This method makes it possible to manipulate and quantify how the cells organize collectively. This is important because we can model pattern in in vitro and disentangle cues tightly coupled in vivo. The technique requires minimal specialist equipment, allowing it to be established in a standard biology lab, and it is quantitative, making possible the discovery of sub-visual patterning events.
This method can easily be applied to any cell system where patterning could emerge and to discover non-random patterns of differentiation, proliferation, and cell-to-cell competition. The main with this method is the systematic optimization of micropatterning parameters for new cell types. For example, pattern size and shape, type of matrix, and cell adhesion time.
Wearing gloves, place a piece of laboratory film into the bottom of a 10-centimeter square Petri dish. Use a 12-millimeter hole punch to cut hydrophobic plastic slides to create 12-millimeter-around cover slips, and place the cover slips in a new Petri dish. Use tweezers to carefully remove the protective film from the cover slips.
Place the photo mask on a clean and stable surface, chrome side up, and add a two-microliter drop of double-distilled water at the position of the desired chip design. Gently press a cover slip onto the drop and place the holder on top of the plastic slides. Carefully fix this sandwich with clamps to maintain the plastic pieces in contact with the photo mask.
Place the assembly in an ultraviolet ozone lamp approximately two centimeters from the light source for a 10-minute illumination. At the end of the illumination period, grasp the sandwich with the photo mask at the bottom and carefully remove the clamps while maintaining pressure with one hand to prevent the slides from moving about while disassembling the sandwich. When all of the clamps have been removed, remove the holder, taking care that all of the plastic pieces are still on the mask and not stuck to the holder, and add double-distilled water to the chips to gently detach the chips from the photo mask.
Place the photo-patterned chips within the matrix deposition chamber, illuminated side up, and add 200 microliters of coating solution onto each chip. Then, add a three-centimeter Petri dish filled with double-distilled water to the chamber to limit evaporation at four degrees Celsius overnight. For seeding of embryonic cells onto the chips, wash the chips with two at least five-minute washes in fresh, sterile PBS per wash, before dispensing one times ten to the fifth cells in 200 microliters of the appropriate cell culture medium onto each chip.
Then close the seeding chamber. Incubate for one hour to allow the cells to adhere to the chips. When the cells have attached, fill the wells of a multiwell plate with 500 microliters of warm medium per well, and use sterile tweezers to transfer the chips into individual plate wells.
Shake the plate vigorously to detach any non-adherent cells, and immediately replace the supernatant with fresh, warm medium. Then check under the microscope to observe whether patterning is visible. 48 hours after their plating, the cells should form dense colonies that rigorously follow the shape of the patterns.
Leaving the chips in the plate, remove all but just enough medium to prevent the chips from drying, and add at least 500 microliters of paraformaldehyde or PFA-based fixation solution per well. After ten minutes, briefly wash the wells three times with washing solution, followed by one wash with 50-millimolar ammonium chloride diluted in washing solution to quench residual PFA crosslinking activity. After the last wash, treat the samples with blocking solution for at least 30 minutes.
For immunostaining of the chip cultures, transfer the chips into a staining chamber cell side up, and immediately add 100 microliters of the primary antibody solution of interest to each chip. After one hour on a rotating platform at room temperature, wash the chips three times with washing solution as demonstrated, and incubate the chips with the appropriate secondary antibodies for one hour on the rotating platform. At the end of the secondary antibody incubation, wash the chips three times in washing solution and mount the chip on a microscopy slide with 20 microliters of any standard mounting medium.
To image the chips, first adjust the scanning speed, image resolution, frame averaging, and detector gains to identify an optimum between the image quality and the imaging time. Then acquire images of each chip. When all of the images have been acquired, install and run PickCells following the documentation available online, create a new experiment, and import the images into the program.
Use the Nessys module to segment the nuclei based on the nuclear envelope signal. Use the basic segmentation module to identify the pattern's autofluorescence signal. Then click Finish"and wait for all of the images to be processed.
To create nuclei objects and to compute basic object features, launch the Intrinsic Features module from the taskbar and close the Ellipsoid Fitter and Surface Extractor panels to keep only the Basic Features panel open. Select nucleus"as the Object Type, and select the provided prefix given for the segmented images. Then click Compute"and wait for all of the images to be processed.
At this stage, additional features need to be written into nuclei before exporting the data for our analysis. For example, to store the name of the image each nucleus belongs to as a Nucleus Attribute, click Data"and New Attribute"and select nucleus"in the pop-up dialogue. Click OK"select Collect data from other objects connected to the node"and click Next"In the left panel, select Image"and double-click on the interrogation mark to set the image node as target of the path.
Expand the Reduction Operation pane and select Get One"Expand the Available Attribute pane and select the Name attribute. Then click Change"and Next"Enter image name, press the Tab key, and click Finish"Then export the data to a tab-separated value file. For our analysis, loop the exported data to the data folder of the R-Script folder.
Open Our Studio"and open the binned map template R-Script"Then set the working directory to the source file location, and run the script to generate density maps. At one hour after cell-seeding, the individual cell culture pattens may not be fully confluent as the cells proliferate over time. However, the patterns will become fully colonized with only very few cells outside the adhesive surfaces.
Patterning that is not clear up to two hours after seeding indicates a failure of the procedure. The spatial confinement of mouse embryonic stem cells on large disks or ring micropatterns guides the patterning of a sub-population of cells expressing the mesodermal brachyury marker. For example, when the mouse embryonic stem cells are grown on large-disk micropatterns, the brachyury-positive cells are preferentially restricted to the periphery of the pattern where the local cell density is the lowest.
This patterning is confirmed by the map of brain-brachyury intensity. These data demonstrate that the method can reveal sub-visual information as the inspection of one colony is not sufficient to identify any form of spatial organization in marker of interest expression. This is notably explained by the important colony-to-colony variability.
The technique also demonstrates that no detectable patterning exists for inhibitor of DNA-binding one which may indicate that T-patterning is not driven by bone morphogenetic protein signaling in this context. Always remember which side of the cover slip has eliminated and coated and also be sure that the cells have properly adhered before washing off the excess cells. This method allows the study of the environmental cues important in guiding self-organization of cells and can also inform mathematical models that help us better under the rudimental patterning.
