We acknowledge L. Brino (IGBMC High Content Screening facility, Illkirch, France) for providing us with the anti-Giantin antibody, M. Labouesse Lab. for C. elegans (IGBMC) and B. S&#233;raphin Lab. for budding yeast (IGBMC), E. Paluch and A. Hyman for fluorescent HeLa cells (MPI-CBG, Dresden), J. Moseley (Dartmouth Medical School) and J.Q. Wu (Ohio State University) for fission yeast cells; A. Ho&#235;l and F. Evenou for experimental help, C. Rick (IBMC, Strasbourg, France) for technical help, and J.C. Jeannot (Femto-st, France) for help in microfabrication. This work was supported by funds from the CNRS, the University of Strasbourg, Conectus, La Fondation pour la Recherche M&#233;dicale and the ci-FRC of Strasbourg.

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</ol>

We have nothing to disclose.

Replica molding was used in order to fabricate the &#8216;eggcups&#8217;. The fabrication process does not need a clean room; it is easy and simple, although some practice may be required. In particular, releasing the PDMS stamp is the most critical step in order to produce a large area of high quality &#8216;eggcups&#8217;. For this reason, special care has to be taken in this step. If this step is repeatedly failing, consider to optimize the plasma cleaner parameters prior to the silanization and plasma binding. Insufficient silanization will lead to strong sticking of the stamp to the PDMS film. If this is observed, the incubation time with the silanizing&#160;reagent can be increased. Note that other techniques and materials can be applied to fabricate the &#8216;eggcups&#8217;, which can be functionalized with a large range of ligands (fibronectin, gelatin, collagen, etc.). In particular, microcavities in polystyrene can be easily fabricated by custom-made hot-embossing technique. This ensures biocompatibility and direct comparison with results obtained in standard culture dishes. Similarly, special care and practice are required in order to optimize the filling percentage. In particular, the rinsing step is critical in order to ensure an appropriate filling with no excess of cells, contributing to noise and background in the signal. If cells are removed easily from cavities, consider to change the size or depth of cavities.
&#8216;Eggcups&#8217; provide 3D-like architecture to cells and high-content screening assays using a simple protocol. Cellular organelles and active processes unknown using standard culture assays can be easily visualized by means of inserting single cells on individual microcavities (&#8216;eggcups&#8217;). Depending on the model system, the size, shape and their dimensions can be easily adapted. In this way mammalian cells, fission yeast, budding yeast and C. elegans can be manipulated and studied, as well as any embryos such as Drosophila, mice or human embryos for in vitro fertilization, or stem cells for example.
In this setup single cells are captured. This is in contrast to epithelial tissues encountered in vivo. However, this environment could be reproduced in our &#8216;eggcups&#8217; by coating the side walls with cadherins to mimic cell-cell contacts using more flexible elastomers. Focal contacts will be promoted by the deposition of fibronectin at the bottom of wells. These respective distributions of adhesion molecules should allow in reproducing the cellular environments encountered in vivo. By this method one would approach the physiological conditions.
Medium exchange in our assay is ensured. Cells in EC do not show any degradation when performing both short- and long-term experiments due to lack of medium exchange. Note also that cells in EC can be cultured until confluence although the main interest is when individual cells or embryos are isolated within the cavities.
Orientation of organelles or entire organisms is revealing new information. We show different dynamics of actin and myosin in the cytokinetic ring. Although the cytokinetic ring in fission yeast and mammalian cells is composed of similar key components, we show with this setup, that their specific dynamics is different 17. This is supporting the result, that the closure mechanism in the two systems is different as well. To develop and investigate such a hypothesis, the orientation of the cell is indispensable. In future studies, this device can be also used to investigate other events related to organelle organization in cells.
Beyond that, this technique can be of great use in developmental biology. Elongated embryos can be easily oriented, observed or further treated in a defined orientation. Probably our assay would not impose polarity of embryos, but the high filling percentage would allow to extract the desired read-out in a reliable manner. Altogether &#8216;eggcups&#8217; could be a good device for high-content screenings.
Other culture assays have been proposed. These methods range from multiple cells in 2D dimensions in multiwell plates, to single cells deposited in micropatterned adhesive motifs with identical shape. However, none of them is appropriate to overcome the limitations detailed above on the observation of cellular organelles and dynamical processes 1.
Future improvements to our system will allow the applicability of &#8216;eggcups&#8217; to industry-oriented purposes. As an example, drug screening applications in pharmaceutical companies require the use of multiwell plates 14,28; implementing &#8216;eggcups&#8217; into such platforms will potentially improve the reliability of tests and results. As such, high&#160;content-screening assays will be performed using the commonly used automatized&#160;processes of pharmaceutical companies (and academic research laboratories) using robots. This will ensure repeatability and reliability with low variability. Some commercial products based on 3D-cell cultured assays have already appeared in the market highlighting the importance of this kind of assays. Finally, these devices open new perspectives for personalized medicine: cells from patient could be placed in &#8216;eggcups&#8217;, and treatment cocktails could be tested in a physiological environment; the biomarker read-out will allow to anticipate an optimal treatment to be given to the patient 29. Altogether the physical shape of the cells and embryos are guiding the architecture of the cavities, and we hope that the device and this method will be widely spread in the future.

