Name: culturing human pluripotent and neural stem cells in an enclosed cell culture system for basic and preclinical research
Uploaded: 2016-06-10T13:00:00.000+00:00
Duration: 8 min 5 s
Description: Watch this Scientific Journal Video about Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research at JoVE.com

作者要感谢在Biospherix工作人员，他们在学习使用Xvivo封闭的细胞培养系统，尤其是马特·弗里曼帮助;的Miles＆凯利建筑公司，公司为他们建立实验室基础设施建设，尤其是拉斯·休斯的工作人员;设施和支持服务工作的奥兰治县科儿童医院在协调实验室改造，尤其是亚当Lukhard和德文Hugie的人员;信息系统为他们建立的数据管理基础设施和远程访问，特别是越南陈德良帮助的奥兰治县科儿童医院的工作人员;奥兰治县执行管理团队的儿童医院为他们的长期支持该项目，特别是玛丽亚博士米农和布伦特Dethlefs的。这项工作是由橙县儿童医院和加州理工学院再生Medicin资助e到补助TR3-05476小灵通。所有作者同样促成了这一工作。

1.初始设置<ol><li>设置气体和温度浓度<ol><li>使用该软件，点击位于图形界面左上角的"来宾"选项卡。登录到使用指定的用户名和密码的系统。确保每个用户都有自己的用户名和密码。 </li><li>单击一个模块来调整（ 图1）。在显示当前设置的新窗口中，点击下面的"设置点"现有的O 2的设定点值并输入该模块所需的O 2浓度水平。输入5％ 的 O 2，如果多能干细胞将生长或此模块中操纵，和9％ 的 O 2，如果神经干细胞将生长或操纵。点击绿色的对勾确认设定点。 注:两个模块没有气体可调:层状罩，并在显微镜室。在层流罩的气体的浓度是大气控制而Ë那些在显微镜室中被动地由在处理室中的气体浓度保持。 </li><li>重复此步骤的CO 2，输入5％的CO 2的设定点为除缓冲室，这是只为O 2可调整的所有模块。 </li><li>监视当前气体浓度，其被标记为"过程值"，以确保它们到达新的设定点。 </li><li>调整所有模块的气体设定点适当的值。匹配的将要暴露于彼此室的CO 2和O 2的水平。例如，调整处理腔室至5％ 的 O 2，5％氧气2.打开，生长的细胞培养箱之前也调整到5％ 的 O 2，用于在此期间将项目添加到处理腔室的任何缓冲室。 </li><li>使用相同的画面气体调整处理室的温度设定为37℃。还设置了工艺室˚Floor温度至37℃。 </li><li>点击孵化模块库，每个孵化器的下面。调整银行的温度至37℃。 </li></ol></li><li>钱伯斯缓冲区操作<ol><li>收集所有对于在开始给定任务（馈送，劈裂，染色等 ），以防止在工作流程的延迟所需要的用品。宽松喷所有材料，用70％的乙醇，并允许他们在层流罩干燥。不要喷瓶或细胞板 - 用70％的乙醇饱和消毒纱布海绵轻轻擦拭。 </li><li>确保两个缓冲室的外部和内部的门关紧。然后打开外门，并将里面的物品。 </li><li>关闭外门。在软件上，选择缓冲模块，然后单击稀释因子选项卡上。输入1到日志因素框。单击开始。 注:"稀释因子"被定义为是指该缓冲器模块的气氛将被疏散并更换1次用干净的HEPA过滤的气体。 </li><li>一旦图形界面停止闪烁的"稀释因子"，打开缓冲室的内门，并把处理室内的材料。 注意:不要让内部和外部的门永远是在同一时间开放，如果缓冲室没有经过稀释因子不要打开内门。 </li><li>为了消除从处理室的项目，首先确保缓冲室发生了稀释因子。然后打开内门，将被删除里面的物品，然后关闭内门。然后打开外门，取出物品。 注意:稀释倍数不需要打开外门之前运行。 </li></ol></li></ol> 2.引进和饲养细胞<ol><li>孵化器安装<ol><li>放置在水盘3的培养皿在每个孵化的底部。填补这些菜肴与不孕辰水。不要直接加满水锅。保持在这些菜水位，因为它们是用于保持相对湿度（RH）设定点在孵化器是至关重要的。 </li><li>在图形界面中，点击孵化器。点击相对湿度（RH）下的设定点中的现有值与输入的85％。点击绿色的对勾接受这个值。 </li></ol></li><li>在电池生产设施的细胞的制备<ol><li>如果不这样做的话，调整缓冲室，处理室，和一个培养箱中细胞被引入的类型所需的气体组分。 </li><li>清洁用无菌纱布和非易燃的消毒剂处理室的表面上。不使用任何过酸的消毒剂，如在一封闭的系统中的强，挥之不去蒸气是有毒的哺乳动物细胞。相反，使用苯扎氯铵为基础的产品。 </li><li>继续消毒。清洁工作的commo手套和曲面NLY感动，如门把手。彻底而有节制地使用的消毒剂，如在处理室过量液体将有助于湿度，冷凝和可能的微生物的生长。 </li><li>清洁层流罩的表面和缓冲用70％乙醇室中，并允许它干燥。将细胞瓶（板）（在我们的情况，物业服务公司或神经干细胞）的引擎盖，并简要用饱和乙醇消毒纱布擦拭海绵其外表面上。 </li><li>把该烧瓶或板中的缓冲室，并运行一个稀释因子（步骤1.2.3）。 </li><li>一旦稀释因子完成后，立即将烧瓶或板到适当的孵化器。作为孵化器在处理腔室的后部，拉架子伸到细胞放置的处理室。