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 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, ‘eggcups’, 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.
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 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 ‘eggcups’ (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 – cylindrical – 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 ‘eggcups’ 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 Petri dishes. Finally, our device will be useful for studying the distributions of cells responses to external stimuli, for example in cancer 18 or in basic research 19.
1. Microfabrication of ‘Eggcups’
2. Introducing Cells into the ‘Eggcups’
In order to introduce mammalian cells inside ‘eggcups’, PDMS surface needs to be functionalized with adhesion proteins of the extracellular matrix. This example uses fibronectin but other proteins of interest, such as collagen, could be used.
3. Observation of Active Cellular Dynamics in ‘Eggcups’: Cytokinetic Ring Closure
NOTE: This example uses HeLa cells which are transfected with MYH10-GFP and Lifeact-mcherry for myosin and actin, respectively, key active molecules involved in the cytokinetic ring closure during cell mitosis. The device is prepared with microcavities of 25 µm in diameter. For their observation, an epifluorescence inverted microscope was used, equipped with a 60X oil objective (1.40 NA, DIC, Plan Apo) and GFP (myosin) and TxRed (actin) filters. Alternatively an upright confocal microscope was used, equipped with a 25X or 63X HCX IR APO L water objective (0.95 NA). For this example, it is highly recommended to synchronize cells by using the double thymidine block, mitotic block or mitotic shake-off method 21-24.
NOTE: The thickness of the PDMS used for the ‘eggcups’ allows the usage of a variety of objectives both in inverted and upright positioned microscopes.
4. Observation of Fixed Cellular Organelles into the ‘Eggcups’
This step can be performed before or after step #3. Cells can be directly fixed after the centrifugation step and stained for the organelle of interest or after the observation in the microscope. This example shows the staining of the Golgi apparatus, nucleus and actin fibers on NIH3T3 fibroblasts in ‘eggcups’.
5. Adaptation for the Observation of Yeast Cells and C. elegans Embryo
The ‘eggcups’ (EC) are a novel high 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 equipment. Figure 1b and 1c shows a large-scale picture and a magnified scanning electron microscope image of ‘eggcups’, respectively. As it can be observed, their shape and size are very regular. This method is very flexible; different shapes and sizes can easily be fabricated and adapted for different model systems. The dimensions of ´eggcups´ were selected in the following manner: dimensions of cells which undergo division were measured on 2D surfaces: they have a spherical shape and their diameter was taken as a good indication for the EC diameter. Cells in ´eggcups´ elongate and orient along their long axis during cell division for example. This dimension depends on the system – cells and embryos – so this dimension should be evaluated in each case.
Figure 2 shows the material needed (Figure 2a) and a step-by-step protocol (Figure 2b) about how to use the ‘eggcups’ (see also Section 2 in the above-described protocol). The filling of the EC with cells of interest (or other model systems) is very simple and fast. Typically, it takes less than 20-30 min, which also includes the time for cell trypsinization. After the filling, samples can be used to study active processes (live imaging) or can be fixed and stained for the visualization of organelles of interest (see also Sections 3 and 4 in the protocol described above).
On flat surfaces, cells show heterogeneous responses and extreme phenotypes of cellular organelles. In fact, it has been suggested that actin stress fibers (and other cellular organelles) are artifacts of the culture conditions 1. In order to prove this hypothesis, we cultured NIH3T3 cells both on 3D ‘eggcups’ and on flat surfaces and compared the phenotypes of different cellular organelles, namely actin stress fibers, Golgi apparatus and nuclei. Figure 3 shows an example of how cells are organized on both configurations. In EC, cells are distributed in an ordered array showing a homogeneous spherical-like phenotype (Figure 3a). On flat surfaces, cells show the typical disordered, spread and heterogeneous morphology (Figure 3b). There are also significant differences in cytoskeleton structures. In particular, cells on ‘eggcups’ show a reduction in the number of stress fibers compared to flat surfaces. This is further confirmed in the 3D reconstructed images where no clear stress fibers are visible (see Figure 3c–d). This confirms that some cellular structures are magnified in 2D cultures. This is also in agreement with observations performed in vivo where stress fibers cannot be identified.