Current in vitro cell-based assays are two-dimensional (2D). This configuration is not natural for mammalian cells and therefore is not physiologically relevant 1; cells show a diversity of shapes, sizes and heterogeneous phenotypes. They present additional serious limitations when applied to screening applications, such as a disordered distribution within the plane and extreme phenotypes of cellular organelles (stress fibers, in particular). This is particularly important in clinical trials for drug testing, where high budgets are spent each year. Most of these drugs though fail when applied to animal models because of the artificial 2D culture condition in early stages of drug screening. In addition, by using this approach, specific cell organelles cannot be properly visualized, such as the cytokinetic actomyosin ring during cell mitosis, and generally structures that are evolving in the plane perpendicular to the plane of observation. Some new 2D assays have been proposed in order to overcome the above-mentioned drawbacks and important insights on cytoskeleton organization have been observed 2,3. However, these assays still present one serious limitation: cells show a very spread phenotype in contrast to what is observed in vivo, where cells present a 3D architecture. These artifacts associated with the culture method may trigger non-physiological features such as enhanced stress fibers 1,4,5.
Three-dimensional cell culture assays provide multiple advantages when compared to 2D environments 6,7. They are physiologically more relevant, and results are therefore meaningful. As an example, cells embedded in hydrogels show 3D-like structures but their morphologies differ from one cell to another 8,9 . However, their morphologies differ from one cell to another, which complicates screening applications. An alternative strategy is to embed single cells in microfabricated cavities 10,11. Cell position, shape, polarity and internal cell organization can then become normalized. Besides providing 3D-like architecture to cells, microcavities also allows for high-content screening studies 10,12-14; single cells can be ordered into microarrays and cellular organelles and their evolutions can be observed in parallel. This regularity provides good statistics with low number of cells and better temporal/spatial resolutions. Useful compounds&#160;are easier to identify reliably.
In this study, we show the fabrication and application of a new 3D-like single cells culture system for high-content-screening applications 10,12,13. The device consists of an array of elastomeric microcavities (105 cavities/cm2), coined &#8216;eggcups&#8217; (EC). Dimensions and total volume of EC in this work are optimized to the typical volume of individual NIH3T3 and HeLa cells during cell division. Morphology of the cavities &#8211; cylindrical &#8211; is selected to properly orient cell shape for the visualization of active processes. Replica molding is used to pattern an array of EC onto a thin polydimethylsiloxane (PDMS) layer adhered on a glass coverslip 15,16. Cells are introduced in the EC by centrifugation. We report here observation and normalization of cellular organelles (actin stress fibers, Golgi apparatus and nucleus) in 3D (EC) in comparison with the same cells on 2D (flat) surfaces. We also report the observation of active dynamical processes such as the closure of the cytokinetic actomyosin ring during cell mitosis 17. Finally, we show results of this methodology on other systems with rigid walls, such as budding yeast, fission yeast and C. elegans embryos which confirms the applicability of our methodology to a wide range of model systems.
We next present a detailed and exhaustive protocol in order to fabricate and apply the &#8216;eggcups&#8217; for 3D microfabrication. Our approach is simple and does not need a clean room. We anticipate that this new methodology will be particularly interesting for drug screening assays and personalized medicine, in replacement of&#160;Petri dishes. Finally, our device will be useful for studying the distributions of cells responses to external stimuli, for example in cancer 18&#160;or in basic research 19.

