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 ...

The overall goal of the following experiment is to observe the morphology and dynamics of cellular organelles on an array of microcavities mimicking the 3D environment encountered in physiological conditions. For cells with rigid walls, such as fission yeast, cavities can organize and orientate cellular structures. This is achieved by first fabricating an array of PDMS pillars from a master to obtain an array of PDMS microcavities, termed egg cups, by replica molding.As a second step, the egg cups are functionalized with proteins of the extracellular matrix and introduced into a tube with a cylindrical custom-made plastic piece, which supports the microcavity sample and allows the introduction of individual mammalian cells on each of the microcavities. Next, the egg cups filled with cells are removed and incubated with the drug or compound of interest in order to study the phenotype and dynamic processes of cells in a 3D confinement. Results are obtained that show differences in cell phenotype and novel dynamic structures observed when compared to standard 2D in vitro assays based on the orientation of cells.The main advantage of this technique over existing methods, like standard 2D cell culture assays, is that mammalian cells in egg cups experience a 3D environment similar to the logical microenvironments but are arranged in an ordered and standardized grade. Order and orientation are also significant improvements in the case of fission yeast. This method can help answer key questions in the cell skeletal and drug discovery field, such as identifying new and anti-cancer drug with high content screening.The implications of this technique extend to a diagnosis of multi-factorial diseases such as cancer, because drug screening assays can easily be performed and analyzed by means of egg cups, making this method particularly interesting for personalized medicine. This method can provide inside key to organized structures and dynamics in individual mammalian cells and fission yeast cells, and it can also be applied to organizing study, developing embryos such as C.elegans and ri-so-flu-a. In this protocol, an array of microcavities or egg cups is patterned onto a polydimethylsiloxane or PDMS layer adhered on a cover slip by replica molding.The first step is the fabrication of the master, which will not be shown, but is described in the text protocol. Once the master has been made, the next step is to fabricate the PDMS replica. In a 50 milliliter tube, thoroughly mix the cross-linker and the prepolymer in a one to 10 weight to weight or volume to volume ratio for a total of 30 grams.Centrifuge the tube at 1, 800 times g for five minutes to remove air bubbles. Gently drop the PDMS on top of the master. If bubbles appear, degas the mix.Place the sample in an oven at 65 degrees Celsius for four hours. After the PDMS is cured, use a scalpel to gently cut the area of interest of about one square centimeter, which includes approximately 10 to the fifth microcavities, or egg cups. Peel the PDMS stamp off gently.To begin the procedure for replica molding, activate the PDMS stamp by oxygen plasma treatment for 30 seconds. Store the activated stamp temporarily in a closed petri dish to prevent deposition of dust. Working in a fume hood, place the activated PDMS stamp with the side with the structures facing up or upside up in a petri dish next to a 15 milliliter cap.Fill the cap with 200 microliters of trimethylchlorosilane. Close the petri dish, and let the stamp salinize for seven minutes. Place the PDMS stamp on the spin coater with the structures upside up.Put a small drop of liquid PDMS on top of the structures. Spin coat at 1500 RPM for 30 seconds to deposit a thin layer of PDMS on top of the structures. Place the stamp in the oven at 65 degrees Celsius for four hours to cure the deposited spin-coated PDMS layer.Next, activate the thin PDMS layer by placing the PDMS stamp upside up together with a glass cover slip in an oxygen plasma cleaner for 30 seconds. Immediately place the stamp in contact with the glass cover slip and gently press all around the surface of the stamp with tweezers for the bonding. After that, keep a constant pressure on top of the stamp for around 10 seconds.30 minutes later, gently peel the stamp out of the cover slip in order to liberate the egg cups. Be very careful not to break the sample. Rinse the egg cups thoroughly with ethanol and dry.If PDMS egg cups are not on the glass cover slip and detached during the unpinning step, consider adjusting the settings of the plasma cleaner and restart the procedure for replica molding. To facilitate the manipulation of the sample later, it is recommended that a small piece of cured PDMS be glued at the edge of the cover slip. In the case of mammalian cells presented in this video, the PDMS surface must be functionalized with adhesion proteins of the extracellular matrix.Hydrophilize the egg cups in the oxygen plasma cleaner for 30 seconds. After that, sterilize the egg cups with ultraviolet light for five minutes. Deposit a small drop of fibronectin solution to cover the entire egg cup area, and incubate for one hour at room temperature.Protect the sample from drying. Gently rinse the egg cups with about one milliliter of PBS, using a plastic dispense pipette. Repeat a total of three times.Next, place a custom-made cylindrical plastic piece into a 50 milliliter tube. Add 13 milliliters of cell culture medium to the tube. The medium should reach at least two centimeters above the