The overall goal of this procedure is to create transient pores in cell membranes using inkjet printing. This is accomplished by first modifying a standard inkjet printer. The second step is to empty and clean a stock printer ink cartridge to allow for the insertion of the bio ink.
Next, the cells of interest are suspended in a solution of fluorescently tagged acton monomers and PBS to create the bio ink solution. The final step is to print the bio ink onto microscope slides using the modified printer and cartridge. Ultimately, fluorescent microscopy can be used to show that the Fluor four conjugated G actin monomers have been internalized by the printed cells through transient membrane pores.
The main advantage of this technique over standard transfection and cell microinjection techniques is that this printing technique is easy to use and yields high cell viability. Though this technique can provide insight into genetics, it can also be applied to other fields of study such as cell biophysics and tissue engineering in cell biophysics. This technique can be used to easily watch the cytoskeleton of a cell during cell migration.
We first had the idea for this method when we wanted to be able to visualize changes in cytoskeleton arrangement due to applied forces on cells. Begin by unlocking the plastic clips from the bottom base of the printer and then slowly lift off the top plastic case. Then detach the button display light panel from the top of the printer, leaving it connected to the printer's motherboard.
Using Kim wipes and ethanol. Clean the inside of the printer, especially the areas where the ink cartridge rests and where printing occurs. Next, locate the cable supplying power to the paper feed mechanisms for the HP desk Jet 500.
They're just below the paper tray to the left of the front and unplug the cables from the motherboard. Now locate the paper detection mechanism a fix a wire loop to the gray plastic lever found above and behind the printing paper feed mechanism to serve as a manual handle, place a stage here. The foam shipping holder for 15 centrifuge tubes is used with several microscope slides taped in place in the printing region in front of the paper feed mechanism to bring the desired printing slides to a level just below the cartridge print head.
Finally, to maintain aseptic technique, place the printer inside a laminar flow hood. Begin by removing the cartridge from its packaging, leaving the protective tape covering the printer contacts and print head. Stabilize the body of the cartridge with a clamp leaving the green top clear of any obstacle using a handheld rotary cutting tool.
Cut the green top off the cartridge, empty any remaining ink from the reservoir, and then remove the plastic protective tape covering the printer contacts and print head. The water will likely leak from the print head during this process, but it will not cause any harm to the cartridge. When the water runs clear, allow the cartridge to dry.
To maintain a clean printing environment, begin by fully submerging the cartridge in a beaker full of deionized water, and then sonicate the cartridge for 15 minutes. After sonication, remove the cartridge and shake out the excess water. Spray 70%ethanol into the cartridge to create a more aseptic environment.
After harvesting the cells of interest, centrifuge the cells in five milliliters of fresh medium in a 15 milliliter conical centrifuge tube for five minutes at 250 times G at room temperature. Next, aspirate the medium and then resuspend the cells at one times 10 to the fifth cells per milliliter in freshly prepared fluorescent G actin stock solution To create bio ink, note that 250 microliters of bio ink prints three cover slips with the line pattern shown here. After the printer is turned on and warmed up, place the desired printing surface here, 22 millimeter by 22 millimeter cover slips are used on the center of the previously assembled stage.
Next, open a drawing software program and select the desired pattern. Now pipette approximately 100 to 120 microliters of the cell suspension into the small circular well at the bottom of the cartridge compartment. Print the file as the printer warms up again, the cartridge will move to the ready position.
When the cartridge moves slightly to the left of the ink drip well and stays there. Pull up on the paper feed mechanism, wire and printing will commence. The printer will print onto the cover slip placed in the center of the printing stage using an HP desk jet 500 printer with a bio ink consisting of a fibroblast cell suspension in a G actin monomer solution.
Inside an HP 26 series, ink cartridge cells were printed onto glass microscope cover slips. This figure illustrates a representative result of printed fibroblast cells that show incorporated fluorescent actin monomers. This pattern was used in the previous figure to create a continuous line of the printing solution across the majority of the microscope slide.
In this figure, the straight line in which the biolink and cells were mutually deposited is shown. Note that immediately after printing, there is an increase of background fluorescence because the biolink solution in which the cells are suspended contains free excess fluorescent actin monomers incubating the cells for about 10 minutes after printing facilitates the adhesion to the slide, allowing excess monomer solution to be washed from the substrate with fresh growth media significantly reducing background fluorescence. Here, a representative image of three T three fibroblasts printed in a localized printing zone, as shown this fluorescent microscopy photo micrograph of a cell taken 15 minutes after printing shows giin in green, the nucleus in blue, and the overlay of both the GI actin and the nuclear dye.
The next two images show fluorescent microscopy of two fibroblasts in a line pattern three hours after printing. This first image shows fluorescently labeled actin monomers inside cells. This second image is an overlay of the fluorescent channel with the background image to show that although there may be some debris on the slide on the bottom left corner, it does not fluoresce.
Finally, a control group of non-print cells incubated for three hours with fluorescently tagged monomers is presented here to demonstrate that monomers can not penetrate the cell membrane without membrane permeation by cell printing. While attempting this procedure, it is important to have several cartridges cleaned and ready for use as printer clogging is one of the problems that slows down this technique. After watching this video, you should have a good understanding of how to convert a standard desktop printer to print living cells, which helps create transient membrane force.
