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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

1. Microfabrication of ‘Eggcups’

1. Fabrication of the Master: Microcavities Array
   1. Heat a 3’’ silicon wafer up to 200 °C to evaporate any presence of humidity.
   2. 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 'eggcups' (EC). For a 30 µm thick layer and SU-8 2025, spin-coat at 2,800 rpm.
   3. Pre-bake the wafer at 65 °C for 1 min (step 1 of 2) for a 30 µm thick SU-8 2025 layer. Adapt the time depending on the photoresist type and thickness desired. Check the manufacturer datasheet for details.
   4. Pre-bake the wafer at 95 °C for 3 min (step 2 of 2) for a 30 µm thick SU-8 2025 layer. Adapt the time depending on the photoresist type and thickness desired. Check the manufacturer datasheet for details.
   5. Load the wafer on the mask aligner for UV exposure. Place the photolithography mask on it. The mask shows a pattern of circular features (disks) of 20 µm in diameter. Ensure a perfect contact between each other.
      NOTE: Different manufacturers offer photolithography masks. The spatial resolution will determine the final cost. Acetate masks provide acceptable resolution (≈10 µm) at low cost. Chromium masks provide better resolution but are more expensive. Adapt the diameters of disks (from the photolithography mask) to the volume of cells. Dimensions of disks on the mask will determine the diameter of cavities in the device. Small diameters will lead to a low filling; too large diameter will not confine the cells. For HeLa and NIH3T3 cells, diameters of 20 µm to 25 µm are suggested.
   6. Check the power of the UV lamp prior exposition and optimize the exposure time accordingly. Irradiate (wavelength = 365 nm) for 41.5 sec (or the optimized exposure time) at 250 mJ/cm².
      NOTE: SU-8 2025 is a negative photoresist, which means that exposed regions to UV will be cured. In this case, the circular features were black and the rest transparent. Positive photoresist work in the opposite way: non-exposed regions are cured. Select the photoresist accordingly, depending on the design and photo-mask.
      NOTE: Protect the eyes from UV light with appropriate safety glasses.
   7. Remove gently the mask from the photoresist layer.
   8. Post-bake the wafer at 65 °C for 1 min (step 1 of 2) for a 30 µm thick SU-8 2025 layer. Post-bake the wafer at 95 °C for 3 min (step 2 of 2) for a 30 µm thick SU-8 2025 layer. Adapt the time depending on the photoresist type and thickness desired. Check the manufacturer datasheet for details. After post-baking, cool the wafer to room temperature on the bench for around 1 min.
   9. Place the wafer in the spin-coater and drop few mm of SU-8 developer to cover the whole wafer area. Develop for 2 min and then spin-coat at 1,000 rpm for 30 sec. Repeat the procedure three times.
   10. Rinse with 2-propanol to ensure the complete removal of undeveloped SU-8. Appearance of white regions is an indication of incomplete development. If so, repeat the developing step an additional time.
   11. Hard-bake the wafer at 200 °C to ensure robustness of the fabricated microstructures. This step is optional.
   12. Store the 3’’ wafer with microstructures inside a 94 mm x 15 mm polystyrene Petri dish.
       NOTE: There is no need for surface treatment, in particular silanization, for the next steps.
2. Fabrication of the Polydimethylsiloxane (PDMS) Replica: Pillars Array
   1. Thoroughly mix in a 1:10 ratio (w/w) the cross-linker and the pre-polymer for a total of 30 g inside a 50 ml tube.
      NOTE: Using a 1:10 (v/v) ratio is also working.
   2. Centrifuge the tube at 1,800 x g for 5 min to remove air bubbles.
   3. Drop gently the PDMS on top of the microstructures.
      NOTE: If air bubbles appear during this step, degas the sample using a vacuum pump for 15-20 min.
   4. Place the sample in an oven at 65 °C for 4 hr.
      NOTE: The curing time varies between users and, together with the cross-linker:pre-polymer proportion. This time will dictate the rigidity of the PDMS. It is recommended to cure more than 2 hr and to stick to a fixed curing time.
   5. Use a scalpel to gently cut the area of interest (stamp) of about 1 cm² which includes around 10⁵ microcavities or ‘eggcups’.
      NOTE: Cut first the PDMS and then, peel it off gently. Check the quality of the PDMS replica with an optical microscope.
3. Fabrication of ‘eggcups’ by replica molding. In the following, two alternative strategies for the fabrication of ‘eggcups’ are described. Both protocols are similar and provide identical results:
   1. Strategy 1
      1. Activate the fabricated PDMS stamp by oxygen plasma treatment for 30 sec. Store temporarily the activated stamps into a closed Petri dish to prevent deposition of dust.
         NOTE: Adjust the exposition time if other gases for the plasma are used.
