我们感谢美国Ganapati mStrawberry OP9细胞株的建立和测试。此外，我们感谢H. Shafifor与她共培养援助。惠普的支持，从国家心脏，肺和血液研究所（F31HL087714）（惠普）的奖学金。这项工作的内容​​完全是作者的责任，并不一定代表的国家心脏，肺和血液研究所或美国国立卫生研究院（NIH）的官方意见。加州大学洛杉矶分校的流式细胞仪核心设施是由美国国立卫生研究院（CA - 16042，AI - 28697），琼森癌症中心，加州大学洛杉矶分校艾滋病研究所，加州大学洛杉矶分校医学学院的支持。

1。 ES细胞的制备<ol><li>根据您的特定细胞系的标准协议的文化ES细胞。请一定要准备足够的ES细胞到0天mStrawberry OP9细胞板块（ES细胞将镀在1 × 10 ^ 3细胞每平方厘米 2 0天） </li></ol> 2。 OP9细胞的制备<ol><li>增长mStrawberry OP9细胞前，准备OP9生长介质 OP - 9细胞培养液中 <table border="1"><tbody><tr><td></td><td> 股票 </td><td> 终浓度。 </td><td> 体积（ml） </td></tr><tr><td> aMEM </td><td></td><td></td><td> 39.5 </td></tr><tr><td>胎牛血清（OP - 9测试） </td><td> 100％ </td><td> 20％ </td><td> 10 </td></tr><tr><td> L -谷氨酰胺</td><td> 100X（200毫米） </td><td> 2MM </td><td> 0.5 </td></tr><tr><td></td><td></td><td></td><td> 50毫升</td></tr></tbody></table> 保存在4 ° C，并在一个星期的准备使用</li><li>为了保持mStrawberry OP9细胞，媒体必须改变，每2天，细胞传代细胞达到（在70-90％汇合的亚文化）汇合前。 mStrawberry OP9细胞培养从未增长超过90％汇合，因为这会导致其分化成脂肪细胞。参见图3。 <ol class="lalpha"><li>由洗涤细胞，用PBS（或1X PBS + 1X 0.1％胰蛋​​白酶- EDTA）2X传代培养细胞</li><li>加入预热的0.1％胰蛋白酶EDTA在37 ° C（5分钟或直至细胞分离） </li><li>当细胞分离，添加OP - 9生长培养基。细胞转移至50ml锥形管。 </li><li>颗粒细胞在室温下5分钟，在300 XG弃上清 。 </li><li>新鲜mStrawberry OP9生长介质中的悬浮细胞</li><li>板细胞密度最小的10 4个细胞/厘米2 </li><li>种植汇合mStrawberry OP9细胞开始前3天共培养的ES细胞（0天，第5天，或Day8 /实验9）需要一天</li></ol></li></ol> 3。 ES / mStrawberry OP9共培养<ol><li>准备共培养分化媒体 <table border="1"><tbody><tr><td></td><td> 股票 </td><td> 决赛 </td><td> 体积（ml） </td></tr><tr><td> aMEM </td><td></td><td></td><td> 39.5 </td></tr><tr><td>胎牛血清</td><td> 100％ </td><td> 20％ </td><td> 10 </td></tr><tr><td> L -谷氨酰胺</td><td> 200MM </td><td> 2MM </td><td> 0.5 </td></tr><tr><td></td><td></td><td></td><td> 50毫升</td></tr></tbody></table></li><li> mStrawberry OP9细胞（日-3） <ol class="lalpha"><li>总是传代OP9日-3细胞，使你有mStrawberry OP9日0共培养细胞融合层。图4b。板mStrawberry OP9细胞在1 × 10 4 /厘米2 0天汇合。一定要为5天的亚文化和合作文化的延续8 / 9日额外细胞。 </li></ol></li><li class="jove_content"> ES / mStrawberry OP9共培养的设置（0天） <ol class="lalpha"><li class="jove_content"> ES细胞洗净，用PBS 2X </li><li> Trypsinize ES细胞与预热的0.25％胰蛋白酶- EDTA，孵化5分钟，37 ° C </li><li>添加细胞共培养分化媒体</li><li>在400xg颗粒胚胎干细胞，5 - 7分钟，RT。弃上清</li><li>悬浮细胞共培养分化媒体</li><li> mStrawberry OP9细胞取出介质</li><li> 共培养分化媒体mStrawberry OP9烧瓶</li><li>板1 × 10 3 ES细胞/厘米2 mStrawberry OP9细胞烧瓶。孵育细胞在37℃，5％的CO 2与湿度。 </li></ol></li><li> mStrawberry OP9细胞（2天） <ol class="lalpha"><li>亚文化mStrawberry OP9，让您有mStrawberry OP9共培养5天的细胞融合层。板mStrawberry OP9细胞汇合在第5天（步骤3.2）在1 × 10 4 /厘米2。务必日8 / 9，如果有必要（在步骤2.2）的额外的传代培养细胞。 </li></ol></li><li> ES / mStrawberry OP9合作文化（3天） <ol class="lalpha"><li>小心取出共培养中等和更换新鲜共培养分化培养基</li></ol></li><li> ES / mStrawberry OP9共培养（5日） <ol class="lalpha"><li>认真洗手合作，文化与PBS 2X </li><li> Trypsinize细胞与预热的0.25％胰蛋白酶EDTA </li><li>洗细胞共培养分化媒体关闭烧瓶</li><li>在400xg颗粒细胞，7分钟，RT。 弃上清 </li><li>共培养分化的媒体加入到细胞。使用21号bluntended针重新悬浮细胞和单细胞悬液，以确保</li><li>使用血球计数细胞。应该有100-150倍胚胎干细胞的数量从0天。 </li><li>重新播种6.48x10 4 /厘米2 - 7.76x10 4 /厘米2到一个新的融合，mStrawberry OP9细胞层细胞共培养</li><li>第5天检测剩余共同培养的细胞可用于流式细胞仪分析，cytopreps（30K细胞），芯片分析等。 </li></ol></li><li> ES / mStrawberry OP9共培养（8 / 9日和12天） <ol class="lalpha"><li>分化媒体从烧瓶中取出并保存。用PBS洗单层1X去除悬浮液中的造血干细胞。不折腾出造血干细胞 。 </li><li> 预热 0.25％胰蛋白酶EDTA Trypsinize贴壁细胞</li><li>洗掉ES分化媒体烧瓶的细胞，并添加到造血干细胞在悬浮液=中发现</li><li>在400xg颗粒细胞，7分钟，RT。 弃上清 </li><li> 添加到细胞共培养分化的媒体或 PBS，使用21号bluntended针重新悬浮细胞和单细胞悬液，计数使用血球细胞，以确保。 </li><li>共培养，可以持续14天的评估重新播种共培养细胞到一个新的融合，mStrawberry OP9细胞在8日或9层的更成熟的造血祖细胞，然后在12天再次。重新优化种子细胞数量的基础上检测每天所需的细胞恢复。 </li></ol></li><li> ES衍生的后代（1-14天）的评价。 ES衍生的后代可以通过流量排序mStrawberry阴性细胞（ES衍生后代）在任何一天共培养评估。 <ol class="lalpha"><li>分化媒体从烧瓶中取出并保存。用PBS洗净单层1X去除悬浮液中的造血干细胞。不折腾出造血干细胞。 </li><li>预热0.25％胰蛋白酶EDTA Trypsinize贴壁细胞</li><li>关闭烧瓶清洗细胞共培养分化媒体，并添加到造血干细胞悬浮液中发现</li><li>颗粒细胞在400xg，7分钟，RT。弃上清</li><li>悬浮细胞21号bluntended针和计数</li><li> ES -衍生的后代（mStrawberry阴性细胞）可从mStrawberry积极OP9细胞有序流动。 ES -衍生的后代，然后可以由流式细胞仪分析，cytopreps，芯片检测等特点</li></ol></li></ol> 4。代表性的成果： <img src="/files/ftp_upload/2559/2559fig1.jpg" alt="图1" /> 图1。 ES / mStrawberry OP9共培养体系 ，我们的实验室使用体外胚胎干细胞共培养体系模型造血发展。在此共培养体系，ES细胞分化成血管细胞在第五天，当上mStrawberry OP9细胞融合层。由于共同文化的收益，造血前体是目前在第8天，而终末分化的造血谱系第14天出现。箭头指示的分化天传代到新的融合层mStrawberry OP9细胞在必要时ES衍生后代。 <img src="/files/ftp_upload/2559/2559fig2.jpg" alt="图2" /> 图2。正确的ES细胞分化。 ES细胞接种到mStrawberry OP9 0天汇合层。 ES衍生的后代开始成为ES衍生后代看上去像一个细胞集群定义的边界，或开始形成一个螺纹模式，在分化的第3天可见。血管细胞，这是常见的中胚层前体的内皮细胞和造血谱系，应该出现在5天的细胞堆积的旋涡。对于共培养时间超过5天，ES衍生后代应该出现造血细胞群。 6 / 7日，两到四个细胞集群，应该出现细胞团成较大的增长，共培养8 / 9日。其他ES衍生后代中也发现贴壁的基质细胞层紧密结合。 <img src="/files/ftp_upload/2559/2559fig3.jpg" alt="图3" /> 图3。从这种合作文化不成功的胚胎干细胞的分化 。结果反映不当的胚胎干细胞分化。注意的ES -衍生的后代不会出现在一个5天的螺纹格局。细胞在螺纹格局的情况下表示hemangioblast形成不当。如果hemangioblast不正常发育，然后继续合作文化将导致造血谱系分化发育不良。不当hemangioblast形成的共同文化应该被丢弃。 <img src="/files/ftp_upload/2559/2559fig4.jpg" alt="图4" /> 图4。 mStrawberry OP9细胞培养（A）mStrawberry OP9细胞传代培养在70-90％汇合。 （二）适当的分化，胚胎干细胞和胚胎干衍生的后代被放置到一个过于mStrawberry OP9细胞融合层。 <img src="/files/ftp_upload/2559/2559fig5.jpg" alt="图5" /> 图5。代表ES / mStrawberry OP9共培养流动剖面。ES衍生后代mStrawberry负的细胞，并且可以从mStrawberry OP9细胞有序流动。 mStrawberry负细胞门是使用传统的ES/OP9合作文化。

