订货单细胞和单胚胎的三维约束：高内涵筛选新设备
Summary
我们报告的设备和新的方法来研究细胞和胚胎。单细胞精确下令微阵列。他们的3D坐月子是朝着在生理条件下遇到的3D环境的一个步骤，并允许细胞器的方向。通过控制细胞的形状，此设置最大限度地减少可变性中的标准试验的报道。
Abstract
生物细胞通常在平面（2D）表面观察。这种条件是不生理和表型和形状是高度可变的。根据在这种环境中的细胞筛选具有因而严重的局限性：细胞器显示极端的表型，细胞形态和尺寸是异质和/或特定的细胞器无法正确显示。此外， 在体内细胞位于3D环境;在这种情况下，细胞表现出不同的表型的，主要是因为它们与组织的周围的细胞外基质相互作用的。为了标准化和产生单个细胞中对基于细胞的测定生理相关的3D环境的次序，我们在这里报告的装置的用于体外三维细胞培养物的微加工和应用。此装置由微腔的2D阵列（通常是10 5的空腔/厘米2）中 ，填充有单细胞或胚胎。细胞POSI化，形状，极性和内部细胞组织变得那么标准化呈现出3D架构。我们使用复制品成型图案的微腔的阵列，'eggcups'，到薄聚二甲基硅氧烷（PDMS）粘附在盖玻片层。腔体上布满了纤维连接蛋白更容易粘附。细胞通过离心插入。填充百分比为每个系统允许高达80％的最优化。细胞和胚胎存活率证实。我们应用此方法对细胞器，如细胞核和高尔基体的可视化，并研究活动进程，如cytokinetic环的细胞有丝分裂期间的关闭。该器件允许cytokinetic闭环和压实表型的高尔基体细胞核对准期间的新功能，如周期性积累和肌球蛋白和肌动蛋白的不均匀性的鉴定。我们的特点的方法哺乳动物细胞，裂殖酵母，芽殖酵母，C.线虫 W¯¯在每种情况下i个具体的适应。最后，该装置的特性使得它特别有趣的药物筛选测定法和个性化的药物。
Introduction
当前在体外基于细胞的测定是二维（2D）。这种构造是不自然的哺乳动物细胞，因此不是生理有关1;细胞显示的形状，大小和异构表型的多样性。当应用于筛选应用，如平面和细胞器的极端的表型（应力纤维，特别是）内的无序分布它们呈现额外严重的局限性。这是在药物测试，在高预算每年花在临床试验尤为重要。当因为在药物筛选的初期阶段人工2D培养条件适用于动物模型大多数这些药物虽然失败。另外，通过使用这种方法，具体细胞器无法正常显现，如细胞有丝分裂期间cytokinetic肌动球蛋白环，一般认为是在平面垂直演进到观察平面结构。一些新的二维测定人为了克服上述缺点和重要的见解上细胞骨架组织已经观察到2,3被提出。但是，这些检测仍然存在一个严重的局限性：细胞表现出非常价差表型相反的是， 在体内 ，其中细胞呈现出3D架构观察。与培养法相关的这些文物可能引发非生理功能，如增强的应力纤维1,4,5。
时相比2D环境6,7-三维细胞培养测定提供多个优点。它们是生理上更相关的，并且结果是因此有意义。作为一个例子，嵌在水凝胶细胞显示3D状结构，但它们的形态从一个小区的不同而不同8,9。然而，它们的形态从一个小区到另一个，其中筛选应用复杂不同。另一种策略是嵌入单细胞微制造的腔10,11。然后细胞位置，形状，极性和内部细胞组织可以成为标准化。除了 提供类似3D的架构，细胞，微腔还允许高内涵筛选研究10,12-14;单电池可以订购成微阵列和细胞器和它们的演进可以并行进行观察。这个规律提供了较低的细胞数量和更好的时间/空间分辨率良好的统计数据。有用的化合物是更容易识别可靠。
在这项研究中，我们显示了高内涵筛选应用10,12,13制作和全新的3D般的单细胞培养系统中的应用。该装置由弹性微腔（10 5腔/厘米2），创造'eggcups“（EC）的阵列。在这项工作中的尺寸和EC的总体积进行优化，个人NIH3T3细胞和HeLa细胞的典型体积在细胞分裂过程。圆柱形 – – 腔形态被选择为活动进程的可视化的正确东方细胞形状。复制品成型用于图案EC的阵列上附着在玻璃的薄聚二甲基硅氧烷（PDMS）层盖玻片15,16。细胞通过离心在欧盟出台。我们在2D使用相同的细胞相比在这里报告的观察和三维（EC）细胞器（应力纤维，高尔基体和胞核）正常化（平）表面。我们还报告，积极动力过程的观察，如cytokinetic肌动球蛋白环的细胞有丝分裂过程中17封。最后，我们表明与刚性壁，如出芽酵母，裂殖酵母和C的其它系统这种方法的结果线虫胚胎这证实了我们的方法是否适用于广泛的模型系统。
接下来我们提出一个详细而详尽的protocol为了制造和应用三维微细加工的“eggcups”。我们的方法是简单，并不需要一个干净的房间。我们预计，这一新方法将成为药物筛选试验和个性化的医学特别有意思，在更换培养皿。最后，我们的设备将用于癌症18或在基础研究19研究的细胞响应的分布对外部刺激，例如有用。
Protocol
1.“Eggcups”的微细法师的制作：微腔阵列热3''的硅晶片到200℃以蒸发湿度的任何存在。 旋涂层的SU-8光致抗蚀剂的薄层。调整树脂及纺丝速度取决于所需的厚度和光致抗蚀剂类型的体积。该厚度将决定'eggcups'（EC）的深度。对于2,800转30微米厚的层和SU-8 2025年，旋涂。 预烘烤在65℃下1分钟（第2步骤1）的晶片为一30μm厚的SU-8 2025层。取决于所需的光致抗蚀剂的?…
Representative Results
该'eggcups'（EC）是一种新型的高含量筛选方法，它允许导向细胞和胚胎的可视化在3D环境。此外，某些细胞过程，这是很难在标准2D（平面）培养物来观察，可以通过这种新的方法观察到的。 图1a显示了EC微细加工步骤的概要（参见章节1中的上述协议）。该方法简便，快捷，高效，无需特殊设备的任何要求。 图1b和1c显示了一个大型的图片和“eggcups”分?…
Discussion
副本成形，为了制造“eggcups”使用。的制造过程中不需要清洁室;它是容易和简单，但也有一些实践可能是需要的。具体地，释放PDMS压模是为了生产高品质'eggcups'的一个大的面积中最关键的步骤。由于这个原因，特别小心在这一步骤才能作出。如果这一步失败反复，考虑到优化之前的硅烷化和等离子结合等离子清洗机的参数。硅烷化不足，会导致邮票的PDMS膜粘着强。如果发生此种情况，培?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
