (like neurospheres do) and eventually flaked off after about 5 days of cultures, leaving only sparse population of OPCs. Have you ever encountered this problem? I was thinking of seeding at a higher cells density than what you describe in the protocol.

    Reply
    Posted by: Brett M.
    December 2, 2012 - 10:21 PM
  22. Hi Brett - sounds like you're doing everything right. Normally I see lots of these "oligospheres" as a result of the overnight shaking protocol. However, I find that these are not the majority of OPCs, as there are many single cells and cells in clumps of ²-4. The oligospheres are difficult to dissociate, and due to this, I generally do not go out of my way to try and break them up. In my hands, these spheres spread out very nicely after about ²4hrs on Laminin-² substrate. You can try to seed at higher densities if you like, but the seeding density should be determined based on the experiment you want to conduct. For example, if you want to perform immunocytochemistry, it may not be ideal to have a very dense cell culture. Also, it's possible the clumps aren't super compatible with your matrix substrate - I always use Laminin. I hope this helps :)

    Reply
    Posted by: Ryan O.
    December 3, 2012 - 11:42 AM
  23. HI Ryan, very informative and nice presentation. I have tried your protocol and everything seem to work fine except at the overnight shaking. There were very few OPCs isolated and it seems there are still many OPCs adhere to the astrocytes. Have you encounter this issue?

    Reply
    Posted by: Esther W.
    December 13, 2012 - 3:25 AM
  24. Hi Esther, Thanks for your comment. How many OPCs are you yielding per mouse? I usually yield on average ²00,000 OPCs per mouse brain, but this is quite variable. At times I have yielded 500,000 or 50,000 per brain, and I'm not sure what accounts for this variability. In any case, you will never dissociate 100% of the OPCs from the monolayer, I've noticed a lot remain stuck. My advice would be to simply give it another shot and see if your yields are a bit higher next time. Hope this helps - RO

    Reply
    Posted by: Ryan O.
    December 16, 2012 - 5:35 PM
  25. Thanks Ryan for your advice. My yield is definitely much lower than yours. I will definitely try another round. :)

    Reply
    Posted by: Esther W.
    December 16, 2012 - 8:24 PM
  26. HI Ryan, I face the problem of getting immature OPC instead of mature OPC after 6 days of OL-enriched culture. What is your view?

    Reply
    Posted by: Esther W.
    January 23, 2013 - 2:51 AM
  27. Hi Esther, you should always expect to have some OPCs that don't differentiate during the timecourse. However, the majority of OLs should at least be expressing MAG, if not MBP at DIV6. My guess would be that your media is not exactly correct. Make sure that all the ingredients are at the correct concentration in your 100x OL-supplement stock - especially tri-iodothyronine and L-thyroxine (4mg per 100mL). I hope this helps, let me know if you still have problems.

    Posted by: Ryan O.
    January 23, 2013 - 12:56 PM
  28. Thanks Ryan for replying. I have added tri-iodothyronine only as there is no mentioned of L-thyronine in the 100x OL-supp recipe. Can i still add L-thyronine when the OL-enriched is on DIV5?

    Posted by: Esther W.
    January 23, 2013 - 8:05 PM
  29. Thanks for notifying me of this mistake, I'll notify the editorial team ASAP. Yes you can try to add it on DIV5, but no guarantees this will drive them any farther since they are aleady 5 days in. Give the protocol a second try, and let me know if the lack of differentiation still occurs. -Ryan

    Reply
    Posted by: Ryan O.
    January 24, 2013 - 12:47 AM
  30. Hi Luisa thanks for your comment :) I don't do lectin panning, as I find that incubation on the culture plates is enough to get rid of most of them, but if you're having a problem with microglia that is an additional option. I usually shake on day 9, but I've found you can shake anywhere from 9 to 1² days, but any longer than that you will get less OPCs. Generally if you see a lot of cells on top of the astrocyte monolayer, they are ready to go. However in my experience this dŒsn't really happen before day 9. Hope this helps RO