<table><tbody><tr><td>ddH&lt;sub&gt;2&lt;/sub&gt;0 (ultrapure)</td><td>Millipore</td><td>-</td><td>Use always fresh water.</td></tr><tr><td>Parafilm (plastic film)</td><td>Bemis</td><td>PM-999</td><td>Adhere Parafilm to the lab bench using some water droplets and ensure a perfect surface flatness.</td></tr><tr><td>Photo-mask</td><td>Selba</td><td>-</td><td>&lt;a href=&quot;http://www.selba.ch/&quot; name=&quot;RANGE!D4&quot;&gt;http://www.selba.ch&lt;/a&gt;</td></tr><tr><td>Silicon wafer</td><td>Siltronix</td><td>-</td><td>&lt;a href=&quot;http://www.siltronix.com/&quot;&gt;http://www.siltronix.com/&lt;/a&gt;</td></tr><tr><td>SU-8 photoresist</td><td>MicroChem</td><td>2000 series</td><td>&lt;a href=&quot;http://www.microchem.com/Prod-SU82000.htm&quot;&gt;http://www.microchem.com/Prod-SU82000.htm&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>working in a fumehood is required; check the data sheet from the manufacturer for more information.</td><td></td><td></td><td></td></tr><tr><td>SU-8 developer</td><td>MicroChem</td><td>-</td><td>&lt;a href=&quot;http://microchem.com/Prod-Ancillaries.htm&quot;&gt;http://microchem.com/Prod-Ancillaries.htm&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>working in a fumehood is required; check the data sheet from the manufacturer for more information</td><td></td><td></td><td></td></tr><tr><td>2-propanol</td><td>Sigma-Aldrich</td><td>19030</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/i9030?lang=en&amp;amp;region=CA&quot;&gt;http://www.sigmaaldrich.com/catalog/product/sial/i9030?lang=en&amp;amp;region=CA&lt;/a&gt;</td></tr><tr><td>Available from multiple companies.</td><td></td><td></td><td></td></tr><tr><td>Sigmacote (siliconizing reagent )</td><td>Sigma-Aldrich</td><td>SL2-25ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/sl2?lang=fr&amp;amp;region=FR&quot;&gt;http://www.sigmaaldrich.com/catalog/product/sigma/sl2?lang=fr&amp;amp;region=FR&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>harmful, working in a fumehood is required; check the data sheet from the manufacturer for more information.&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Chlorotrimethylsilane (TMCS)</td><td>Sigma-Aldrich</td><td>386529-100ML</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/386529?lang=fr&amp;amp;region=FR&amp;nbsp;&amp;nbsp;</td></tr><tr><td colspan="4">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</td></tr><tr><td>Nitrile gloves</td><td>Kleenguard</td><td>57372</td><td>&lt;a href=&quot;http://www.kcprofessional.com/products/ppe/hand-gloves/thin-mil-/57372-kleenguard-g10-blue-nitrile-gloves-m&quot;&gt;http://www.kcprofessional.com/products/ppe/hand-gloves/thin-mil-/57372-kleenguard-g10-blue-nitrile-gloves-m&lt;/a&gt;</td></tr><tr><td>Available from multiple companies.</td><td></td><td></td><td></td></tr><tr><td>Glass coverslips #0</td><td>Knittel glass</td><td>KN00010022593</td><td>&lt;a href=&quot;http://www.knittelglass.com/index_e.htm&quot;&gt;http://www.knittelglass.com/index_e.htm&lt;/a&gt;</td></tr><tr><td>Very fragile. Manipulate gently.</td><td></td><td></td><td></td></tr><tr><td>Sharp straight tweezers</td><td>SPI</td><td>0WSSS-XD</td><td>http://www.2spi.com/catalog/tweezers/t/elec7</td></tr><tr><td>50 ml tube</td><td>BD Falcon</td><td>352070</td><td>http://www.bdbiosciences.com/cellculture/tubes/features/index.jsp</td></tr><tr><td>Available from multiple companies.