我们承认L. Brino（IGBMC高内涵筛选设备，伊尔基希，法国）为我们提供了抗Giantin抗体，M. Labouesse实验室。为C.线虫 （IGBMC）和B塞拉芬实验室。对芽殖酵母（IGBMC），E帕卢赫和A.海曼荧光灯HeLa细胞（MPI-CBG，德累斯顿），J·莫斯利（达特茅斯医学院）和JQ吴（俄亥俄州立大学）的裂殖酵母细胞; A.饭店Hoel和F Evenou实验的帮助下，里克C.（IBMC，斯特拉斯堡，法国）的技术的帮助下，和JC JEANNOT（飞秒日，法国）在微细的帮助。这项工作是由来自法国国家科学研究中心，斯特拉斯堡大学，Conectus资金的支持，拉基金会倒拉RECHERCHE MEDICALE和斯特拉斯堡的CI-FRC。
Materials
| Name of Material | Company | Catalog Number | Comments/Description |
| ddH20 (ultrapure) | Millipore | – | Use always fresh water. |
| Parafilm (plastic film) | Bemis | PM-999 | Adhere Parafilm to the lab bench using some water droplets and ensure a perfect surface flatness. |
| Photo-mask | Selba | – | http://www.selba.ch |
| Silicon wafer | Siltronix | – | http://www.siltronix.com/ |
| SU-8 photoresist | MicroChem | 2000 series | http://www.microchem.com/Prod-SU82000.htm |
| working in a fumehood is required; check the data sheet from the manufacturer for more information. | |||
| SU-8 developer | MicroChem | – | http://microchem.com/Prod-Ancillaries.htm |
| working in a fumehood is required; check the data sheet from the manufacturer for more information | |||
| 2-propanol | Sigma-Aldrich | 19030 | http://www.sigmaaldrich.com/catalog/product/sial/i9030?lang=en®ion=CA |
| Available from multiple companies. | |||
| Sigmacote (siliconizing reagent ) | Sigma-Aldrich | SL2-25ML | http://www.sigmaaldrich.com/catalog/product/sigma/sl2?lang=fr®ion=FR |
| harmful, working in a fumehood is required; check the data sheet from the manufacturer for more information. | |||
| Chlorotrimethylsilane (TMCS) | Sigma-Aldrich | 386529-100ML | http://www.sigmaaldrich.com/catalog/product/aldrich/386529?lang=fr®ion=FR |
| TMCS produces acute inhalation and dermal toxicity, and is highly flammable (with ignition flashback able to occur across considerable distances), consequently it should be used in a fume cupboard away from sources of ignition | |||
| Nitrile gloves | Kleenguard | 57372 | http://www.kcprofessional.com/products/ppe/hand-gloves/thin-mil-/57372-kleenguard-g10-blue-nitrile-gloves-m |
| Available from multiple companies. | |||
| Glass coverslips #0 | Knittel glass | KN00010022593 | http://www.knittelglass.com/index_e.htm |
| Very fragile. Manipulate gently. | |||
| Sharp straight tweezers | SPI | 0WSSS-XD | http://www.2spi.com/catalog/tweezers/t/elec7 |
| 50 ml tube | BD Falcon | 352070 | http://www.bdbiosciences.com/cellculture/tubes/features/index.jsp |
| Available from multiple companies. | |||
| PDMS | Dow Corning | Sylgard 184 kit | http://www.dowcorning.com/applications/search/default.aspx?R=131EN |
| The package contains both PDMS base and curing agent. Similar elastomers are available from multiple companies. | |||
| Microscope glass slides | Dutscher | 100001 | http://www.dutscher.com/frontoffice/search |
| Available from multiple companies. | |||