    Reply
    Posted by: Ryan O.
    April 10, 2013 - 2:28 PM
  31. Hi Ryan.Thanks for this protocol. I did exactly what you have described in this protocol, except the shaking step. I did it without CO2, but the caps are tightly sealed, how is it possible that CO2 will effect pH of tightly sealed flask? My yield was very low, besides I had fibroblast contamination as well. I used uncoated flasks as well, because once during the shaking all the layer came off the bottom of the flask, what do you think would be the reason for this?.And in my experience when FBS is used in OPC culture media it made OPC's to differentiate to astrocytes rather then oligodendrocytes.Overall I couldnt replicate this procedure unfortunately and trying to find other methods..

    Reply
    Posted by: Aysel J.
    February 20, 2014 - 5:33 PM
  32. Hi Aysel - from what you describe in your message, you didn't follow my protocol.

    1) So you didn't shake the flasks? Or you didn't use the same method of shaking?

    2) As far as I know, all standard tissue culture incubators have some level of CO2 buffering (usually 5%). Strictly speaking, if you tighten the cap of your flasks, there will be no gas exchange between the incubator and the flask, if you are using plugged caps. I use vent-capped flasks (0.22um pore), so gas exchange occurs between the flask and the incubator when the caps are tight. To answer your question, CO2 buffering will have little-to-no effect on the pH of the media if you are using tightly-screwed plugged flask caps.

    3) I haven't experienced fibroblast contamination in my cultures, I imagine this would be a dissection issue. The only source of fibroblasts I can imagine would be from the meninges, so be sure to fully remove this prior to dissociation of the cortex. Unless you are confusing fibroblasts for astrocytes. Too many astrocytes is a result of too much agitation at the differential adhesion step.

    4) I have always used coated flasks, in my experience OPCs will not adhere to uncoated surfaces. I am willing to bet you'll get almost no OPCs if you continue to use uncoated flasks. If you want you can try to use other substrates such as PDL, poly-O-ornithine, collagen etc.

    5) I haven't noticed any significant transformation of OPCs into astrocytes in the presence of FBS. I imagine this might occur more frequently in immortalized OPC cell lines. Perhaps that's what you are thinking of.

    I hope this helps, otherwise best of luck in your research.

    Reply
    Posted by: Ryan O.
    February 20, 2014 - 8:52 PM
  33. Hi Ryan, thank you ver much for detailed answer. Sorry for my english, i couldnt explain good.
    I did the shaking of course but without putting shaker inside the CO2 incubator, I did shaking at 37 degree incubation only and at 180 rpm. Because the flasks are uncoated(I have used uncoated flasks for rat OPC"s without problem, that is why I tried this way with mouse as well) I hesitated to increase rpm to 240 or so, to prevent astrocyte detachment, and I also think that fibroblast contamination is because of the meninges. Second time I will prevent it.
    It is good to know that I must use coated flasks, I will try again, if you say so.
    Thank you so much, good luck to too..

    Reply
    Posted by: Aysel J.
    February 21, 2014 - 6:22 AM
  34. Hi Again,
    Alright - I strongly suggest to put the shaker in the incubator for the overnight shake, as I have found this to be a crucial step in maintaining pH balance. The media on your cells should always be a rich red colour (indicative of a balanced pH) all the time. Mouse OPCs are very sensitive to pH, and will die with too much fluctuation. If during your shaking, the media becomes at all purple-ish or yellow-ish, your OPC yield will drastically reduce. I wouldn't be surprised either if this was a contributing factor to the monolayer detachment you experienced previously. There is a significant difference in the in vitro behaviour/viability of mouse OPCs as compared to rat, hence the generation of this protocol. Your best bet is to simply follow this protocol exactly as is.

    Thanks for your comment, and good luck,
    Ryan

    Reply
    Posted by: Ryan O.
    February 21, 2014 - 12:25 PM
  35. Hi.
    One more question Ryan, about amount of the brains seeded, I was seeding 2 rat brains/ T75 flask , in the mouse case to obtain same amount of cells seeded I was using 6 brains per flask. Would you suggest to use 3-4 instead of six? and if i seed 6 animals should I increase concentration of insulin added to mixed culture media? Overall what would be optimum amount of brains per T75 flask in your opinion? Does it matter to much? Because here you use 1 brainper T25 flask.
    Thanks again..