</td><td></td><td></td><td></td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>Sylgard 184 kit</td><td>&lt;a href=&quot;http://www.dowcorning.com/applications/search/default.aspx?R=131EN&quot;&gt;http://www.dowcorning.com/applications/search/default.aspx?R=131EN&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>The package contains both PDMS base and curing agent. Similar elastomers are available from multiple companies.</td><td></td><td></td><td></td></tr><tr><td>Microscope glass slides</td><td>Dutscher</td><td>100001</td><td>&lt;a href=&quot;http://www.dutscher.com/frontoffice/search&quot;&gt;http://www.dutscher.com/frontoffice/search&lt;/a&gt;</td></tr><tr><td>Available from multiple companies.</td><td></td><td></td><td></td></tr><tr><td>DMEM high-glucose medium</td><td>Fisher Scientific</td><td>41965-039</td><td>&lt;a href=&quot;http://www.fishersci.com/ecomm/servlet/Search?LBCID=12301479&amp;amp;keyWord=41965-039&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&quot;&gt;http://www.fishersci.com/ecomm/servlet/Search?LBCID=12301479&amp;amp;keyWord=41965-039&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&lt;/a&gt;</td></tr><tr><td>Bovine calf serum</td><td>Sigma-Aldrich</td><td>C8056-500ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/c8056?lang=en&amp;amp;region=CA&quot;&gt;http://www.sigmaaldrich.com/catalog/product/sigma/c8056?lang=en&amp;amp;region=CA&lt;/a&gt;</td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Fisher Scientific</td><td>25200-072</td><td>&lt;a href=&quot;http://www.fishersci.com/ecomm/servlet/Search?keyWord=25200-072&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&quot;&gt;http://www.fishersci.com/ecomm/servlet/Search?keyWord=25200-072&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&lt;/a&gt;</td></tr><tr><td>PBS 1x</td><td>Fisher Scientific</td><td>14200-067</td><td>&lt;a href=&quot;http://www.fishersci.com/ecomm/servlet/Search?keyWord=14200-067&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&quot;&gt;http://www.fishersci.com/ecomm/servlet/Search?keyWord=14200-067&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&lt;/a&gt;</td></tr><tr><td>PBS is at 10x and should be diluted to 1x using ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>L-15 medium</td><td>Fisher Scientific</td><td>21083-027</td><td>&lt;a href=&quot;http://www.fishersci.com/ecomm/servlet/Search?keyWord=21083-027&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&quot;&gt;http://www.fishersci.com/ecomm/servlet/Search?keyWord=21083-027&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&lt;/a&gt;</td></tr><tr><td>Medium for atmospheres without CO&lt;sub&gt;2&lt;/sub&gt; control</td><td></td><td></td><td></td></tr><tr><td>Fibronectin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F1141-5MG</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=F1141-5MG&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&quot;&gt;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=F1141-5MG&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&lt;/a&gt;</td></tr><tr><td>Penicillin &amp;amp; Streptomycin</td><td>Fisher Scientific</td><td>15140-122</td><td>&lt;a href=&quot;http://www.fishersci.com/ecomm/servlet/Search?keyWord=15140-122&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=CHEM&quot;&gt;http://www.fishersci.com/ecomm/servlet/Search?keyWord=15140-122&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=CHEM&lt;/a&gt;</td></tr><tr><td>Petri