      2. Place the activated PDMS stamp upside up (the side with the structures) in a Petri dish next to a 15 ml tube cap. Fill the cap with 200 µl of Trimethylchlorosilane (TMCS). Close the Petri dish and let the stamp silanize for 7 min.
         NOTE: Some temporary deformation on the stamp and/or change in color (white) may be observed. The stamp will recover its original shape in short time and the structures will not be affected.
         NOTE: 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.
      3. Place the PDMS stamp on the spin-coater with the structures upside up. Put a small drop of few microliters (around 20 µl) of liquid PDMS (1:10 w/w cross-linker:pre-polymer) on top of the structures. Spin-coat at 1,500 rpm for 30 sec to deposit a thin layer of PDMS on top of the structures.
         NOTE: If the stamp does not fit the spin-coater chuck place the stamp on top of a Petri dish lid with a small hole at its center.
      4. Place the stamp in the oven at 65 °C for 4 hr to cure the deposited spin-coated PDMS layer.
      5. Activate the thin PDMS layer by placing the PDMS stamp upside up, together with a glass coverslip #0 of 25 mm in diameter, using oxygen plasma cleaner for 30 sec. Proceed quickly to the next step.
         NOTE: Coverslips with other thicknesses, shapes, and dimensions can be used as well. However, some cellular structures could be difficult to visualize depending on the selected coverslip thickness and objective magnification and/or NA and/or working distance. Check the objective data sheet.
      6. Place in contact the stamp (the side with the thin spin-coated layer) with the glass coverslip. Press gently all around the surface of the stamp with tweezers to make the ‘bonding’. Finally, keep a constant pressure on top of the stamp for around 10 sec.
      7. After 30 min gently ‘peel’ the stamp out of the coverslip in order to ‘liberate’ ‘eggcups’ (see Figure 1). Rinse thoroughly with ethanol and dry. If PDMS ‘eggcups’ are not well adhered on the glass coverslip (i.e. they detach during the ‘unpeeling’ step), consider adjusting the settings of the plasma cleaner and restart at step 1.3.1.5.
         NOTE: This step is delicate. Pay attention in order to avoid breakage of the coverslip and/or detachment of the thin PDMS layer.
      8. Glue a small piece (handle) of cured PDMS of 1 mm x 1 mm x 3 mm in volume at the edge of the coverslip with a small drop of liquid PDMS and cure the PDMS as before. This will facilitate the manipulation of the sample afterwards (see Figure 1). This step is optional.
   2. Strategy 2
      1. Hydrophilize the fabricated PDMS stamps by oxygen plasma treatment for 30 sec. Store temporarily the activated stamps in a closed Petri dish to prevent deposition of dust.
         NOTE: Adjust the exposition time if other gases for the plasma are used.
      2. Hydrophilize the 25 mm diameter glass coverslips #0 oxygen plasma treatment at 15 W for 30 sec. Proceed quickly to the next step.
         NOTE: Coverslips with other thicknesses, shapes, and dimensions can be used as well. However, cellular structures will be difficult to visualize depending on the selected coverslip thickness and objective characteristics (see note above).
      3. Spin-coat a small drop of PDMS (1:10 w/w cross-linker:pre-polymer) of few microliters onto the glass coverslips. Spin-coat at 1,500 rpm for 30 sec for a final thickness of around 50 µm.
      4. Glue a small piece (handle) of cured PDMS of 1 mm x 1 mm x 3 mm in volume at the edge of the coverslip with a small drop of liquid PDMS and cure the PDMS as before. This will facilitate the manipulation of the sample afterwards (see Figure 1). This step is optional.
      5. Store temporarily the PDMS-coated coverslips onto a clean wipe inside a Petri dish to protect from dust deposition.
      6. Put a drop of silanizing reagent on top of each stamp and let it evaporate for 1-2 min. Then, dry them under a stream of N₂.
         NOTE: In this step, a temporary deformation of the stamp can be observed during evaporation. The stamp will recover its original shape after drying with N₂ without any permanent deformation of microstructures.
      7. Drop very gently the silanized stamp on top of the PDMS-spin-coated glass coverslip stored in the Petri dish. Make sure that both sides are completely parallel during the contact. Avoid pressing or moving the stamp after placing it onto the PDMS-coated coverslip.
      8. Place the Petri dish with samples in the vacuum for 1-2 hr to remove air bubbles.
         NOTE: Ensure that samples are totally horizontal to avoid stamp displacement. Avoid also vibration potentially caused by the vacuum pump.
      9. Place the samples in the oven at 65 °C for 4 hr.
      10. Gently, peel off the stamp to reveal ‘eggcups’. Rinse thoroughly with ethanol and dry.
          NOTE: Practice at this point is needed. Pay attention in avoiding breakage of the coverslip and/or detachment of the thin PDMS layer.