| DMEM high-glucose medium | Fisher Scientific | 41965-039 | http://www.fishersci.com/ecomm/servlet/Search?LBCID=12301479&keyWord=41965-039&store=Scientific&nav=0&offSet=0&storeId=10652&langId=-1&fromSearchPage=1&searchType=PROD |
| Bovine calf serum | Sigma-Aldrich | C8056-500ML | http://www.sigmaaldrich.com/catalog/product/sigma/c8056?lang=en®ion=CA |
| 0,25% Trypsin-EDTA | Fisher Scientific | 25200-072 | http://www.fishersci.com/ecomm/servlet/Search?keyWord=25200-072&store=Scientific&nav=0&offSet=0&storeId=10652&langId=-1&fromSearchPage=1&searchType=PROD |
| PBS 1X | Fisher Scientific | 14200-067 | http://www.fishersci.com/ecomm/servlet/Search?keyWord=14200-067&store=Scientific&nav=0&offSet=0&storeId=10652&langId=-1&fromSearchPage=1&searchType=PROD |
| PBS is at 10X and should be diluted to 1X using ddH2O | |||
| L-15 medium | Fisher Scientific | 21083-027 | http://www.fishersci.com/ecomm/servlet/Search?keyWord=21083-027&store=Scientific&nav=0&offSet=0&storeId=10652&langId=-1&fromSearchPage=1&searchType=PROD |
| Medium for atmospheres without CO2 control | |||
| Fibronectin | Sigma-Aldrich | F1141-5MG | http://www.sigmaaldrich.com/catalog/search?interface=All&term=F1141-5MG&lang=en®ion=CA&focus=product&N=0+220003048+219853082+219853286&mode=match%20partialmax |
| Penicillin & Streptomycin | Fisher Scientific | 15140-122 | http://www.fishersci.com/ecomm/servlet/Search?keyWord=15140-122&store=Scientific&nav=0&offSet=0&storeId=10652&langId=-1&fromSearchPage=1&searchType=CHEM |
| Petri dish P35 | Greiner | 627102 | http://www.greinerbioone.com/en/row/articles/catalogue/article/144_11/12885/ |
| Petri dish P60 | Greiner | 628163 | http://www.greinerbioone.com/nl/belgium/articles/catalogue/article/145_8_bl/24872/ |
| Petri dish P94 | Greiner | 633179 | http://www.greinerbioone.com/nl/belgium/articles/catalogue/article/146_8_bl/24882/ |
| Paraformaldehyde 3 % | Sigma-Aldrich | P6148-500G | http://www.sigmaaldrich.com/catalog/product/sial/p6148?lang=fr®ion=FR |
| Harmful in-particular for the eyes, working in a fumehood is required; check the data sheet from the manufacturer for more information. | |||
| Triton 0.5 % | Sigma-Aldrich | 93443-100ML | http://www.sigmaaldrich.com/catalog/search?interface=All&term=93443-100ML&lang=en®ion=CA&focus=product&N=0+220003048+219853082+219853286&mode=match%20partialmax |
| Phallodin-Green Fluorescent Alexa Fluor 488 | InVitrogen | A12379 | http://www.lifetechnologies.com/order/catalog/product/A12379?CID=search-a12379 |
| dissolve powder in 1.5 ml methanol | |||
| Alexa Fluor 647 | InVitrogen | A21245 | 1:200 dilution in PBS 1X |
| rabbit polyclonal anti-Giantin | Abcam | ab24586 | 1:500 dilution in PBS 1X |
| http://www.abcam.com/giantin-antibody-ab24586.html | |||