避免不必要地打开培养箱的门或延长的时间周期相比，该孵化器的处理室的空气很干燥，并会导致一个显著在孵化器湿度的损失。 </li></ol></li><li>饲养细胞培养<ol><li>收集培养基成分，血清学移液器，电子移液器，纱布和消毒。还聚集花费细胞培养基中的废弃物容器，但不要使用漂白剂填充它。如在第1.2节中所述，通过一个缓冲室使物料进入处理室。制备细胞培养基中的处理室，如先前所描述9,10-（也见材料表 ） </li><li>安排在这样的方式的材料，以使最佳的工作空间。避免将不被在处理室的中心使用的物品。 </li><li>允许与离开媒体容器稍微不封顶系统中存在的CO 2和O 2的水平达到平衡的媒介。有些PSC中厂商表示不会在37℃到温暖的媒体;如果是这样的情况下，使用一个缓冲室以平衡介质。让我dium下平衡20分钟。 </li><li>取出从水井使用适当的体视移液管和电子移液器旧的网上平台。对于物业服务公司，删除所有，但旧的介质的小型剩余量，足以防止在饲养过程中干燥。对于神经干细胞，取出一半的旧介质。吸取废为废物容器。 </li><li>放置用于吸管放回原始包装，并将其移动到处理室的一侧，或成指定用于清除废物的缓冲室。用新鲜的无菌血清吸管，添加到细胞培养板和板放回其指定的孵化器新鲜培养基。 </li><li>在进料结束时，喷洒消毒纱布和彻底清洁处理室的底板和门把手。如果在系统正在生长多个细胞系，消毒在馈送每个细胞系之间。这有助于减少交叉污染的风险。 </li><li>项目建成后，排除废物通过缓冲室中的一个或其他不需要的物品。 </li></ol></li></ol> 3.分裂细胞培养<ol><li>物业服务公司或神经干细胞传代酶<ol><li>收集所需传代的所有项目。这包括媒体，酶或非酶细胞解离缓冲液（后者为神经干细胞只），DPBS，板或烧瓶，细胞外基质（ECM），锥形管，和血清移液管。使其使用缓冲室系统。 </li><li>外套新鲜平板或瓶中的ECM，如前所述9,10。一些节能措施 - 如那些来自小鼠肉瘤细胞系的 - 只有4和15℃之间的液体，所以使用无菌冷块或凝胶冰袋，以保持ECM冷加热工艺室工作时。 </li><li>吸取了从培养基中用过的培养基和废物容器处置。 </li><li>冲洗用1ml DPBS /井或烧瓶表面良好，DPBS处置。 </li><li>添加1 - 2毫升温酶向每个孔中。只有非常密集的文化需要2毫升</li><li>立即将培养皿或瓶显微镜室，并仔细观察的文化。留意开始松动了的菜单个细胞的迹象。不要等到电池飘进悬挂在进行下一步之前。 </li><li>返回单元到主处理腔室。使用5ml的血清吸管，加入4毫升的DPBS为每1ml酶，然后有力地吸管上下以从井表面移去细胞。如果一个多层板内传代多口井，以及从各井撞出细胞前加入DPBS到每个。 </li><li>转移酶和PBS细胞悬浮到合适大小的锥形管中。 </li><li>如果离心机是不可用的电池生产工厂，封紧锥形管，将其放置在缓冲室和密封门关闭。 </li><li>从buf中取出锥形管从层流侧FER室和自旋细胞在100×g离心在RT 5分钟。 </li><li>喷用70％乙醇的锥形管，并使其通过一个缓冲室和稀释因子处理室。 </li><li>吸管掉上清，重悬细胞于2ml PSC或NSC培养基。如果物业服务公司传代，一定要包括在10μM的浓度介质ROCK抑制剂，如前面所述9,10。 </li><li>算使用血球细胞，并确定所需的井或板的数量。板的PSCs在5×10 4 - 1 10 5细胞/ cm 2以下。 2比在1拆分神经干细胞。 注意:神经干细胞经常聚集并用血细胞计数器抗蚀准确计数。 </li><li>如果需要的细胞的冷冻保存，按照相应的协议9,10，但确保所有的用品和冷冻培养基在预先准备和放置在处理腔室内部。 DMSO是在37到细胞毒性很大6; C，所以工作非常快。保持异丙醇夹套冷冻集装箱在RT在层流罩。一旦冷冻小瓶填充和密封后，立即通过使它们通过一缓冲室移除处理室的小瓶中。 </li></ol></li><li>物业服务公司的手册传代<ol><li>确认该培养需要传代，如前所述8。如果是这样，准备新鲜的板或涂有细胞外基质瓶。然后完全改变介质。 </li><li>清洁用一个备用量不燃消毒剂湿润无菌纱布显微镜室 - 太多会导致在腔室过量起雾。只有清洁手套，建筑面积，和舞台。带来了几款200微升或1000微升单独包装的吸管提示进入室内。 </li><li>物业服务公司带来的板进入显微镜室，揭开盖子，然后将其正面朝下放置在刚清洁过的地板上。离开了盖子暴露的底部目录ectly到气流，以及潜在的污染。 </li><li>解开一个枪头，并使用显微镜可视化培养，挑开有使用吸管的尖端良好的形态菌落。 注意:如果该个体用户发现这更舒适的吸管尖可以附着到移液管。 </li><li>上盘更换盖子，和移动板回处理室。 </li><li>吸管关闭从新板过量的ECM，和来自旧板（含有在悬浮液中的菌落件）到新的板传送介质。如果老盘仍含有殖民地，它们没有准备好采摘，喂养它。 </li></ol></li></ol> 4.专业培养技术<ol><li>成纤维细胞的仙台转成产品分成合同<ol><li>内的小区的生产设施，展开人皮肤成纤维1中的成纤维细胞培养基含有DMEM / F12，10％FBS和20毫微克/毫升FGF2，以5％ 的 O 2。使用6孔培养disheš涂有0.1％明胶。 </li><li>通过用1ml 0.25％胰蛋白酶的解离它们每孔被转导制备的细胞。灭活用1 ml成纤维细胞培养基中胰蛋白酶。