dish P35</td><td>Greiner</td><td>627102</td><td>&lt;a href=&quot;http://www.greinerbioone.com/&quot;&gt;http://www.greinerbioone.com/en/row/articles/catalogue/article/144_11/12885/&lt;/a&gt;</td></tr><tr><td>Petri dish P60</td><td>Greiner</td><td>628163</td><td>&lt;a href=&quot;http://www.greinerbioone.com/&quot;&gt;http://www.greinerbioone.com/nl/belgium/articles/catalogue/article/145_8_bl/24872/&lt;/a&gt;</td></tr><tr><td>Petri dish P94</td><td>Greiner</td><td>633179</td><td>&lt;a href=&quot;http://www.greinerbioone.com/&quot;&gt;http://www.greinerbioone.com/nl/belgium/articles/catalogue/article/146_8_bl/24882/&lt;/a&gt;</td></tr><tr><td>Paraformaldehyde 3 %</td><td>Sigma-Aldrich</td><td>P6148-500G</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/p6148?lang=fr&amp;amp;region=FR&quot;&gt;http://www.sigmaaldrich.com/catalog/product/sial/p6148?lang=fr&amp;amp;region=FR&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>Harmful in-particular for the eyes, working in a fumehood is required; check the data sheet from the manufacturer for more information.</td><td></td><td></td><td></td></tr><tr><td>Triton 0.5 %</td><td>Sigma-Aldrich</td><td>93443-100ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=93443-100ML&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&quot;&gt;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=93443-100ML&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&lt;/a&gt;</td></tr><tr><td>Phallodin-Green Fluorescent Alexa Fluor 488</td><td>InVitrogen</td><td>A12379</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/A12379?CID=search-a12379&quot;&gt;http://www.lifetechnologies.com/order/catalog/product/A12379?CID=search-a12379&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>dissolve powder in 1.5 ml methanol</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 647</td><td>InVitrogen</td><td>A21245</td><td>1:200 dilution in PBS 1x</td></tr><tr><td>rabbit polyclonal anti-Giantin</td><td>Abcam</td><td>ab24586</td><td>1:500 dilution in PBS 1x</td></tr><tr><td>&lt;a href=&quot;http://www.abcam.com/giantin-antibody-ab24586.html&quot;&gt;http://www.abcam.com/giantin-antibody-ab24586.html&amp;nbsp;&lt;/a&gt;</td><td></td><td></td><td></td></tr><tr><td>rabbit anti-anillin</td><td>Courtesy of M. Glotzer, Published in Piekny, A. J. &amp;amp; Glotzer, M. Anillin is a scaffold protein that links RhoA, actin, and myosin during cytokinesis. Current biology 18, 30&amp;ndash;6 (2008).</td><td></td><td>1:500 dilution in PBS 1x</td></tr><tr><td>Anti-phosphotyrosine</td><td>Transduction Lab</td><td>610000</td><td>http://www.bdbiosciences.com/ptProduct.jsp?ccn=610000</td></tr><tr><td>Cy3 goat anti-rabbit&amp;nbsp;</td><td>Jackson Immunoresearch</td><td>111-166-047</td><td>&lt;a href=&quot;http://www.jacksonimmuno.com/catalog/catpages/fab-rab.asp&quot;&gt;http://www.jacksonimmuno.com/catalog/catpages/fab-rab.asp&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>1:1,000 dilution in PBS 1x</td><td></td><td></td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D8417&amp;nbsp;</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/d8417?lang=fr&amp;amp;region=FR&quot;&gt;http://www.sigmaaldrich.com/catalog/product/sigma/d8417?lang=fr&amp;amp;region=FR&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>1 mg/ml for 1 