The Golgi apparatus also shows significant variation in their phenotype depending on the culture condition (see Figure 4). The Golgi apparatus on 2D cultures typically shows an extended phenotype ‘embracing’ the nucleus periphery whereas in ‘eggcups’ it shows a more compacted phenotype (see Figure 4a–b). In order to simulate a drug screening manipulation, we also evaluated the effect of drugs on cells cultured on both environments. We selected Blebbistatin mainly because it disrupts the actin stress fibers and could have an effect on Golgi morphology (see Figure 3c–d). Since the Golgi is located next to the cell nucleus, this drug could also have an effect on its architecture. We first observed that cells treated with this drug showed a less regular and uniform morphology compared to wild type (WT) cells (see Figure 3c–d). We then compared and quantified the Golgi phenotype observed on ‘eggcups’ and on flat surfaces (see Figure 4c). We observed that on 2D surfaces cells showed mostly an extended phenotype whereas on ‘eggcups’ cells showed a more compacted phenotype. We did not observe though a striking difference between WT and Blebbistatin-treated cells.
Finally, on 2D surfaces the cell nucleus is randomly oriented whereas for cells in EC it is orthogonally oriented with respect to the XY plane in both WT and Blebbistatin treated cells (see Figure 5a–c). This highlights the strength of the device to orient cellular organelles, similar to a former application of the method for orienting the plane of observation of the cytokinetic ring in yeast and mammalian cells 10,12,13. We finally studied how the nucleus sphericity(defined as ψ = [π1⁄3 6Vn2⁄3]/An , where Vn is the volume of the nucleus and An its surface area) was affected depending on the culturing condition and upon the treatment of cells with Blebbistatin. Figure 5d shows the corresponding distributions of ψ. We did not observe a difference for WTflatvs WTEC, which reveals that the EC are not affecting the normal sphericity of cells. However, we observed a difference when comparing WTEC to BlebbEC suggesting that the EC are revealing a real effect of the drug that is masked in 2D.
Live cell studies using ‘eggcups’ allow also identification of novel active processes which are not visible in standard cultures. We plated cells in EC and visualized cell division. Figure 6 shows a sequence of images of the cytokinetic ring closure during cell mitosis. The ‘eggcups’ device allows a complete visualization of the ring, whereas standard 2D cultures only shows two areas which corresponds to one single plane 10. Reconstruction of the ring from a sequence of z-stack images using 2D cultures can be done 27, but important information is lost. The quality is diminished due to low z resolution and dynamic processes cannot be resolved. Actin and myosin are the key proteins in the force generation of cell division. Their dynamics cannot be imaged and studied in 2D culture (Figure 6a), whereas with ‘eggcups’ it is immediately revealed. We have identified novel structures and processes: in HeLa cells we find periodic accumulations of myosin 17. These accumulations move radially as the ring is closing (Figure 6b). In fission yeast we also find inhomogeneities in myosin and actin (Figure 6c, right) 17. In contrast to what we see in HeLa cells, they rotate on the ring during closure. The speed is in the range of µm min-1 and would not be resolvable by z reconstruction with standard microscopes. Finally the cytokinetic ring can be further studied by staining for its components. We find that there is an accumulation of phosphotyrosine in the vicinity of the ring (Figure 6d). We can also show that anillin is colocalizing in the ring (Figure 6e). By staining the cells in this orientation, we reveal that anillin shows also an inhomogeneous distribution.
The ‘eggcups’ were also applied to different model systems: we reported mammalian cells, fission yeast, but we also tested budding yeast and C. elegans (see Figure 7a–e). In this case, the protocol was adapted for each specific system in terms of culture media, cavities size and morphology (see Table 1). As an example, conical V-shaped ‘eggcups’ were the optimal morphology for immobilizing fission yeast efficiently 12, instead of completely cylindrical (or U-shaped) shape used for mammalian cells 13. This allowed testing the effect of different cytoskeleton drugs with potential application in Life Science research. This demonstrates the flexibility and reliability of the developed methodology.
Furthermore, the highly ordered arrangement of cells allows an easy, automated read-out of the fluorescence of single cells. We illustrate this by inserting NIH3T3 cells expressing GFP in ‘eggcups’ (Figure 8a). The cell position can be easily recognized and the corresponding expression level measured. Figure 8b shows the distribution of fluorescence signals. This can be applied to any read-out (immunofluorescence, fluorescent reporters in cells for example).