旋在200 xg离心，重悬在1ml的成纤维细胞培养基的细胞，并使用血细胞计数器计数。 <ol><li>悬浮2.0×10 5个细胞在2 ml成纤维细胞培养基中，将细胞悬浮液添加到1以及一个明胶包被6孔板（2.1×10 4个细胞/ cm 2）的。把细胞放回孵化器。 </li></ol></li><li>允许细胞附着到板的底部24小时。 </li><li>放置在处理室内部的新鲜的成纤维细胞培养基（1毫升，每细胞的孔中的要转导），并允许它平衡至气体。 </li><li>消毒用70％乙醇的预冷却冷块，并将其放置在一个缓冲室。使用凝胶冰袋与削减它，如果一个商业冷块不可用孔。 注:有3 - 4个独立仙台病毒（取决于转导试剂盒的版本），它必须迅速解冻，但保持在低温块一旦解冻。 </li><li>浸管的下半部分在37℃的热水浴中15秒（不浸没管），然后立即清洁管外墙用70％的乙醇。放置在缓冲腔室中的灭菌冷块上的管，并运行一个稀释因子。 </li><li>工作迅速，添加每个仙台病毒重新编程所必需的量的平衡介质2。细胞的数目，每种病毒的滴度，以及感染的每个病毒所需的多重 - 该体积是由三个变量决定。请咨询推荐MOI试剂盒说明书。其计算公式为: [（细胞数转染）（感染复数）（1000）] /（病毒效价）=微升病毒的所需 </li><li>移除细胞的阱（多个）将上清液进行再编程。对于每个孔，加入1毫升的MEDI嗯含有病毒。轻轻摇动平板，小心不要溅出媒体和板放回5％O 2孵化器。 </li><li> 2小时后再次轻轻摇动平板又经过2小时再重复。 </li><li>后（第1天后转导）24小时，进料孔用1ml多能干细胞培养基。重复第2天，但不从很好地去除网上平台。 </li><li>天3和4，改变的一半培养基。 </li><li>第5及以后的一天，改变整个网上平台。 </li><li>当菌落出现，弄脏使用无菌的抗体，以确认该菌落确实多能干细胞，如前面2中所述的培养。作为喂养细胞，确保包含所述抗体对PSC媒体已被平衡到在细胞生产设施的气体。 注意:在成功的重新编程，殖民地的iPSC应转1后出现约两周。 </li><li>当殖民地长大大够了，用显微镜来标记和机械通过他们到一个ECM-涂层板，如第3.2节所述。 </li><li>手动或酶促展开菌落，如前所述9,10。 </li></ol></li></ol> 5.清理后日用品<ol><li>将所有废物材料，包括液体废物烧瓶中，到无菌缓冲室。擦拭处理室的表面上，并已通过用无菌纱布和非易燃的消毒用品接触的所有区域。 </li><li>从缓冲室中取出所有的固体废料，并在适当的废物容器丢弃。 </li><li>丢弃废液适当的废物容器，或者干脆用漂白剂混合，倒入血本无归。清洁用肥皂和刷子废物容器。消毒用70％乙醇的容器，并经由缓冲室将其移回处理室。 注意:不建议使用漂白剂废物容器中，同时它是系统内部的，如漂白烟雾可能再循环和损害的细胞培养物。 </li><li>对于卫生考虑，擦拭系统的用70％乙醇处理室的前塑料表面。这有助于从用户的前额清理关闭汗水和皮肤油。 </li><li>如果冷凝已建立的过程中室内，运行稀释因子清除凝结。留出至少1小时这个来完成。 </li></ol> 6.常规非日常任务<ol><li>根据需要用无菌蒸馏水补充在细胞培养孵化器中的水的菜肴。一个星期顶掉的菜至少一次。然而，蒸发速度会波动基于所述培养箱的门是如何经常​​打开，液体培养物中的体积，以及培养箱的设置。 </li><li>每月一次，重新调整各室的O 2和CO 2的设置。这需要使用跨度（校准）气体，其续的AINS称为O 2和CO 2浓度水平作为参考标准。确保有开始校准前SPAN充足的天然气供应。该系统使用特定气体传感器，位于每个模块中，以检测气体的浓度;因此，使用高品质的SPAN气体确保了传感器可靠地产生精确的气体浓度。 </li><li>定期替换在处理室和显微镜室的手套。不管系统是如何经常​​使用更换手套每2个月。安装前，灭菌消毒剂新手套。在处理室中运行的稀释因子的手套已更换后。 注意:当绷在该系统的袖口，丁腈手套必须开发在暴露于物理应力和热区的孔的倾向。这是正常的和不可避免的磨损。 </li><li>全室变出注射器过滤器每6个月。 </li><li>更换或清洗Ť他尽快预过滤的层流罩，因为它开始变脏。 </li><li>经常是指制造商的文档有关更换主要部件的信息。 </li></ol> 7.系统的清洗<ol><li>在影响到大多数的细胞培养物的一个主要污染事件的情况下，移动任何尚存的细胞培养物从系统中取出，或（如果可能的话）转化为未受影响孵化器。 </li><li>关闭除了将不受影响孵化器的所有室的气体控制。 </li><li>去除装置的柔性面板，并且从受影响的细胞培养孵化器中移除的不锈钢架。同时去除水中的锅。 </li><li>高压灭菌孵化器的货架和泛水，并把它们放回孵化器。擦拭用70％乙醇的保育箱的内表面。允许乙醇蒸发，并关闭车门孵化器。 </li><li>擦拭过程和显微镜室的表面用70％等hanol。更换装置的前面板。 </li><li>擦拭装置的内表面，包括受影响的培养箱，于不易燃的消毒剂。离开受影响的培养箱打开的门，并且在处理室中运行的稀释因子。确保显微镜室门也打开了。 </li><li>对于细胞培养孵化器的常规清洗，确保培养箱和处理室在同一大气设置。打开培养箱门，并将所有的细胞培养皿处理室的表面上。 </li><li>擦拭培养箱货架表面和内表面用不可燃的消毒剂。允许消毒剂蒸发，并移动至细胞培养物回培养箱。尽可能快地工作，以避免细胞培养dessication。 </li></ol>