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正确mStrawberry OP9细胞的Flk1 +血管细胞共培养5天的生产结果的汇合层上的ES细胞的分化。血管细胞应该会出现堆积起来的，在图1观察细胞的旋涡。其余共培养，可见ES衍生的后代应该出现的造血干细胞群（图1）。在第六天/ 7两到四个细胞团应该会出现和生长成较大的细胞团（壁和悬浮）共培养8 / 9日。其他ES衍生后代中也发现，紧紧地贴壁的基质细胞层内的约束。 虽然没有在协议中列出，这是预测试的ES生长介质，mStrawberry OP9增长媒体和OP9分化的ES / mStrawberry媒体使用的胎牛血清的关键。正确的测试血清应确保mStrawberry OP9细胞的正常生长，以及在联合培养的ES细胞的正常分化，直到5日的分化。 OP9细胞作为替代方案，除了mStrawberry OP9细胞，可照射，在8000拉德，前汇合播种（7.8x10 4个细胞/ 厘米 2） 。 OP9细胞照射可以消除重复replating步骤，以及允许，几乎完全消除旧，污染下游检测OP9细胞。 OP9细胞工程3ug/mL了CMV启动子控制下的mStrawberry（pRRLsin CMV载体，加州大学洛杉矶分校的载体核心）的慢病毒表达载体转导与OP9细胞表达mStrawberry的蛋白质。 标准ES/OP9文化合作协议涉及到一个融合层OP9细胞胚胎干细胞的位置，以及一个5天replating步骤，以减少老污染OP9细胞传代细胞。此外，只有造血干细胞悬浮液中发现评价ES衍生后代都将被删除。然而，一个replating一步，只有悬浮细胞的收获之一可能错过了发展中的大量ES衍生的造血细胞。这个问题就变得很重要，以解决尝试，表征与基因敲除ES线相关的造血缺陷时。为了规避这个问题，我们的实验室使用了修改后的共同文化，通过使用mStrawberry OP9细胞表达。这可以保证所有的ES后代在5天的继续合作文化，并为所有下游的检测ES衍生后代的收获。此修改提供一个有用的工具，从人类和小鼠ES线衍生的造血后代的评价和表征。 这ES - mStrawberry OP9共培养模型概括的各个阶段，原始的和明确的造血。在研究造血发育的早期阶段，许多人使用的BL - CFC（爆炸般的集落形成细胞）检测，评估ES细胞hemangioblast的发展。虽然BL - CFC检测是一个有用的工具，在调查早期造血发展，共培养系统的mStrawberry - ES展品增加实用工具，因为它允许hemangioblast评估，以及，更多的成人造血谱系。 以前的工作，使用传统OP9/ES和EB分化协议表明，也许有必要，如HOXB4过度表达，为了额外的因素得到长期的复育造血干细胞在体外。更多的工作是必要的，以评估是否mStrawberry负，排序ES衍生的人群中真正的造血干细胞。