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.

1. Hydrophilize the ‘eggcups’ in the oxygen plasma cleaner for 30 sec.
   NOTE: Optimize the parameters if needed.
2. Prepare a solution in PBS 1x of 20 µg ml^-1 fibronectin from Bovine sources.
3. Sterilize the 'eggcups' with UV for 5 min. Deposit a small drop (around 20-50 µl) of fibronectin solution to cover the entire 'eggcups' area and incubate for 1 hr at RT. Protect the sample from drying.
4. Rinse gently the ‘eggcups’ with PBS 1x. Repeat 3 times.
   NOTE: The sample is ready to use immediately or stored at 4 °C in the dark for several weeks.
5. Introduce a cylindrical custom-made plastic piece of 63 mm in height, 26 mm of external radius and 7 mm of internal radius dimensions into a 50 ml tube (see Figure 2) ²⁰
   CAUTION: Use UV-sterilized items or sterilize them prior use.
   NOTE: This piece can be easily fabricated in the lab. or by any available machine shop.
6. Put 13 ml of cell culture medium inside the tube (see Table 1). The medium should fill at least 2 cm above the cylindrical piece to ensure complete immersion of the sample.
   NOTE: For details of specific cell types, and other model systems such as yeast or C. elegans embryos, and the corresponding medium used, refer to section 5 and Table 1. The described protocol was optimized for HeLa, NIH3T3 cells, and other cell lines (see Table 1).
7. Introduce very gently the ‘eggcups’ inside the tube and parallel to the upper side of the plastic piece. Use sharp tweezers to hold the sample using the PDMS handle. Press gently the coverslip until it lies on top of the upper side of the plastic piece, until it is fully immersed (see Figure 2).
   NOTE: It is recommended to use sharp and straight tweezers. With curved tweezers, the manipulation of the sample is difficult and may cause breakage.
8. Culture cells until 80-100% confluence in a P60 Petri dish and collect them by trypsinization.
   NOTE: Cells can be wild-type, transfected or treated with any drug of interest.
   NOTE: Avoid the formation of cell aggregates which will avoid single cells to enter the ‘eggcups’. To optimize this step, pipette up and down thoroughly after trypsinization.
9. Re-suspend cells into 5 ml culture medium. Pipette 200 µl of cells on top of the ‘eggcups’.
   NOTE: Drop cells as centered as possible on top of the ‘eggcups’ but avoiding physical contact. This will prevent breakage and/or damage of the sample.
10. Centrifuge at 1,800 x g for 2 min.
    NOTE: After the first centrifugation, check in a microscope the filling percentage of the ‘eggcups’.
11. Pipette again 200 µl of cells on top of the ‘eggcups’ and centrifuge at 1,800 x g for 2 min. Repeat for a total of three times in order to optimize the filling percentage.
    NOTE: After the last centrifugation, check with a microscope the filling percentage of ‘eggcups’. If necessary, repeat the filling + centrifugation steps until reaching the desired filling percentage.
12. Remove the sample from the tube using the sharp tweezers holding the PDMS handle. Make sure to be careful in not ‘disturbing’ cells which are held in the ‘eggcups’ (see Figure 2).
13. Place the sample in a Petri dish with medium. Rinse to remove the excess of cells which are not in the ‘eggcups’ by pipetting up and down three times gently next to each side (total 4 sides) of the microstructure array.
    NOTE: Pipetting too strongly may release some cells out from the ‘eggcups’.
14. Replace the medium with fresh medium to remove nonattached cells.
    NOTE: In this step a drug of interest can be added.
15. Fix cells or prepare them for time-lapse imaging.See step 4.1.