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	<li>Brick, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Autism+Spectrum+Disorders+Stem+Cell+Resource+at+Children's+Hospital+of+Orange+County:+Implications+for+Disease+Modeling+and+Drug+Discovery.">The Autism Spectrum Disorders Stem Cell Resource at Children's Hospital of Orange County: Implications for Disease Modeling and Drug Discovery.</a> Stem Cells Transl.Med. 3 (11), 1275-1286 (2014).</li><li>Nethercott, H. E., Brick, D. J., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+induced+pluripotent+stem+cells+by+lentiviral+transduction.">Derivation of induced pluripotent stem cells by lentiviral transduction.</a> Methods Mol.Biol. 767, 67-85 (2011).</li><li>Pistollato, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+HIF1alpha+repress+the+differentiative+effects+of+BMPs+in+high-grade+glioma.">Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma.</a> Stem Cells. 27 (1), 7-17 (2009).</li><li>Pistollato, F., Chen, H. L., Schwartz, P. H., Basso, G., Panchision, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+tension+controls+the+expansion+of+human+CNS+precursors+and+the+generation+of+astrocytes+and+oligodendrocytes.">Oxygen tension controls the expansion of human CNS precursors and the generation of astrocytes and oligodendrocytes.</a> Mol.Cell Neurosci. 35, 424-435 (2007).</li><li>Raval, J. S., Koch, E., Donnenberg, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+non-viable+airborne+particles+correlates+with+airborne+colonies+and+represents+an+acceptable+surrogate+for+daily+assessment+of+cell-processing+cleanroom+performance.">Real-time monitoring of non-viable airborne particles correlates with airborne colonies and represents an acceptable surrogate for daily assessment of cell-processing cleanroom performance.</a> Cytotherapy. 14 (9), 1144-1150 (2012).</li><li>Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+stem+cell+therapies+for+neurological+diseases.">The potential of stem cell therapies for neurological diseases.</a> Expert.Rev.Neurother. 6, 153-161 (2006).</li><li>Schwartz, P. H., Brick, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+therapies+for+the+lysosomal+storage+diseases+-+the+quintessential+neurodegenerative+diseases.">Stem cell therapies for the lysosomal storage diseases - the quintessential neurodegenerative diseases.</a> Curr.Stem Cell Res.Ther. 3 (2), 88-98 (2008).</li><li>Schwartz, P. H., Brick, D. J., Nethercott, H. E., Stover, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+human+embryonic+stem+cell+culture.">Traditional human embryonic stem cell culture.</a> Methods Mol.Biol. 767, 107-123 (2011).</li><li>Stover, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process-based+expansion+and+neural+differentiation+of+human+pluripotent+stem+cells+for+transplantation+and+disease+modeling.">Process-based expansion and neural differentiation of human pluripotent stem cells for transplantation and disease modeling.</a> J Neurosci.Res. 91 (10), 1247-1262 (2013).</li><li>Stover, A. E., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+human+pluripotent+stem+cells+to+feeder-free+conditions+in+chemically+defined+medium+with+enzymatic+single-cell+passaging.">Adaptation of human pluripotent stem cells to feeder-free conditions in chemically defined medium with enzymatic single-cell passaging.</a> Methods Mol.Biol. 767, 137-146 (2011).</li><li>Yoshida, Y., Takahashi, K., Okita, K., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+enhances+the+generation+of+induced+pluripotent+stem+cells.">Hypoxia enhances the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 5 (3), 237-241 (2009).</li></ol>