OP - 9细胞维持液联合组件<table border="1"><tbody><tr><td>试剂名称</td><td>公司</td><td>产品编号</td><td>评论</td></tr><tr><td> aMEM </td><td> Invitrogen公司</td><td> 12571-063 </td><td></td></tr><tr><td>胎牛血清</td><td>欧米茄科学</td><td> FB01 </td><td>经过预测试</td></tr><tr><td> L -谷氨酰胺</td><td> Cellgro </td><td> 25 - 005 - CI </td><td> 200nm的 </td></tr><tr><td>胰蛋白酶EDTA </td><td>干细胞技术</td><td> 07901 </td><td></td></tr></tbody></table> ES/OP-9细胞分化培养基联合组件<table border="1"><tbody><tr><td>试剂名称</td><td>公司</td><td>产品编号</td><td>评论</td></tr><tr><td> aMEM </td><td> Invitrogen公司</td><td> 12571-063 </td><td></td></tr><tr><td>胎牛血清</td><td> Hyclone公司</td><td> SH30070 </td><td>经过预测试</td></tr><tr><td> L -谷氨酰胺</td><td> Cellgro </td><td> 25 - 005 - CI </td><td> 200MM </td></tr><tr><td> PBS（没有的Ca 2 +和Mg 2 +） </td><td> Cellgro </td><td> 21 - 031 - CV </td><td></td></tr><tr><td>胰蛋白酶EDTA </td><td>干细胞技术</td><td> 07901 </td><td></td></tr></tbody></table>

modified es / op9 co-culture protocol provides enhanced characterization of hematopoietic progeny