Figure 1: Fabrication of ‘eggcups’. (a) Schematic description of the fabrication procedure of ‘eggcups’ by replica molding: (i) Pour liquid PDMS on the SU-8 mold and cure it. (ii) Cut out the stamp and remove it carefully from the surface, then plasma activate it to silanize it. (iii) Pour liquid PDMS on the silanized stamp and centrifuge it to obtain a thin PDMS layer. (iv) After curing the PDMS layer, plasma activate both, the PDMS covered stamp and a glass coverslip. (v) Plasma bind both by applying a gentle, homogeneous pressure. (vi) After plasma bonding, remove carefully the stamp to uncover the ‘eggcups’ surface. (vii) To simplify the handling in the next steps, add a small PDMS handle piece. Bind the PDMS piece to the coverslip by gluing it with liquid PDMS and (viii) cure it then in the oven. (b) Image of a 25 mm coverslip with PDMS ‘eggcups’ and a handle. (c) Scanning electron microscope images of PDMS ‘eggcups’. The distance between centers of ‘eggcups’ is 30 μm, and their diameter about 25 μm. (Left) Top view. (Right) ‘Eggcups’ are cut to image the inner part. Please click here to view a larger version of this figure.
Figure 2: (a) Elements needed for the EC filling. (1) 50 ml tube; (2) cylindrical piece (top and side view); (3) cell culture medium; (4) ‘eggcups’; (5) sharp tweezers. (b) Schematic of the EC filling procedure. (i) A cylindrical piece is first introduced into a 50 ml tube and filled with 13 ml of cell culture medium. Next, (ii) the ‘eggcups’ are gently deposited on top of the cylindrical piece using sharp tweezers to manipulate the EC using the small PDMS piece. (iii) Cells at the proper density are pipetted on top of the EC. (iv) Cells are introduced in the ‘eggcups’ by centrifugation. (v) Finally, the sample is gently released out from the tube and it is ready to use. Please click here to view a larger version of this figure.
Figure 3: Comparison of cell phenotypes on 3D ‘eggcups’ and 2D flat surfaces. Confocal microscopy (25X water objective, 0.95 NA, Leica) image of NIH3T3 cells on (a) EC forming an ordered array, and showing a homogeneous spherical phenotype, and on (b) standard 2D flat culture, randomly distributed with heterogeneous phenotypes. Cells were stained for actin (in green), Golgi (in orange) and nucleus (in blue). Scale bars = 100 µm. (c) 3D reconstruction of cells on EC and (d) on flat surfaces for WT and Blebbistatin-treated cells. Scale bars = 20 µm. Please click here to view a larger version of this figure.
Figure 4: Study of NIH3T3 Golgi apparatus phenotype. Schematic and sample image of Golgi phenotype classification for cells on (a) flat and (b) EC. Cells were classified as compacted, extended or fragmented depending on the α-value. (c) Quantification of Golgi phenotypes. Scale bars = 10 µm. Please click here to view a larger version of this figure.
Figure 5: Study of NIH3T3 nucleus phenotype. (a) (Left) Confocal microscopy image of a NIH3T3 cell inside an EC and stained for actin (in green), Golgi (in orange) and nucleus (in blue). (Right) Scheme of nuclei orientation inside EC. (b) Angular distribution of nuclei inside EC for WT and (c) Blebbistatin-treated cells. (d) Nucleus sphericity values for WT and Blebbistatin-treated cells both for EC and flat surfaces (P[WTEC-BlebbEC] <0.001, P[Blebbflat-BlebbEC] <000.1; nWTflat=47, nWTEC=94, nBlebbflat=59, nBlebbEC=141 cells). Scale bar = 10 µm. Please click here to view a larger version of this figure.
