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。

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	<li>Avellana-Adalid, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+rat+oligodendrocyte+progenitors+into+proliferative+"oligospheres"+that+retain+differentiation+potential.">Expansion of rat oligodendrocyte progenitors into proliferative "oligospheres" that retain differentiation potential.</a> J Neurosci Res. 45, 558-570 (1996).</li><li>McCarthy, K. D., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+separate+astroglial+and+oligodendroglial+cell+cultures+from+rat+cerebral+tissue.">Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.</a> J Cell Biol. 85, 890-902 (1980).</li><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+and+control+of+cell+survival+in+the+oligodendrocyte+lineage.">Cell death and control of cell survival in the oligodendrocyte lineage.</a> Cell. 70, 31-46 (1992).</li><li>Chan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGF+controls+axonal+receptivity+to+myelination+by+Schwann+cells+or+oligodendrocytes.">NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes.</a> Neuron. 43, 183-1891 (2004).</li><li>Camara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-mediated+axoglial+interactions+initiate+myelination+in+the+central+nervous+system.">Integrin-mediated axoglial interactions initiate myelination in the central nervous system.</a> J Cell Biol. 185, 699-712 (2009).</li><li>Ishibashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+promote+myelination+in+response+to+electrical+impulses.">Astrocytes promote myelination in response to electrical impulses.</a> Neuron. 49, 823-832 (2006).</li><li>Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+and+mouse+oligodendrocyte+precursor+cells.">Isolation and culture of rat and mouse oligodendrocyte precursor cells.</a> Nat Protoc. 2, 1044-1051 (2007).</li><li>Pedraza, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production,+characterization,+and+efficient+transfection+of+highly+pure+oligodendrocyte+precursor+cultures+from+mouse+embryonic+neural+progenitors.">Production, characterization, and efficient transfection of highly pure oligodendrocyte precursor cultures from mouse embryonic neural progenitors.</a> Glia. 56, 1339-1352 (2008).</li><li>Lewin, G. R., Ritter, A. M., Mendell, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+role+of+nerve+growth+factor+in+the+development+of+myelinated+nociceptors.">On the role of nerve growth factor in the development of myelinated nociceptors.</a> J Neurosci. 12, 1896-1905 (1992).</li><li>Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+vitro+studies+on+the+nerve+growth+factor+(NGF)+requirement+of+neurons.+II.+Sensory+neurons.">Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons. II. Sensory neurons.</a> Dev Biol. 58, 106-113 (1977).</li></ol>