The authors declare that they have no competing financial interests.

在CPF内生长的细胞看到在氧气或二氧化碳的浓度没有变化，因为它们从培养箱移动到处理室对显微镜室和背部。至关重要的是，在每一个腔室的条件匹配的特定培养箱，其中从培养箱除去细胞之前将细胞保持。该装置内的气氛是连续HEPA过滤并是可定制对于氧气和二氧化碳的浓度。细胞可以在标准浓度的PSCs或神经干细胞，5％和9％，中生长;或替代浓度可以选择为不同的细胞类型或特定的实验。因此，该装置与医疗级氧气，二氧化碳，和氮（ 图4）的常数源供给。所有这三种气体是由特定气体歧管系统来确保恒定的供应提供。该装置还与一校准气体混合物包括供应10％（±0.01％）二氧化碳的氧气。歧管系统被安置在电池生产设施外和气体管道输送到通过天花板设施。校准气体被容纳在设施内。该装置还与房子的真空供给，也通过在天花板上。使用电子监控系统和无线发送装置，全部歧管的输出压力不断监测。在任何压力下降超出范围的情况下，电池生产工厂运营商都将自动打电话并通知等适当的行为采取行动。 该装置的功率要求是由​​六个专用120伏的电路从天花板降会见并连接到医院的备用发电机，以确保恒定的供应。该设备的操作是通过软件来通过不间断电源供电的基于PC的计算机上进行控制。这些电源和电脑安排确保系统的功能连续地甚至在一个公共电力系统故障的情况下。该软件控制装置具有用户友好的图形界面（ 图1），它允许对氧气和二氧化碳浓度的控制以及温度，湿度，以及腔室压力。所有这些参数的值连续记录提供的所有设备参数的运行记录。每天晚上这个数据备份到远程服务器上，以保护其完整性。计算机和软件可以远程管理通过用户访问，以评估和/或更改任何参数。此外，计算机和软件可以远程访问，允许设备参数互动的评估，并与当地用户的故障诊断。一个附加的警报发送单元被连接到该装置，使得细胞生产设施人员收到通知的装置的任何超出范围状态。远程访问Çapabilities允许登录并超出范围的条件的具体的评估。 该装置被设计成一个模块化系统无论在宏观和微观意义。个别的细胞培养的模块，如孵化器和处理室，可以在考虑到它们的尺寸和要求，以及在它们的布局相对于彼此进行定制。此外，大多数的单个模块的控制功能本身模块化使得个体气氛气体控制器，例如，可以容易地不显著中断系统取代。 专门处理室，例如一个用于显微镜可视化和细胞培养物的处理，很容易适应系统。两者相差和荧光显微镜是系统（ 图6），使得细胞可被活染色内部，菌落可以在相同的大气条件被解剖作为内部吨他的孵化器。通过在处理室的侧壁密封垫圈电缆的路由允许设备如电源和计算机要保持该装置的外面，通常在一个购物（ 图6）。 在细胞生产设施处理室具有比常规的BSC不同气流模式。在常规的BSC，气流从中央排气口向下流动并分裂成两个单独的流，然后将其由两个不同的进风口在机壳的底板的前端和后部吸收。相比之下，CPF在顶棚的前部的单个排气口。空气流向下和朝向腔室，在那里，然后向上抽吸到进气通风口的背面。虽然公积金本身是很干净，这种独特的气流模式意味着，技术人员需要稍微调整自己的技术，以减少污染的风险。与传统的BSC，实验室工人小号HOULD避免将自己的双手张开细胞培养板和媒体瓶的上游。然而，这是上游方向已经在CPF改变该电池生产工厂实验室本身是相当标准，并配备了-20℃的冰柜，-80℃的冰柜，4℃冰箱，离心机和水浴。实验室还具有对方便免提操作脚控制一个接收器。为了使该实验室成为官能临床细胞生产设施，然而，几个另外​​的修饰仍必须进行。首先，该装置本身必须升级为具有监视挥发性有机化合物，颗粒，和二氧化氯是用于去污的浓度的能力。其次，含在FACS机器的处理腔室可以被容纳并且经由缓冲器模块连接到该设备的其余部分。这将允许细胞分选和TR的纯化适当的环境条件下ansplantable细胞群。最后，整个装置必须在软墙洁净室之内被安置。这提供了一个国际标准化组织（ISO）8级环境，为设备5。 森林合作伙伴关系的高度不育和计算机控制的特性使得它与细胞治疗和良好的制造工艺的未来应用的理想系统。污染的风险被大大减轻，但更重要的是，细胞扩增的条件，自动记录和由计算机系统归档。在气体浓度，温度，湿度，和接入到系统中的所有事件偏差严格记载。这可以调查产品出现质量问题时有很大帮助。然而，仍然有限制。任何和所有试剂和耗材的使用（ 例如，媒体组件，移液管，板）必须单独记录。加itionally，也有可能出现，这是完全无关的CPF的监控系统记录的变量可能出现的问题（包括人为错误的多种形式），众说纷纭。因此，需要对训练有素的人员和任务的详细手册文档保持在原位。