在体外诱导分化的ES细胞向造血细胞的命运是非常有用的，学习时，很难在体内和表征最早的基因参与造血，而无需处理与胚胎lethalities访问的细胞群。 ES/OP9共培养体系最初的设计到生产没有对生产的巨噬细胞的造血后代，OP9基质细胞系是从颅骨骨质突变小鼠缺乏功能的M - CSF所得。可用于以总结早期造血发展，在体外ES/OP9共培养体系。 OP9基质细胞培养时，ES细胞分化成FLK - 1 +血管细胞，造血祖细胞，并终于成熟了，终末分化谱系。标准ES/OP9文化合作协议涉及到OP9细胞融合层的ES细胞的位置，以及定期replating的步骤，以删除旧的，污染OP9细胞。此外，目前的协议涉及评估，悬浮液中发现的的造血干细胞和分化的每一天不ES衍生后代的评价优化。然而，随着replating步骤和收获的只有悬浮细胞，可能错过了大量的ES衍生的后代和发展造血干细胞的部分。这个问题就变得很重要，以解决尝试，表征与基因敲除ES线相关的造血缺陷时。在这里，我们描述了修改后的ES / mStrawberry OP9共同的文化，可以消除污染下游检测OP9细胞。这种方法允许所有天共培养ES衍生后代的完整的评估，导致造血分化格局，更直接对应于观察胚胎内的造血分化格局。

Watch this Scientific Journal Video about Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny at JoVE.com

Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

mStrawberry OP9细胞允许所有从联合培养的ES衍生后代的完整的评价。

修改ES / OP9共培养协议，提供增强造血后代的表征

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo ...

modified-es-op9-co-culture-protocol-provides-enhanced

Research

JoVE Journal

Biology

20.6K 次观看.  University of California, Los Angeles. mStrawberry OP9细胞允许所有从联合培养的ES衍生后代的完整的评价。

视频: Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

冻结人类胚胎干细胞

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

科研

生物学

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

人类胚胎干细胞：启动文化冷冻细胞

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES细胞源性神经上皮细胞培养

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

人类胚胎干细胞（ES细胞）的繁殖

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

一个多室中枢神经系统的神经胶质细胞共培养的微流体平台

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

微流体调查可溶性信号介导的相互作用的上皮细胞和细菌的共同文化

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

从三维混合细胞球体共培养的乳腺上皮细胞的隔离

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

在细胞间接触缺失的两个蜂窝型的插入共培养系统的开发

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. Deviations from what is considered a healthy microbiota composition (dysbiosis) may impair vital functions leading to pathologic conditions. Recent and ongoing research efforts have been directed toward the characterization of associations between microbial composition and human health and disease.
Advances in high-throughput sequencing technologies enable characterization of the gut microbial composition. These methods include 16S rRNA-amplicon sequencing and shotgun sequencing. 16S rRNA-amplicon sequencing is used to profile taxonomical composition, while shotgun sequencing provides additional information about gene predictions and functional annotation. An advantage in using a targeted sequencing method of the 16S rRNA gene variable region is its substantially lower cost compared to shotgun sequencing. Sequence differences in the 16S rRNA gene are used as a microbial fingerprint to identify and quantify different taxa within an individual sample.
Major international efforts have enlisted standards for 16S rRNA-amplicon sequencing. However, several studies report a common source of variation caused by batch effect. To minimize this effect, uniformed protocols for sample collection, processing, and sequencing must be implemented. This protocol proposes the integration of broadly used protocols starting from fecal sample collection to data analyses. This protocol includes a column-free, direct-PCR approach that enables simultaneous handling and DNA extraction of large numbers of fecal samples, along with PCR amplification of the V4 region. In addition, the protocol describes the analysis pipeline and provides a script using the latest version of QIIME (QIIME 2 version 2017.7.0 and DADA2). This step-by-step protocol is aimed to guide those interested in initiating the use of 16S rRNA-amplicon sequencing in a robust, reproductive, easy to use, detailed way.

This manuscript describes a detailed standardized protocol of high-throughput 16S rRNA-amplicon sequencing. The protocol introduces an integrated, uniformed, feasible, and inexpensive protocol starting from fecal sample collection through data analyses. This protocol enables analysis of large numbers of samples with rigorous standards and several controls.

16S rRNA-扩增子测序法对粪便微生物特性的指导协议

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. ...