没有利益冲突的声明。

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。

<html><table border="1"><tbody><tr><td> 产品名称 </td><td> 公司 </td><td> 产品编号 </td></tr><tr><td>贝科改良Eagle培养基（DMEM） </td><td>多节</td><td> 319 - 005 - CL </td></tr><tr><td>汉克的平衡盐溶液（HBSS） </td><td> Invitrogen公司</td><td> 14170-112 </td></tr><tr><td>最小的重要媒体（MEM） </td><td> GIBCO公司</td><td> 12360-038 </td></tr><tr><td>胎牛血清（FBS） </td><td> GIBCO公司</td><td> 10091-148 </td></tr><tr><td>青霉素，链霉素（PEN /链球菌） </td><td> GIBCO公司</td><td> 15140-122 </td></tr><tr><td> GlutaMAX </td><td> Invitrogen公司</td><td> 35050-061 </td></tr><tr><td>聚- L -赖氨酸</td><td> Sigma - Aldrich公司</td><td> P2636 </td></tr><tr><td>牛血清白蛋白（BSA） </td><td> Sigma - Aldrich公司</td><td> A4503 </td></tr><tr><td>人力merosin纯化蛋白（液氮） </td><td> Millipore公司</td><td> CC085 </td></tr><tr><td>重组大鼠睫状神经营养因子（CNTF） </td><td> PeproTech </td><td> 450-50 </td></tr><tr><td> L -甲状腺素</td><td> Biochemika </td><td> 89430 </td></tr><tr><td>全息转</td><td> Sigma - Aldrich公司</td><td> T0665 </td></tr><tr><td> B27的补充</td><td> GIBCO公司</td><td> 0080085 - SA </td></tr><tr><td>牛胰岛素</td><td> Sigma - Aldrich公司</td><td> I6634 </td></tr><tr><td> 3,3&#39;,5 -三碘- L -甲状腺素</td><td> Sigma - Aldrich公司</td><td> I6634 </td></tr><tr><td>孕酮</td><td> Sigma - Aldrich公司</td><td> P8783 </td></tr><tr><td>腐胺</td><td> Sigma - Aldrich公司</td><td> P7505 </td></tr><tr><td>亚硒酸钠</td><td> Sigma - Aldrich公司</td><td> S5261 </td></tr><tr><td> 5 - 氟-2&#39; - 脱氧尿苷（FUDR） </td><td> Sigma - Aldrich公司</td><td> F0503 </td></tr><tr><td>木瓜蛋白酶溶液</td><td>沃辛顿</td><td> LS003126 </td></tr><tr><td> DNaseI </td><td>罗氏</td><td> 1010159001 </td></tr><tr><td> L -半胱氨酸</td><td> Sigma - Aldrich公司</td><td> C7352 </td></tr><tr><td> 24孔组织培养皿</td><td>蜂星</td><td> 662-160 </td></tr><tr><td>组织培养瓶发泄第T25 - </td><td>康宁</td><td> 430639 </td></tr><tr><td> 10厘米的组织培养皿</td><td>康宁</td><td> 430167 </td></tr><tr><td> 10厘米的培养皿</td><td> Fisher Scientific则</td><td> 0875713 </td></tr><tr><td>胶原酶一个</td><td>罗氏</td><td> 103578 </td></tr><tr><td> CellTrics 50微米过滤器（可选） </td><td> PARTEC </td><td> 04-004-2327 </td></tr><tr><td>髓鞘碱性蛋白（MBP）抗体</td><td> ABD Serotec </td><td> MCA409S </td></tr><tr><td> NG2抗体</td><td> Millipore公司</td><td> AB5320 </td></tr><tr><td>髓鞘相关糖蛋白（MAG）抗体</td><td> Millipore公司</td><td> MAB1567 </td></tr><tr><td> 2&#39;，3&#39; -环核苷酸的3&#39; -磷酸二酯酶（CNP）的抗体</td><td> Covance公司</td><td> SMI - 91R - 100 </td></tr><tr><td>肌动蛋白泛AB - 5抗体</td><td>杰拉德</td><td> 10R - A106AX </td></tr><tr><td>神经丝- 200（NF）抗体</td><td> Sigma - Aldrich公司</td><td> N4142 </td></tr><tr><td>微管蛋白β- 3链（Tuj1）抗体</td><td> Millipore公司</td><td> MAB5544 </td></tr><tr><td>的Alexa Fluor 488山羊抗兔IgG（H + L）二级抗体</td><td> Invitrogen公司</td><td> A11008 </td></tr><tr><td>的Alexa Fluor 555山羊抗小鼠IgG（H + L）二级抗体</td><td> Invitrogen公司</td><td> A21422 </td></tr><tr><td>的Alexa Fluor 647山羊抗大鼠（IgG抗体）（H + L）二级抗体</td><td> Invitrogen公司</td><td> A21247 </td></tr><tr><td>羊抗鼠IgG（H + L）HRP标记二抗</td><td> BioRad公司</td><td> 170-6516 </td></tr><tr><td>羊抗鼠IgG（H + L）HRP标记二抗</td><td>圣克鲁斯生物技术</td><td> SC - 2065 </td></tr><tr><td> 4&#39;,6 - diamidino 2 -苯基吲哚（DAPI） </td><td> Sigma - Aldrich公司</td><td> D9542 </td></tr></tbody></table>媒体食谱 100X的OL -补编 <table border="1"><tbody><tr><td> 成分 </td><td> 添加的金额</td></tr><tr><td> DMEM培养液</td><td> 100毫升</td></tr><tr><td>牛血清白蛋白</td><td> 1.02摹</td></tr><tr><td>孕酮</td><td> 0.6毫克</td></tr><tr><td>腐胺</td><td> 161毫克</td></tr><tr><td>亚硒酸钠</td><td> 0.05毫克</td></tr><tr><td> 3,3&#39;,5 -三碘- L -甲状腺素</td><td> 4毫克</td></tr></tbody></table> *贮存于-80 ° C在250μL，分装T“&gt; OL媒体 <table border="1"><tbody><tr><td> 成分 </td><td> 添加的金额</td></tr><tr><td> DMEM培养液</td><td> 23.75毫升</td></tr><tr><td> 100X的OL补充</td><td> 250μL </td></tr><tr><td>牛胰岛素（1毫克/毫升的股票） </td><td> 125微升</td></tr><tr><td> GlutaMAX </td><td> 250μL </td></tr><tr><td>全息转（从33毫克/毫升的股票） </td><td> 37.5微升</td></tr><tr><td> B27的补充</td><td> 500μL， </td></tr><tr><td>胎牛血清</td><td> 125微升</td></tr><tr><td>睫状神经营养因子（从50毫微克/μL股票） </td><td> 25μL </td></tr></tbody></table> 混合胶质文化传媒（在DMEM） <table border="1"><tbody><tr><td> 成分 </td><td> 终浓度 </td></tr><tr><td>胎牛血清</td><td> 10％ </td></tr><tr><td> PEN /链球菌，从股票（0.33％） </td><td> 33单位/ ml青霉素和33μg/ mL链霉素</td></tr><tr><td> GlutaMAX </td><td> 1％ </td></tr></tbody></table> DRGN媒体（在DMEM） <table border="1"><tbody><tr><td> 成分 </td><td> 终浓度 </td></tr><tr><td>胎牛血清</td><td> 10％ </td></tr><tr><td> PEN /链球菌（从1％的股份） </td><td> 100单位/ ml青霉素和100μg/ mL链霉素</td></tr></tbody></table>消解液配方： OPC木瓜蛋白酶溶液（在MEM） <table border="1"><tbody><tr><td> 成分 </td><td> 终浓度 </td></tr><tr><td>木瓜蛋白酶溶液</td><td> 1.54毫克/毫升</td></tr><tr><td> L -半胱氨酸</td><td> 360微克/毫升</td></tr><tr><td> DNaseI </td><td> 60微克/毫升</td></tr></tbody></table> DRG的木瓜蛋白酶溶液（在HBSS中） <table border="1"><tbody><tr><td> 成分 </td><td> 终浓度 </td></tr><tr><td>木瓜蛋白酶</td><td> 1.54毫克/毫升</td></tr><tr><td> L -半胱氨酸</td><td> 360微克/毫升</td></tr></tbody></table> DRG的胶原酶A溶液（HBSS在） <table border="1"><tbody><tr><td> 成分 </td><td> 终浓度 </td></tr><tr><td>胶原酶一个</td><td> 4毫克/毫升。 </td></tr></tbody></table>