Figure 6: Detailed study of the cytokinetic ring in live and fixed samples and in two systems using ‘eggcups’. (a) Time sequence of the cytokinetic ring using standard 2D in vitro culture. Only two bright spots in actin (Lifeact-mcherry, red) and myosin (GFP tagged, green) are visible in the cleavage furrow of the HeLa cells (Scale bar = 10 μm). (b) Time sequence of the closure for the cytokinetic ring in HeLa cells during mitosis using ‘eggcups’. The images show actin (in red) and myosin (green). ‘Eggcups’ allow the identification of still myosin accumulations. One example is highlighted with an arrowhead. (Scale bar = 5 μm). (c) The cytokinetic ring can also be visualized in fission yeast. (Left) Cells lie on a flat surface, the cytokinetic ring is only visible as two dots. (Right) Cells in ‘eggcups’: the entire closure can be captured. Actin is labeled with CHD-GFP (Scale bars = 2 μm). Time in min:sec. (d–e) Examples of stained cytokinetic rings. (d) Actin-GFP expressing HeLa cells are stained for phosphotyrosine (PY) which also shows signal in the ring (Scale bar = 5 µm). (e) HeLa cells expressing GFP tagged myosin and Lifeact-mcherry (actin) are stained for anillin. Anillin is revealed to localize in the cytokinetic ring and less concentrated in the cortex. It shows co-localization with actin and myosin (Scale bar = 5 µm). Please click here to view a larger version of this figure.
Figure 7: Application of the ‘eggcups’ to other cell types and model systems. (a) U2OS (human osteosarcoma). The inset shows a dividing cell. (Scale bar = 20 μm). (b) NIH3T3 cells expressing GFP. Difference in expression levels can be easily read out (Scale bar = 20 μm). (c) SW480 cells (Scale bar = 20 μm). (d) Budding yeast; their cycle time is unchanged. (Scale bar = 10 μm). (e) C. elegans worms; (Left) on a flat surface. (Right) In ‘eggcups’, embryo is seen from an otherwise hidden perspective. (Scale bars = 10 μm). Time in min:sec. Please click here to view a larger version of this figure.
Figure 8: The organization in an array of ‘eggcups’ allows an automated analysis of cell population. (a) NIH3T3 cells in EC (Scale bar = 20 μm). They have different expression levels of GFP. (b) Automated recognition of cell position allows an individual analysis of the expression level. It is summarized in the histogram of the GFP expression of the cell population. Please click here to view a larger version of this figure.
Model System | Type | Culture Medium | Observation Medium | 'eggcups' diameter (µm) | Comments/Description |
Mammalian cells | NIH3T3 | 10 % BCS high-glucose DMEM | 10 % BCS L-15 | 20 | Other stable cell lines, such as REF52 or MDCK, as well as primary cell lines, cancerous cells and/or stem cells can also be inserted in the 'eggcups'. |
HeLa | 10 % FCS high-glucose DMEM | 10 % FCS L-15 | 25 | Available from many different sources. | |
U2OS | 10 % FCS high-glucose DMEM | 10 % FCS L-15 | 20-25 | Available from many different sources. | |
SW480 | 10 % FCS high-glucose DMEM | 10 % FCS L-15 | 17-20 | Available from many different sources. | |
Yeast | Fission Yeast | Agar plate (YE5S) and liquid media (YE5S and EMM5S) | Filter sterilized EMM media (see the list of materials) | 5 | The surface does not need to be functionalized with adhesive proteins. |
Budding Yeast | Agar plate (YPD) and liquid media (YEPD and SD) | SD media | 5 | The surface does not need to be functionalized with adhesive proteins. | |
Embryo | C. elegans | NGM plate | ultrapure water | 25 | Alternatively M9 medium can be used for long-term experiments. The recipe of this salted solution can be found here: http://cshprotocols.cshlp.org/content/2009/5/pdb.rec11798.full?text_only=true |
Table 1: Culture conditions in ‘eggcups’ for different model systems. The above-related protocol can easily be adapted by just replacing the described culture conditions and the size of ‘eggcups’.
Replica molding was used in order to fabricate the ‘eggcups’. 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 ‘eggcups’. 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 reagent can be increased. Note that other techniques and materials can be applied to fabricate the ‘eggcups’, 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.
‘Eggcups’ 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 (‘eggcups’). 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 ‘eggcups’ 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 ‘eggcups’ 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 ‘eggcups’ to industry-oriented purposes. As an example, drug screening applications in pharmaceutical companies require the use of multiwell plates 14,28; implementing ‘eggcups’ into such platforms will potentially improve the reliability of tests and results. As such, high content-screening assays will be performed using the commonly used automatized 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 ‘eggcups’, 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.
The authors have nothing to disclose.
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. Séraphin 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. Hoël 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 Médicale and the ci-FRC of Strasbourg.
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 |