Three-dimensional Patterning of Engineered Biofilms with a Do-it-yourself Bioprinter

3D bioprinting with bacteria is a newly developed technique. This protocol provides an easy way of constructing engineered biofilms 3D printed with bacteria. The main advantage of this technique is the ability to produce 3D printed biofilms using an inexpensive home-built 3D printer.

One possible application of our 3D printer is to create reproducible model biofilms that can be used to develop new antibacterial therapies. Our 3D printing approach can be applied to any type of bacteria that is compatible with our alginate based bioink. Preparation of the bioink and of the printing substrates are fairly standard procedures, while the 3D printing process, especially the calibration of the Z-axis is a crucial step that requires some practice.

Calibration of the set access height will influence the resolution of our 3D printer, and is strongly dependent on personal experience. This procedure requires manual adjustments, and it's difficult to be described in a written format. Connect a 200 microliter pipette tip to a length of silicone tubing, and mount the pipette tip onto the 3D printer's extruder head as a replacement for the original extruder.

Next, add four milliliters of a five molar calcium chloride solution to 400 milliliters of 1%agar dissolved in Luria-Bertani broth, and supplement it with the appropriate antibiotics and inducers. Then, dispense 20 milliliters of the LB agar solution into each 150 millimeter by 15 millimeter Petri dish. Dry the dish for 30 minutes at room temperature, with the lid half open.

Prepare a 3%sodium alginate solution, and heat it to the boiling point three times to sterilize the solution. Then, store the sterile solution at four degrees Celsius until it is used. To prepare the bacterial component of the bioink, grow E.coli bacteria carrying plasmids for constitutive GFP expression in 50 milliliters of LB medium containing antibiotics.

Shake the culture at 250 RPM and 37 degrees Celsius overnight. After overnight growth of the culture, pellet the bacteria for five minutes at 3, 220 times gravity and then remove the supernatant. Resuspend the bacteria pellet in 10 milliliters of LB medium, and add 10 milliliters of 3%sodium alginate.

Connect the 3D printer to a computer, and open the 3D printing software. Click the Home button for the X, Y, and Z axes, to move the print head to its home position. For each print, place a prepared printing substrate onto a particular location on the printing bed.

Raise the print head to a height of over 22 millimeters under manual control, so that it will not collide with the edge of the Petri dish during movement. Position the print head over the top of the plate and move it down until the pipette tip contacts the printing surface. Assign this Z-axis position as Z1, the height of the printing surface.

Next, raise the print head and manually move it outside of the plate area. If the working distance between the print head and the plate surface is defined as Z2, enter the height of the printing surface plus the working distance into the printing program as the Z value during printing. Load a pre-programmed G-code file containing commands for printing the desired shape.

At each command line, the position of the print head may be changed in the X, Y, and or Z axes. Be sure to input the Z value during all printing steps as the height of printing surface plus the working distance. Load the liquid bioink into syringes and mount them in the syringe pump of the 3D bioprinter.

Then, set the extrusion speed to 0.3 milliliters per hour. Print the bioink onto the printing substrate by clicking the Print button. Wait to start the syringe pump until after the printing has started, and before the print head comes into contact with the printing surface.

During printing, control the print head movement entirely by the software. Stop the syringe pump as soon as the print head arrives at the last point of printing, otherwise excess bioink will drop onto the printing substrate and reduce the printing resolution. For the construction of 3D structures, all the movements of the print head are controlled in the G-code editor.

To increase the printing height for the second layer, type in the printing height of the first layer, and increase the Z value in the code by 0.2 millimeters. Thereafter, increase the Z value by 0.1 millimeter, when moving to a higher layer. Incubate the printed samples at room temperature for three to six days to allow the production of the biofilm components such as Curli fibers.

Then, place the plate on a fluorescent scanner and image the plates. To dissolve the alginate matrix, add 20 milliliters of a 0.5 molar sodium citrate solution at pH 7, to the printed substrate. Incubate the plate at room temperature for two hours while shaking at 30 rpm.

Then, discard the liquid and image the plates again, to compare with the images of the plates before and after the citrate treatment. The 3D bioprinter can create bacteria encapsulating hydrogels in a variety of two-dimensional and three-dimensional shapes. These printed shapes can then be used to assess whether the formation of biofilm was successful or if the alginate matrix is completely dissolved using a sodium citrate solution.

In the case of bioink without the inducible Curli production plasmid, the printed pattern was completely dissolved after the sodium citrate treatment, signifying that no biofilm Curli network had formed. The bacteria containing the inducible Curli production plasmid was not dissolved after sodium citrate treatment, indicating that the printed bacteria were able to form a Curli network extensive enough to stabilize the printed pattern of bacteria. To construct multi-layered structures, additional layers can be printed by controlling the G-code editor.

Increasing the number of printed layers in a sample caused the width and the height of the printed structures to increase incrementally. When E.coli engineered to inducibly produce Curli proteins were printed into multi-layered structures, sodium citrate treatment did not dissolve the samples, whereas multilayer structures containing non Curli producing E.coli were dissolved. The most critical parts of the 3D printing procedure are the calibration of the Z-axis, and the coordination of starting the print and starting the syringe pump.

The bioink developed for this process is fairly soft, with low toughness. Further modification could be made to the bioink, in order to provide and improve mechanical stability. This 3D printing technique enables production of biofilms with excellent mechanical properties, which may enable fabricating biomimetic materials.

When handling these bacteria, wear proper protection, such as gloves.