    Reply
    Posted by: Aysel J.
    February 26, 2014 - 4:20 PM
  36. Hi Aysel,
    Again, your best bet for success is to follow the protocol exactly as is - T25 flasks are what this method has been optimized for. I suppose T75 flasks have 3 times the surface area, so in theory 3 mice would have to be seeded to a T75 to yield the same cell density. I've never tried this protocol with T75 flasks, and I know that different culture vessels have different impact on growth and survival of OLs.
    Best - RO

    Reply
    Posted by: Ryan O.
    February 27, 2014 - 10:47 AM
  37. Hi Ryan . I want to say that I replicated the procedure exactly as here, and have got very good yield, but I have 1 more question to you, majority of OPCs die in the differentiation media after 6-7 days of incubation. You indicated that it is important not to distrub them until fixation, but how do you do the medium chage, I put 1 ml media first day and did 2/3 media change each 3 days, I seeded all cells to one 24 well format plate, at each time points(3 d, 6 d, 9 d) I transfer the slides to new plate and fix them there and do medium change for rest of the slides. By the way I culture OPCs at 5% CO2, becaue I have only 2 options 5% and 10%, i cant adjust ph of the incubators at this time point. Is it the contributing factor for death of the cells after 4-5 days of incubation?

    Reply
    Posted by: Aysel J.
    March 19, 2014 - 2:43 PM
  38. Hi Aysel - I'm glad to hear things are looking up -

    1. I don't suggest differentiating the OLs for longer than 6 days. If you're finding they are not differentiating fast enough, you can add 4 mg of L-Thyroxine to the 100X OL-supplement (see the media recipe list above).

    2. It is normal to lose OLs over the time course of differentiation. That being said, you don't want to encourage death by moving the cells or culturing at low pH. You should not be changing the media of the purified OL cultures at any point - they do not require media changes. The protocol also says to culture the OLs at 8.5% CO2. Since you have access to both 5 and 10% CO2 incubators, and 8.5 is closer to 10 than it is to 5, I suggest culturing them at 10%.

    3. Rather than culturing all of your time points in one dish, use 3 dishes (one for 3 days, one for 6 etc). That way, you won't have to disturb your other time points.

    4. Do not remove the coverslips from the wells of your 24 well dish during fixation - simply add concentrated PFA directly into the media and allow to fix for about 15 mins at room temp.

    Sounds like you're almost there, Best of luck - Ryan

    Reply
    Posted by: Ryan O.
    March 19, 2014 - 3:58 PM
  39. Let me write it again to see if I have understood you correct, you say dont do any media change ever, neither at 3 days or 6 days or further? The other things I will try what you suggest, culturing and seeding in different plates at 10% CO2
    I want to thank you a lot for your help and all of the explanations.
    Thanks ..
    Best,
    Aysel

    Reply
    Posted by: Aysel J.
    March 19, 2014 - 4:22 PM
  40. Thats correct :)
    You're very welcome,

    Ryan

    Posted by: Ryan O.
    March 19, 2014 - 5:49 PM
  41. Hi Ryan,

    Thanks for sharing this protocol, looks really good and will try it out. I have a couple of questions regarding your protocol and was hoping you could shed some light on some problem I am experiencing at the moment.

    With regards to what you have just described, we actually perform a very similar mixed glial culture on P0-P3 rat brains. Our isolation stages are very similar with the exception we use DNAse1 and L-cysteine along with Papain. Upon plating our cell suspensions the flasks are densely populated with microglia on top, then opc's below and then astrocytes. We basically shake off our microglia before doing the overnight shake for the OPC's.

    My question is how do you get rid/ not see any microglia in your mouse cultures? Is it also possible to obtain microglia from your method?