cylindrical piece to ensure complete immersion of the sample.Using sharp and straight tweezers to hold the sample by the PDMS handle, carefully place the egg cups inside the tube, gently pressing the cover slip until it lies on top of the upper side of the plastic piece and is fully immersed. The cells to be introduced into the egg cups should be cultured to 80 to 100%confluency and resuspended in five milliliters of culture medium. Pipette 200 microliters of cells on top of the egg cups, dropping cells as centered as possible on top of the egg cups, but avoiding physical contact.This will prevent breakage and/or damage of the sample. Centrifuge the 50 milliliter tube at 1, 800 times g for two minutes. Pipette another 200 microliters of cells on top of the egg cups and centrifuge again at 1, 800 times g for two minutes.Repeat for a total of three times in order to optimize the filling percentage. After the last centrifugation, check the filling percentage of egg cups with a microscope. Using the tweezers to hold the PDMS handle, remove the sample carefully from the tube without disturbing the cells in the egg cups, and place the sample in a petri dish with medium.To remove the excess cells that are not in the egg cups, rinse by gently pipetting up and down three times next to each side of the microstructure array. Lastly, replace the medium with fresh medium to remove nonattached cells. To observe fixed cellular organelles, place the egg cups with a fixed sample on the stage of an epifluorescence inverted microscope and select the 60x oil objective.In this example, the sample is NIH 3T3 fibroblasts, where the Golgi apparatus, nucleus, and actin fibers have been stained. Focus carefully using bright field light until the egg cups and cells are in the plane of observation. Select and focus the region of interest and start the image capture.Begin this procedure by placing the egg cups into a microscope holder and filling it with one milliliter of 10%FCS L15 cell culture medium. Install the holder on the stage of the microscope. To avoid evaporation, place a glass lid on top of the holder.Observe the cells with an epifluorescence inverted microscope equipped with a 60x oil objective. This example uses HeLa cells that express fluorescently tagged myosin and actin, two key molecules involved in the cytokinetic ring closure during mitosis. Select the region of interest and look for a cytokinetic ring, using either the GFP or Texas Red channel.Focus accurately. Run the automatic acquisition in both channels until the ring is completely closed. Scanning electron microscope images of egg cups show that their shape and size are very regular.For cells with rigid walls such as fission yeast, the egg cups can orient the cytokinetic ring. For mammalian cells, a comparison between cells on 3D egg cups and 2D flat surfaces reveals that the former form an ordered array with the homogeneous spherical-like phenotype, while the latter show a disordered and heterogeneous morphology. Cells on egg cups also show a reduction in the number of stress fibers, as indicated by the number of modest effect of treatment with Blebbistatin.The Golgi apparatus on 2D cultures typically shows an extended phenotype, whereas in egg cups, it show a more compacted phenotype. This was confirmed by a quantification of the Golgi phenotypes. Lastly, on 2D surfaces, the cell nucleus is randomly orientated, whereas on egg cups, it is orthogonally oriented on the xy-plane, in both wild type and Blebbistatin-treated cells.A determination of nucleus sphericity values reveals that the egg cups do not affect the normal sphericity of the cell nucleus. However, the difference between wild type and Blebbistatin-treated cells on egg cups suggests that the egg cups are revealing a real effect of the drug that is masked in 2D. Live cell studies using egg cups enable the identification of novel active processes, which are not visible in standard cultures.For example, periodic accumulations of myosin that move radially as the cytokinetic ring is closing during mitosis were observed in HeLa cells. Once mastered, the egg cups can be prepared from a replica in less than six hours, and the filling of the cells can be done in about 15 minutes. While attempting this process, it's important to have the size of the egg cups to the cells under study.This procedure can be further adapted to high content screening platforms in pharmaceutical companies for mass screening activities and also in personalized diagnostic assays of patients in assessing individual specific treatments. After watching this video, you should have a good understanding of how to fabricate and use the egg cups microcavities. The egg cups standardize and generate order to a variety of similar systems, ranging from individual cell without walls, such as mammalian cells, to system with walls, such as yeast and embryo.

Research

JoVE Journal

Bioengineering

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

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Nanomanipulation of Single RNA Molecules by Optical Tweezers

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Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

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A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

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3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

A Protocol for Real-time 3D Single Particle Tracking

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Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...