Standard stem cell culture techniques suffer from several environmental constraints that place undue stresses on the cells and expose the cells to unacceptable risks of contamination. Among the stresses that cells may endure under standard cell culture conditions are precipitous changes in the levels of carbon dioxide and oxygen concentrations3,4. This occurs when the cells are moved from the incubator to the biosafety cabinet and/or microscope which may not be optimal for the cells. Previous studies have confirmed the advantages of culturing both pluripotent and neural stem cells in hypoxic conditions4,11, and for best results, these conditions need to be continuous. Moreover, risks of cellular contamination are higher as the laboratory environment and personnel impinge upon the cells at almost every step of their culture and manipulation. Traditional clean rooms comprise one effective method to greatly decrease contamination risks but they are expensive, have a large footprint and fail to address stressors related to carbon dioxide and oxygen concentrations. 
A cell production facility that can address both contamination risks and gas concentrations and that can be qualified to meet cGMP criteria9 provides high quality cells for basic science research as well as clinical applications1,6,7. Such a cell production facility consists, at a minimum, of the following components: a process chamber, which acts as a heated workspace for the feeding and manipulation of cell cultures; a laminar flow hood, for the initial sterilization of reagents, tubes, and tools; two buffering airlock chambers in between the hood and the process chamber; two cell culture incubators accessible from the process chamber; a microscope chamber adjacent to the process chamber; and finally, computer software to set and monitor the conditions within these modules. Using this basic infrastructure, a wide variety of tasks can be performed, such as standard feeding and passaging of pluripotent stem cells and multipotent neural stem cells, as well as more specialized methods like Sendai virus-based reprogramming, in vitro migration studies, and differentiation of neural stem cells for electrophysiological characterization.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;i&gt;Xvivo System&lt;/i&gt;</td><td>Biospherix</td><td>custom made</td><td></td></tr><tr><td>&lt;i&gt;Xvivo&lt;/i&gt; Software</td><td>Biospherix</td><td>version i.o.2.1.2.1</td><td></td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt; Manifold</td><td>Amico</td><td>P-M2H-C3-S-U-OXY</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Manifold</td><td>Amico</td><td>M2H-C3-D-U-CO2</td><td></td></tr><tr><td>N&lt;sub&gt;2&lt;/sub&gt; Manifold</td><td>Western Innovator</td><td>CTM75-7-2-2-BM</td><td></td></tr><tr><td>Microscope with DP21 camera and fluorescence</td><td>Olympus Corporation</td><td>CKX41</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM/F12 Glutamax</td><td>Life Technologies</td><td>10565-018</td><td></td></tr><tr><td>StemPro hESC Supplement</td><td>Life Technologies</td><td>A100006-01</td><td></td></tr><tr><td>Accutase</td><td>Millipore</td><td>SCR005</td><td></td></tr><tr><td>Phosphate-Buffered Sodium</td><td>Hyclone</td><td>9236</td><td></td></tr><tr><td>Fibroblast Growth Factor 2</td><td>R&amp;amp;D Systems</td><td>AFL233</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Protide</td><td>PP1130</td><td></td></tr><tr><td>Hank&#039;s-based Cell dissociation Buffer</td><td>Life Technologies</td><td>13150-016</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Life Technologies</td><td>21985-023</td><td></td></tr><tr><td>Epidermal Growth Factor</td><td>R&amp;amp;D Systems</td><td>AFL236</td><td></td></tr><tr><td>Oct-3/4 Antibody</td><td>Millipore</td><td>AB3209</td><td></td></tr><tr><td>TRA-1-60 Antibody</td><td>Millipore</td><td>MAB4260</td><td></td></tr><tr><td>SSEA4 Antibody</td><td>Millipore</td><td>MAB4304</td><td></td></tr><tr><td>BIT-9500 Serum Supplement</td><td>Stemcell Technologies</td><td>9500</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Consumable Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2 ml Serological pipet</td><td>VWR</td><td>89130-894</td><td></td></tr><tr><td>5 ml Serological pipet</td><td>Olympus Plastics</td><td>12-102</td><td></td></tr><tr><td>10 ml Serological pipet</td><td>Olympus Plastics</td><td>12-104</td><td></td></tr><tr><td>25 ml Serological pipet</td><td>Olympus Plastics</td><td>12-106</td><td></td></tr><tr><td>50 ml Serological pipet</td><td>Olympus Plastics</td><td>12-107</td><td></td></tr><tr><td>6-well plate</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>12-well plate</td><td>Corning</td><td>353043</td><td></td></tr><tr><td>T25 flask</td><td>TPP</td><td>90026</td><td></td></tr><tr><td>T-75 flask</td><td>TPP</td><td>90076</td><td></td></tr><tr><td>20 &amp;micro;l pipet tips</td><td>Eppendorf</td><td>22491130</td><td></td></tr><tr><td>200 &amp;micro;l pipet tips</td><td>Eppendorf</td><td>22491148</td><td></td></tr><tr><td>1,000 pipet tips</td><td>Eppendorf</td><td>22491156</td><td></td></tr><tr><td>Cryovials</td><td>Thermo Scientific</td><td>5000.102</td><td></td></tr><tr><td>70% ethanol</td><td>BDH</td><td>BDH1164-4LP</td><td></td></tr><tr><td>Sanimaster 4</td><td>Ecolab</td><td>65332960</td><td></td></tr><tr><td>Bleach</td><td>Clorox</td><td>A714239</td><td></td></tr></tbody></table>

