Name: ordering single cells and single embryos in 3d confinement: a new device for high content screening
Uploaded: 2016-09-18T13:00:00
Description: 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

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.

1. Microfabrication of &#8216;Eggcups&#8217;
<ol>
	<li>Fabrication of the Master: Microcavities Array
	<ol>
		<li>Heat a 3&#8217;&#8217; silicon wafer up to 200 &#176;C to evaporate any presence of humidity.</li>
		<li>Spin-coat a thin layer of SU-8 photoresist. Adjust the volume of resin and spinning speed depending on the desired thickness and photoresist type. This thickness will dictate the depth of the &#39;eggcups&#39; (EC). For a 30 &#181;m thick layer and SU-8 2025, spin-coat at 2,800 rpm.</li>
		<li>Pre-bake t...

<ol>
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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. Insuff...

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.

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.

The &#8216;eggcups&#8217; (EC) are a novel high&#160;content-screening methodology which allows the visualization of oriented cells and embryos in a 3D environment. Additionally, some cellular processes, which are difficult to observe in standard 2D (flat) cultures, can be observed by this new method. Figure 1a shows a summary of the procedure for the EC microfabrication (see also Section 1 in the above-described protocol). The method is simple, fast, efficient and without any requirement of special equi...

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 ...

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