min</td><td></td><td></td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G2025</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=G2025&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&quot;&gt;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=G2025&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&lt;/a&gt;</td></tr><tr><td>Mineral oil</td><td>Sigma-Aldrich</td><td>M8410-500ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=M8410-500ML&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&quot;&gt;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=M8410-500ML&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&lt;/a&gt;</td></tr><tr><td>HeLa cells</td><td>-</td><td>-</td><td>Mammalian cells are available from many companies. See also &lt;strong&gt;Table 1&lt;/strong&gt;</td></tr><tr><td>NIH3T3 cells</td><td>ATCC</td><td>-</td><td>Mammalian cells are available from many companies. See also &lt;strong&gt;Table 1&lt;/strong&gt;</td></tr><tr><td>Fission yeast&amp;nbsp;</td><td>-</td><td>-</td><td>For details on strains, contact the corresponding author.&amp;nbsp; See also &lt;strong&gt;Table 1&lt;/strong&gt;</td></tr><tr><td>&lt;em&gt;C. elegans&lt;/em&gt; worms</td><td>-</td><td>-</td><td>For details, contact the corresponding author.&amp;nbsp; See also &lt;strong&gt;Table 1&lt;/strong&gt;</td></tr><tr><td>YES (Agar) + 5 Supplements included</td><td>MP Biomedicals</td><td>4101-732</td><td>&lt;a href=&quot;http://www.mpbio.com/search.php?q=4101-732&amp;amp;s=Search&quot;&gt;http://www.mpbio.com/search.php?q=4101-732&amp;amp;s=Search&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>For preparation: follow&amp;nbsp; instructions as given on the box</td><td></td><td></td><td></td></tr><tr><td>YES (Media) + 5 Supplements included</td><td>MP Biomedicals</td><td>4101-522</td><td>&lt;a href=&quot;http://www.mpbio.com/search.php?q=4101-522&amp;amp;s=Search&quot;&gt;http://www.mpbio.com/search.php?q=4101-522&amp;amp;s=Search&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>For preparation: follow the instructions as given on the box</td><td></td><td></td><td></td></tr><tr><td>EMM (Media)</td><td>MP Biomedicals</td><td>4110-012</td><td>&lt;a href=&quot;http://www.mpbio.com/search.php?q=4110-012&amp;amp;s=Search&quot;&gt;http://www.mpbio.com/search.php?q=4110-012&amp;amp;s=Search&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>For preparation: follow&amp;nbsp; instructions as given on the box</td><td></td><td></td><td></td></tr><tr><td>Filter sterilized EMM (Media) - Only for imaging</td><td>MP Biomedicals</td><td>4110-012</td><td>For preparation: follow instructions as given on the box. Filter sterilize the media using a 0.22 &amp;micro;m filter instead of autoclaving. This gives transparency to the media and reduces the autofluorescence.</td></tr><tr><td>Supplements (for EMM)</td><td>MP Biomedicals</td><td>4104-012</td><td>&lt;a href=&quot;http://www.mpbio.com/search.php?q=4104-012&amp;amp;s=Search&quot;&gt;http://www.mpbio.com/search.php?q=4104-012&amp;amp;s=Search&lt;/a&gt;</td></tr><tr><td>&amp;nbsp;(Add 225 mg/L into the EMM media before autoclaving or filtering)</td><td></td><td></td><td></td></tr><tr><td>Stericup and Steritop Vaccum driven sterile filters</td><td>Millipore</td><td>-</td><td>&lt;a href=&quot;http://www.millipore.com/cellbiology/flx4/cellculture_prepare&amp;amp;tab1=2&amp;amp;tab2=1#tab2=1:tab1=2&quot;&gt;http://www.millipore.com/cellbiology/flx4/cellculture_prepare&amp;amp;tab1=2&amp;amp;tab2=1#tab2=1:tab1=2&lt;/a&gt;</td></tr></tbody></table>

