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.