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.

1. Place ‘eggcups’ into a microscope holder and fill it with 1 ml of 10 % FCS L-15 observation medium. To avoid evaporation, place a glass lid on top of the holder or apply a thin layer of mineral oil on top of the medium.Select the 60X oil objective.
   NOTE: L-15 medium is adequate for non-CO₂ atmospheres. Note also that some compounds of DMEM are auto-fluorescent. When using this medium, it is recommended to photobleach the fluorescent compounds by illuminating it with a high intensity lamp for 1-2 hr.
   NOTE: Avoid using plastic lids when working with DIC imaging.
2. Place the holder with ‘eggcups’ and observation medium in the microscope. Focus carefully using brightfield light until the ‘eggcups’, and cells are in the same plane of observation.
3. Open the software and adjust the parameters. Select the filters TxRed and GFP for actin and myosin; adjust the exposition time for each channel. A typical acquisition rate is 5 sec for both channels.
   NOTE: The exposition time may have to be adjusted depending on the setup used, dye or other cellular organelles of interest.
4. Select the region of interest and seek for a cytokinetic ring using either the GFP or TxRed channel. Focus accurately.
   NOTE: The ring is sharper in myosin and easier to recognize.
5. Run the automatic acquisition in both channels until the ring is completely closed.
   NOTE: Some photobleaching may be observed. Adjust the microscope parameters in order to reduce it.

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’.

1. Fixation of Cells in the ‘Eggcups’
   1. Prepare 3% paraformaldehyde (PFA) and warm at 37 °C. Remove the ‘eggcups’ sample from the 50 ml tube (or the microscope holder) and place it inside a P35 Petri dish. Rinse once with PBS 1x.
      NOTE: Protocols for the preparation of 3 % paraformaldehyde are widely available elsewhere.
      CAUTION: Use nitrile gloves and eye protection during the preparation of PFA.
   2. Remove completely the PBS and drop 1 ml of 3% PFA and incubate for 17 min. Remove the PFA and rinse twice with 1 ml of PBS 1X. Permeabilize cells using 1 ml of 0.5% Triton for 3 min and wash twice with PBS 1x for 5 min.
2. Staining of Cells in ‘Eggcups’
   1. Incubate cells for Golgi apparatus staining with the primary antibody rabbit polyclonal anti-Giantin in a 1:500 dilution in PBS. Place a 100 µl drop of antibody solution onto a plastic film sheet and incubate the cells inside the ‘eggcups’ upside down for 45 min.
      NOTE: Protect the sample with a cover to prevent drying.
   2. Release carefully the ‘Eggcups’ and place them into a P35 Petri dish. Rinse 3 times, 5 min each, with PBS 1x.
   3. Prepare a cocktail in PBS with the secondary antibody Cy3 goat anti-rabbit (1:1,000) and with Phalloidin Alexa Fluor 488 (1:200) for staining actin stress fibers.
   4. Incubate cells with a 100 µl drop of antibody solution onto a plastic film sheet and incubate cells inside the ‘eggcups’ upside down for 45 min.
   5. Release carefully the ‘eggcups’ and place them into a P35 Petri dish. Rinse 3 times, 5 min each, with PBS 1x.
   6. Incubate cells for nucleus staining placing a 100 µl drop of 1 µg ml^-1 DAPI in PBS onto a plastic film sheet and incubate cells inside the ‘eggcups’ upside down for 45 min. This step can be performed with step 4.2.3.
   7. Release carefully the ‘eggcups’ and place them into a P35 Petri dish. Rinse 3 times, 5 min each, with PBS 1x.
   8. Mount cells using a 15 µl Glycerol:PBS (1:1 v/v) on a standard microscope glass slide and seal the sample with nail polish to avoid drying.
      NOTE: Depending on the ‘eggcups’ thickness, mounting may be difficult. It is recommended to store then the sample into a P35 Petri dish in PBS, protected from drying. 
3. Microscope Observation
   NOTE: For this example an upright confocal microscope is used, equipped with PMT and Hybrid detectors. A 25X or 63X HCX IR APO L water objective (0.95 NA) was selected to provide a wide field of the sample and show the applicability of the device for high-content-screening applications.
   1. Select the 25X or 63X water objective.
      NOTE: Different objectives can be used depending on the application and signal. But usage of high numerical aperture objectives is recommended.
   2. Place the fixed sample with the 'eggcups' and focus carefully using brightfield light (phase contrast or DIC) until the 'eggcups' and cells are in the plane of observation.
   3. Open the software and adjust the parameters. Select the filters GFP, Cy3 and DAPI for actin, Golgi and nucleus observation, respectively; adjust the exposition time for all channels.
      NOTE: The exposition time may have to be adjusted depending on the setup.
   4. Select and focus the region of interest; start image capture (see Figure 3).