ordering single cells and single embryos in 3d confinement: a new device for high content screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

Watch this Scientific Journal Video about Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening at JoVE.com

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

ordering-single-cells-single-embryos-3d-confinement-new-device-for

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.5K Views.  Institut de Science et d'Ingénierie Supramoléculaires (ISIS), CNRS and Université de Strasbourg. We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays. 

Video: Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Developmental Biology

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and computational modeling to classify and compare biological processes. Although cell culture model systems are great for high content imaging, high throughput studies of cell morphology suggest that ex vivo cultures are limited in recapitulating the morphological complexity found in cells within living organisms. As such, there is a need for a scalable high throughput model system to image living cells within an intact organism. Described here is a protocol for using a high content image analyzer to simultaneously acquire multiple time-lapse videos of embryonic Drosophila melanogaster development during the syncytial blastoderm stage. The syncytial blastoderm has traditionally served as a great in vivo model for imaging biological events; however, obtaining a significant number of experimental replicates for quantitative and high-throughput approaches has been labor intensive and limited by the imaging of a single embryo per experimental repeat. Presented here is a method to adapt imaging and microinjection approaches to suit a high content imaging system, or any inverted microscope capable of automated multipoint acquisition. This approach enables the simultaneous acquisition of 6-12 embryos, depending on desired acquisition factors, within a single imaging session.

Presented here is a protocol to microinject and simultaneously image multiple Drosophila embryos during embryonic development using a plate-based, high content imager.

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and ...

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Biology

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...

Immunology and Infection

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do not allow for accurate interpretation of this interplay. Using a quantitative imaging-based approach, we present a high-content protocol for characterizing the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations to changes in environmental stimuli. We highlight our ability to distinguish between cell types based upon either fluorescence intensity or inherent morphology features depending on the application. This platform allows for a more comprehensive characterization of subpopulation response to perturbation while utilizing shorter time, smaller amounts of reagents, and lower likelihood of error than traditional cell biology assays. However, in some cases, cell populations may be difficult to identify and quantitate based on complex cellular features and will require additional troubleshooting; we highlight some of these circumstances in the protocol. We demonstrate this application using response to drug in a cancer model; however, it can easily be applied more broadly to other physiological processes. This protocol allows one to identify subpopulations within a co-culture system and characterize the particular response of each to external stimuli.

We have developed a novel protocol for studying heterocellular population dynamics in response to perturbations. This manuscript describes an imaging-based platform that produces quantitative datasets for simultaneous characterization of multiple cellular phenotypes of heterocellular populations in a robust manner.

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and disease modeling, drug discovery, and regenerative medicine. To fully exploit these models, it is crucial to study them at cellular and subcellular levels. However, characterizing such in vitro 3D cell culture models can be technically challenging and requires specific expertise to perform effective analyses. Here, this paper provides detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed in vitro 3D cell culture models ranging from 100 &#181;m to several millimeters. These protocols are applicable to a wide variety of organoids and spheroids that differ in their cell-of-origin, morphology, and culture conditions. From 3D structure harvesting to image analysis, these protocols can be completed within 4-5 days. Briefly, 3D structures are collected, fixed, and can then be processed either through paraffin-embedding and histological/immunohistochemical staining, or directly immunolabeled and prepared for optical clearing and 3D reconstruction (200 &#181;m depth) by confocal microscopy.

Here, we provide detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed three-dimensional cell culture models ranging from 100 &#181;m to several millimeters, thus enabling the visualization of their morphology, cell-type composition, and interactions.

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Summary

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Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Upright Imaging of Drosophila Embryos

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models