culturing human pluripotent and neural stem cells in an enclosed cell culture system for basic and preclinical research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

该原稿在一些细节描述了一种细胞生产设施，以及一个封闭的系统内培养细胞的独特方面。细胞生产设施的本体包括一个定做，封闭的，细胞培养物具有小尺寸和可容易地容纳在6×7.5米2室（ 图2）内装置。在任何时候都不会暴露于实验室人员或环境中的装置内的细胞。该装置由几个模块组成:一个处理室，一个层流罩，在该罩和处理室，两个细胞培养孵化器，以及邻近于所述处理室的显微镜室2之间的缓冲气锁腔室（ 图3）。该系统是由软件允许环境变量来进行控制和监视（ 图1）控制。运行该软件的计算机上的不间断电源。气体被送入第由一组歧管为氧气，氮气和二氧化碳（ 图4）电子系统。活动集和一个备用组 - 该设备的气体是由有两套坦克每个歧管系统提供。当活动集拉下来，歧管会自动切换至备用设置和工作人员为了更换坦克。电源是备用发电机系统。 多能干和神经干细胞可以在低氧条件下，使用先前开发的协议9，具有固有的新颖设备添加的并发症可以生长在这个设施。多能干细胞可以使用仙台病毒中获得，使用特定的多能性的抗体，以确定完全转染的菌落，然后将其分离并展开。 （ 图5A和B）中 ，从这些iPS细胞，神经干细胞和神经元可以使用双SMAD抑制衍生，和它们的表型验证USI<html>纳克特定NSC-抗体（ 图5C和D）。 <img alt="图1" src="/files/ftp_upload/53685/53685fig1.jpg" /> 图1.图形界面的电池生产基地。（ 一 ）中央公积金的图形表示是默认的屏幕和图3所示 。鼠标在缓冲模块1咔嗒所示的CAD图纸相符（2F 图1）打开其中O 2的值被设置到处理室（3在图3中）相匹配的控制屏幕（B）。同样，点击处理室或孵化器1带来了各自的控制屏幕（C和D，分别地），其中值可以被改变或监测适当。<html> rget ="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/53685/53685fig2.jpg" /> 图2.电池生产设施。（A）从正面右视图中看到的系统。注意在天花板和用显微镜附件和计算机右侧的电源和气体连接购物。 （B）中的层流罩与所述接入门在右侧看到的缓冲器模块。 （C）与两孵化（黑色门）的处理室的后部看到。 （ 四 ）通过系统的右侧视图显示了显微镜，并与门的距离看到缓冲室监控。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53685/53685fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> T"FO:保together.within页="1"&gt; <img alt="图3" src="/files/ftp_upload/53685/53685fig3.jpg" /> 在电池生产设备的图3. CAD绘图。进入设备的所有项目都先擦拭用酒精和空气层流罩干燥（1）。然后项目被传递到UCPC模块之前转换到前台缓存模块（2F）在适当的气氛下，或处理室（3）。细胞是在两个保温箱（4,5）中培养。活细胞染色，菌落采摘，一般细胞形态和迁移分析评估开出的UPC模块代替，或显微镜室（6）。最后，废物排出通过后缓冲模块（2R）系统。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53685/53685fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> &lt;<html> / P&gt; <img alt="图4" src="/files/ftp_upload/53685/53685fig4.jpg" /> 图4.源气体和动力的。（ 一 ） 二氧化碳气歧管是一个4×4设置为它提供了实验室的所有孵化器。 （ 二 ）O 2歧管为2×2的设置和耗材只有电池生产设施。在一个蒸发器中产生的氮为系统（℃，略低于退出符号）附加到液氮歧管（右侧）也自动地（左下角）填充cryofreezers。歧管是2对:160L液氮罐被提供2×2的设置。 CO 2，O 2和N 2从天花板通过关闭阀（D）提供。室内真空也是在这个位置提供。刚刚看到供气关闭阀的后面，是一对给系统供电的电力电缆。F ="htt​​ps://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53685/53685fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/53685/53685fig5.jpg" /> 在缺氧衍生出仙台病毒图5.多能干细胞。（A）SC187-SF4-2I0-E3 iPS细胞iPS细胞（命名在Stover 等9中），相差10倍。 （B）SC88.1-UH1-2I0 iPS细胞活沾上AF-594标记TRA-1-60，1:100稀释媒体。所有iPS细胞在仙台病毒在5％O 2的电池生产工厂转导。 （C）SC68.1-UH0-2I0-M0S13-N2G6源自iPSC的神经干细胞的相衬，在玻片在5％O 2增长。然后将细胞固定在4％多聚甲醛，并用抗人巢蛋白第一抗体染色，和Alex-488的二级抗体。所有比例尺都在1001;:M <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53685/53685fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research at JoVE.com