5. Adaptation for the Observation of Yeast Cells and C. elegans Embryo

1. Fission and Budding Yeast cells
   NOTE: This example uses fission yeast cells which are tagged with RLC1-mcherry and CHD-GFP for myosin and actin, respectively. The budding yeast cells are not fluorescently labeled here. For fission yeast observation an inverted spinning disk confocal microscope was used. A 100X HCX PL APO CS oil objective (1.4 NA) was used for all acquisitions. Alternatively, cells were also observed using an inverted phase-contrast microscope equipped with a 20X phase contrast air objective LCPlanFl (0.4 NA). In this example, the protocol is identical for both cell types.
   1. Prepare the ‘eggcups’ surface as described above. For fission and budding yeast cells, prepare cavities of 5 µm in diameter (see Table 1). In this case, the surface does not need to be functionalized with adhesion proteins.
      NOTE: The filling can be optimized by using conical ‘eggcups’. This shape captures and retains the cells avoiding releasing during the rinsing step after centrifugation. Filling percentage is optimum at about 80%. These conical ‘eggcups’ can be fabricated by means of Deep Reactive Ion Etching ¹³.
   2. Culture yeast cells in the proper culture media (see Table 1) until reaching an optical density (OD) in the range of 0.2 and 0.8. Sonicate the culture of yeast cells to remove aggregates.
   3. Insert yeast cells in ‘eggcups’ by centrifugation. For centrifugation, 4 ml of cultured cells in the appropriate OD is added onto the tube with ‘eggcups’. After the first centrifugation, gently shake the tube to re-suspend cells which are not in the ‘eggcups’, while cells in the ‘eggcups’ are not disturbed. Without opening the tube, centrifuge again and repeat this step twice. This ensures the deposition of cells from the culture into the empty microcavities and will increase the filling percentage.
      NOTE: When working with yeast, it is recommended to pre-heat the centrifuge to the working temperature during experiments
      NOTE: The protocol can be paused here and continued up to 12 hr later. In this case, store the sample at the working temperature and cover it to prevent evaporation.
   4. Place ‘eggcups’ in a microscope holder and fill the holder with filter sterilized medium for imaging. Now rinse the cells with the same media until the floating yeast cells are removed efficiently. Take care not to disturb cells in ‘eggcups’ during the rinsing process.
   5. Select the 100X oil objective and focus carefully. Open the software and adjust the parameters. For fission yeast, select the filters GFP and TxRed for actin and myosin and adjust the exposition time for both channels. A typical acquisition rate is 3 sec.
      NOTE: Depending on the fluorophore, type of tagging and the set-up, exposure time varies for other systems.
2. C. elegans Embryo
   NOTE: This example uses C. elegans embryos 25-30 µm wide and 50-55 µm long. Embryos were cultured as indicated in ²⁵. A simple visual protocol of how to manipulate C. elegans can be found in ²⁶. The observation was performed using an inverted phase-contrast microscope equipped with a 40X air objective 0.55 NA.
   1. Prepare the ‘eggcups’ surface as described above and 25 µm in diameter (see Table 1). In this case, the surface does not need to be functionalized with adhesion proteins.
   2. Culture the C. elegans embryos in the proper culture media (see Table 1).
   3. Insert embryos in ‘eggcups’ by centrifugation as described above (see section 2.6 to 2.12) using ultrapure water as culture medium.
      NOTE: Embryos were ‘behaving’ normally in ultrapure water for the duration of the experiment. Alternatively use a physiological M9 buffer for long-term experiments.
   4. Rinse the sample as described above (see section 2.14). Place the ‘eggcups’ into a microscope holder. Select the 40X air objective and focus carefully. Open the software and adjust the parameters. Select an acquisition rate of 3 sec. 

Results

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. Fi...

Discussion

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. Insuff...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
1:1,000 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 225 mg/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