derivation of enriched oligodendrocyte cultures and oligodendrocyte/neuron myelinating co-cultures from post-natal murine tissues

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。

Watch this Scientific Journal Video about Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues at JoVE.com

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

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。

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

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

derivation-enriched-oligodendrocyte-cultures-oligodendrocyteneuron

Research

JoVE Journal

Neuroscience

36.0K 次观看.  Ottawa Hospital Research Institute. 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。

视频: Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

生成高度丰富的神经文化皮层组织块冷冻保存

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

科研

神经科学

Derivation of Glial Restricted Precursors from E13 mice

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

从E13小鼠胶质限制前体的推导

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

来自祖，人脑少突胶质细胞培养系统

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

同时前和突触后电生理记录<em&gt;爪蟾</em神经肌肉文化

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

神经元共培养与单细胞精度的制备

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

器官切片文化研究少突胶质细胞动力学和髓鞘

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

调查器官脊髓联合培养物中生长多电极阵列功能再生

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of Parkinson&#39;s disease is not pathologically confirmed in up to 25% of clinically diagnosed Parkinson&#39;s disease. Therefore, tissue collected from clinically diagnosed patients with idiopathic Parkinson&#39;s disease can have a high rate of misdiagnosis; hence in vitro studies from such tissues to study Parkinson&#39;s disease as a preclinical model can become futile.
By collecting postmortem human leptomeninges with a confirmed neuropathological diagnosis of Parkinson&#39;s disease and characterized by nigrostriatal cell loss and intracellular protein inclusions called Lewy bodies, one can be certain that clinically observed parkinsonism is not caused by another underlying disease process (e.g. tumor, arteriosclerosis).
This protocol presents the dissection and preparation of postmortem human leptomeninges for derivation of a meningeal fibroblast culture. This procedure is robust and has a high success rate. The challenge of the culture is sterility as the brain procurement is generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B.
The derivation of meningeal fibroblasts from autopsy-confirmed cases with Parkinson&#39;s disease provides the foundation for in vitro modeling of Parkinson&#39;s disease. Meningeal fibroblasts appear 3-9 days after sample preparation and about 20-30 million cells can be cryopreserved in 6-8 weeks. The meningeal fibroblast culture is homogenous and the cells express fibronectin, a commonly used marker to identify meninges.

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

从尸检人脑软脑膜捐助外植体培养的推导

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which macrophages are induced to produce conditioned medium (CM) that promotes neurite outgrowth. Adult dorsal root ganglion (DRG) neurons are acutely dissociated and plated. After the neurons are stably attached, peritoneal macrophages are co-cultured on a cell culture insert overlaid on the same well. Dibutyryl cyclic AMP (db-cAMP) is applied to the co-cultures for 24 h, after which the cell culture insert containing the macrophages is moved to another well to collect CM for 72 h. The CM from the co-cultures treated with db-cAMP, when applied to a separate adult DRG neuron culture, exhibits robust neurite outgrowth activity. The CM obtained from the db-cAMP-treated cultures consisting of single cell type alone, either DRG neuron or peritoneal macrophage, did not exhibit neurite outgrowth activity. This indicates that the interaction between neurons and macrophages is indispensable for the activation of macrophages secreting molecular factors with neurite outgrowth activity into CM. Thus, our co-culture paradigm will also be useful to study intercellular signaling in the neuron-macrophage interaction to stimulate the macrophages to be endowed with a pro-regenerative phenotype.

The current protocol presents experimental procedures to stimulate cultured macrophages to be endowed with capacity to release molecular factors that promote neurite outgrowth. Treatment of cAMP to the neuron-macrophage co-cultures induces the macrophages to produce conditioned medium that possesses strong neurite outgrowth activity.

神经元-巨噬细胞共培养活化巨噬细胞分泌突起活性的分子因子

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

髓鞘有机小脑片培养的制备及免疫染色

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...