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

culturing-human-pluripotent-neural-stem-cells-an-enclosed-cell

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

10.2K 次观看.  Childrens Hospital of Orange County Research Institute. Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

视频: Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

一种新的文化模式的人多能干明胶胎盘条件培养基细胞增殖

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

科研

发育生物学

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

从废弃的人类胎儿大脑皮质组织神经干细胞的生成

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

神经科学

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

利用人类胚胎干细胞的单细胞培养进行高效神经分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

使用自动化培养系统诱导多能干细胞分化

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, including the elucidation of rare disease pathologies, the development of new medicines, and regenerative medicine aiming to restore damaged organs. Despite this, the social implementation of hiPSCs is still limited. This is partly because of the difficulty of reproducing differentiation in culture, even with advanced knowledge and sophisticated technical skills, due to the high sensitivity of iPSCs to minute environmental changes. The application of an automated culture system can solve this issue. Experiments with high reproducibility independent of a researcher's skill can be expected according to a shared procedure across various institutes. Although several automated culture systems that can maintain iPSC cultures and induce differentiation have been developed previously, these systems are heavy, large, and costly because they make use of humanized, multi-articulated robotic arms. To improve on the above issues, we developed a new system using a simple x-y-z axis slide rail system, allowing it to be more compact, lighter, and cheaper. Furthermore, the user can easily modify parameters in the new system to develop new handling tasks. Once a task is established, all the user needs to do is prepare the iPSC, supply the reagents and consumables needed for the desired task in advance, select the task number, and specify the time. We confirmed that the system could maintain iPSCs in an undifferentiated state through several passages without feeder cells and differentiate into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. The system will enable highly reproducible experiments across institutions without the need for skilled researchers and will facilitate the social implementation of hiPSCs in a wider range of research fields by diminishing the obstacles for new entries.

作者聚焦:自动化 iPSC 培养以提高可重复性 

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of cells.

用于维持和分化人诱导的多能干细胞的自动化培养系统

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

人类多能干细胞的神经元在一个Multititre的板格式的快速和高效地生成

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

生物学

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

另类文化的人多能干细胞的生产，维护和遗传分析

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6&#8210;8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC)&#160;transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

通过集成活细胞监测，自动生产人类诱导多能干细胞衍生皮质神经元和多巴胺神经元

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are ...

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

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此视频中的章节

一种新的文化模式的人多能干明胶胎盘条件培养基细胞增殖

从废弃的人类胎儿大脑皮质组织神经干细胞的生成

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

利用人类胚胎干细胞的单细胞培养进行高效神经分化

用于维持和分化人诱导的多能干细胞的自动化培养系统

用于维持和分化人诱导的多能干细胞的自动化培养系统

用于维持和分化人诱导的多能干细胞的自动化培养系统

人类多能干细胞的神经元在一个Multititre的板格式的快速和高效地生成

另类文化的人多能干细胞的生产，维护和遗传分析

通过集成活细胞监测，自动生产人类诱导多能干细胞衍生皮质神经元和多巴胺神经元