    We are currently experiencing problems with obtaining OPC's from our overnight shake (in rat). The cells look healthy when in culture with the microglia but once the microglia are removed and we perform the steps required to plate the OPC's we have a lot of contaminants in with the cells, almost like long fibrous structures that are stained by trypan blue. We still obtain + 5 million OPC's but the cells do not look healthy and the morphology is not consistent with OPC's. Upon plating these cells they fail to show characteristics of OPCs and a lot die off. I will mention we do not shake in an incubator but in an orbital shaker at 250rpm with the caps sealed tight and parafilm. Have you ever encountered this?

    Many thanks .

    Reply
    Posted by: Graeme I.
    February 9, 2015 - 1:06 PM
  42. Hi Graeme, thanks for your question.
    We do in fact get microglia in our mixed cultures, but we remove them prior to the overnight shake with a slow short shake (as in the rat method). These shaken off microglia are a very pure population, and can be cultured separately if desired.
    With regard to the problems you’re encountering shaking your rat cultures, I can’t say I’ve experienced anything quite like what you’re describing. It almost sounds like your cultures are suffering from contamination (maybe yeast?). We’ve experienced contamination a couple of times, and it resulted in much of the monolayer dissociating from the base of the flask during the overnight shake. If you like, you can send me a pic of the fibrous structures in the hopes that I could recognize what they are (romea077@uottawa.ca).
    We have never had luck shaking flasks in an orbital shaker – we exclusively use tissue culture incubators with vented caps. One risk of using an orbital shaker is they pose a threat for contamination, as they are often used for bacterial cultures.
    I hope this helps and best of luck,
    Ryan

    Reply
    Posted by: Anonymous
    February 20, 2015 - 11:12 AM
  43. Hi Ryan,

    Thanks for your reply.

    I tried the protocol and achieved very nice OPC's so everything seems ok there.

    With regards to what I was seeing after my rat OPC shake, I believe it not to be of any contamination but more something coming out of solution in our media. As a test I shook DMEM10% in a T75 flask, 37 degrees, 250rpm overnight. The next day I took an aliquot and looked at it with trypan blue. To my surprise I saw the same fibrous clumps that I was seeing after the OPC shake

    . My DMEM only contains FBS and pen-step so this has to be something in FBS. My most recent shake off I decided to shake off 5 flasks with DMEM 10% and 5 flasks with OPC media (our OPC media contains 2.5ml FBS vs 50ml in DMEM). The next day after following normal protocol I counted the cells from both conditions. The OPC media flasks had almost none of the fibrous clumps whereas the DMEM flasks had alot. I can now partially get round this problem by filtering with 40uM strainer but have you ever heard or seen this kind of thing. We have used several batches if FBS so it cannot be that every batch has precipitated proteins in it but perhaps what were doing is causing precipitation of FBS proteins that are affecting the OPC's?

    Thanks

    Graeme

    Reply
    Posted by: Graeme I.
    March 15, 2015 - 4:16 PM
  44. Hi Ryan,

    Is your FBS from Invitrogen decomplemented ? If so did you buy decomplemented or did you heat it at 56*C and for how long.

    Thanks

    Reply
    Posted by: Ludovic D.
    February 24, 2017 - 10:40 AM
  45. Hi Ludovic,
    I've used FBS that has been heat-inactivated by the manufacturer, and I've also used FBS that I've heat inactivated myself. For the latter, if memory serves, about 30 minutes at roughly 50 degrees C should do the trick (water bath).
    Good luck, Ryan

    Reply
    Posted by: Anonymous
    February 26, 2017 - 11:49 AM
  46. Hi Ryan,
    I am wondering if you have any experience with adding Cre Recombinase Adenovirus to the cultures? And if you think a particular day would be best to add it? I was thinking of adding it on D8 (1 day before the full media change) but wasn't sure if adding virus and then shaking the cells would cause too much stress?
    Thanks,
    Megan

    Reply
    Posted by: Megan R.
    April 30, 2018 - 2:39 PM

Post a Question / Comment / Request

You must be signed in to post a comment. Please or create an account.

Usage Statistics