| rabbit anti-anillin | Courtesy of M. Glotzer, Published in Piekny, A. J. & Glotzer, M. Anillin is a scaffold protein that links RhoA, actin, and myosin during cytokinesis. Current biology 18, 30–6 (2008). | 1:500 dilution in PBS 1X | |
| Anti-phosphotyrosine | Transduction Lab | 610000 | http://www.bdbiosciences.com/ptProduct.jsp?ccn=610000 |
| Cy3 goat anti-rabbit | Jackson Immunoresearch | 111-166-047 | http://www.jacksonimmuno.com/catalog/catpages/fab-rab.asp |
| 1:1000 dilution in PBS 1X | |||
| DAPI | Sigma-Aldrich | D8417 | http://www.sigmaaldrich.com/catalog/product/sigma/d8417?lang=fr®ion=FR |
| 1 mg/ml for 1 min | |||
| Glycerol | Sigma-Aldrich | G2025 | http://www.sigmaaldrich.com/catalog/search?interface=All&term=G2025&lang=en®ion=CA&focus=product&N=0+220003048+219853082+219853286&mode=match%20partialmax |
| Mineral oil | Sigma-Aldrich | M8410-500ML | http://www.sigmaaldrich.com/catalog/search?interface=All&term=M8410-500ML&lang=en®ion=CA&focus=product&N=0+220003048+219853082+219853286&mode=match%20partialmax |
| HeLa cells | – | – | Mammalian cells are available from many companies. See also Table 1 |
| NIH3T3 cells | ATCC | – | Mammalian cells are available from many companies. See also Table 1 |
| Fission yeast | – | – | For details on strains, contact the corresponding author. See also Table 1 |
| C. elegans worms | – | – | For details, contact the corresponding author. See also Table 1 |
| YES (Agar) + 5 Supplements included | MP Biomedicals | 4101-732 | http://www.mpbio.com/search.php?q=4101-732&s=Search |
| For preparation: follow instructions as given on the box | |||
| YES (Media) + 5 Supplements included | MP Biomedicals | 4101-522 | http://www.mpbio.com/search.php?q=4101-522&s=Search |
| For preparation: follow the instructions as given on the box | |||
| EMM (Media) | MP Biomedicals | 4110-012 | http://www.mpbio.com/search.php?q=4110-012&s=Search |
| For preparation: follow instructions as given on the box | |||
| Filter sterilized EMM (Media) – Only for imaging | MP Biomedicals | 4110-012 | For preparation: follow instructions as given on the box. Filter sterilize the media using a 0.22 µm filter instead of autoclaving. This gives transparency to the media and reduces the autofluorescence. |
| Supplements (for EMM) | MP Biomedicals | 4104-012 | http://www.mpbio.com/search.php?q=4104-012&s=Search |
| (Add 225mg/l into the EMM media before autoclaving or filtering) | |||
| Stericup and Steritop Vaccum driven sterile filters | Millipore | – | http://www.millipore.com/cellbiology/flx4/cellculture_prepare&tab1=2&tab2=1#tab2=1:tab1=2 |
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Citar este artículo
Wollrab, V., Caballero, D., Thiagarajan, R., Riveline, D. Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening. J. Vis. Exp. (115), e51880, doi:10.3791/51880 (2016).