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

Summary

This article describes a method of transforming a low-cost commercial 3D printer into a bacterial 3D printer that can facilitate printing of patterned biofilms. All necessary aspects of preparing the bioprinter and bio-ink are described, as well as verification methods to assess the formation of biofilms.

Abstract

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced antibiotic resistance, which poses potential health dangers, but can also be beneficial for environmental applications such as purification of drinking water. The further development of anti-bacterial therapeutics and biofilm-inspired applications will require the development of reproducible, engineerable methods for biofilm creation. Recently, a novel method of biofilm preparation using a modified three-dimensional (3D) printer with a bacterial ink has been developed. This article describes the steps necessary to build this efficient, low-cost 3D bioprinter that offers multiple applications in bacterially-induced materials processing. The protocol begins with an adapted commercial 3D printer in which the extruder has been replaced with a bio-ink dispenser connected to a syringe pump system enabling a controllable, continuous flow of bio-ink. To develop a bio-ink suitable for biofilm printing, engineered Escherichia coli bacteria were suspended in a solution of alginate, so that they solidify in contact with a surface containing calcium. The inclusion of an inducer chemical within the printing substrate drives expression of biofilm proteins within the printed bio-ink. This method enables 3D printing of various spatial patterns composed of discrete layers of printed biofilms. Such spatially-controlled biofilms can serve as model systems and can find applications in multiple fields that have a wide-ranging impact on society, including antibiotic resistance prevention or drinking water purification, among others.

Introduction

There is currently an increasing need to develop environmentally-friendly, sustainable solutions for the production of spatially-patterned materials, due to the expanding number of markets for such materials¹. This article presents a simple, economical method for the production of such materials and therefore offers a large spectrum of future applications. The method presented here allows three-dimensional (3D) printing of spatially-patterned structures using a bio-ink containing living bacteria. Bacteria remain viable within the printed structures for over one week, enabling the bacteria to perform natural or engineered metabolic activities. Printed bacteria can thereby produce and deposit desired components within the printed structure, for example creating a functional cross-linked biofilm².

Traditional methods for the production of advanced materials involve high energy expenditures (e.g., high temperatures and/or pressures) and can produce large quantities of chemical waste, often toxic substances that require cost-extensive utilization^3,4. In contrast, multiple bacterial species are able to produce materials that can be readily applicable in various industries. These materials include polymers such as polyhydroxyalkanoates (PHA)⁵ or poly(glycolide-co-lactide) (PGLA)⁶, bacterial cellulose⁷, bacterial concrete materials⁸, biomimetic composites⁹, amyloid-based adhesives¹⁰, or bio-based electrical switches¹¹, among others. Moreover, bacterial production of valuable materials typically takes place at near-ambient temperatures and pressures and in aqueous environments, without requiring or producing toxic compounds. While producing materials with bacteria has been demonstrated in the literature and some industrial applications have already emerged^12,13, a reliable method for spatial patterning of such materials remains a challenge.

This article demonstrates a straight-forward method of converting a low-cost commercial 3D printer into a 3D bacterial printer. The protocol shows how to prepare a bio-ink containing and sustaining the living bacteria, as well as how to prepare substrates onto which the 3D printing can be performed. This method is appropriate to use with a variety of natural and engineered bacterial strains able to produce materials. These bacteria can be spatially distributed within a 3D printed structure and still continue their metabolic activity, which will result in a spatial distribution of the desired materials produced by the bacteria.

This printing method enables additive manufacturing of biofilms, aggregates of bacteria surrounded by a self-produced extracellular matrix. Biofilms are heterogeneous 3D networks in which proteins, polymers, bacterial cells, oxygen, and nutrients are all spatially structured¹⁴. While in the form of a biofilm, bacteria exhibit an increased antibiotic resistance and structural robustness, making them difficult to eradicate from surfaces including medical catheters and implants. The key to biofilm properties, and also the largest challenge to biofilm research, seems to be the heterogeneity of biofilms^15,16,17. Production of spatially-controlled model biofilms is of special interest as it would allow for either reproducing or tuning the spatial patterns of biofilm components, aiding the understanding of the stable deposition of biofilms on virtually any surface in nature.

This article presents a method for the production of biofilms using 3D-printed hydrogels containing engineered E. coli bacteria that produce biofilm proteins in the presence of an inducer, as well as methods of verification of biofilm formation². The major extracellular matrix components of these biofilms are curli amyloid fibers¹⁸ that contain self-assembled CsgA proteins. When engineered E. coli bacteria are induced to express CsgA proteins, they form a stable model biofilm that protects the cells against being washed off of the printing surface. Such a 3D printed biofilm can be spatially controlled and can serve as a useful research tool for the investigation of multiscale biofilm structure-function mechanics or materiomics¹⁹. These bespoke biofilms will aid the understanding of the principles of biofilm formation and their mechanical properties, enabling further research into the mechanisms of antibiotic resistance among other applications.

Protocol

1. Conversion of a commercial 3D printer into a 3D bioprinter

1. Remove the extruder and the heater of a commercial 3D printer (Table of Materials) from the printer frame, and unplug the wiring controlling these elements from the main circuit board (Figure 1A). Since the sensor that controls the operational temperature of the printer needs to be functional to communicate with the printer software, remove from the printing software the algorithm that delays printing until operational temperature is reached.
2. Connect a pipette tip (200 µL tip) via silicon tubing (inner diameter of 1 mm) to a 5 mL syringe loaded into a syringe pump. Mount the pipette tip onto the 3D printer extruder head as a replacement for the original extruder (Figure 1B).
3. If more than one type of bio-ink will be used, mount additional tubing system(s) and pipette tip(s) to the printer.

2. Substrate preparation for 3D printing

1. Add 4 mL of 5 M CaCl₂ solution to 400 mL of 1% w/v agar dissolved in Luria-Bertani broth (LB) medium, supplemented with appropriate antibiotics and inducers (here 34 µg/mL chloramphenicol and 0.5% rhamnose).
2. Dispense 20 mL of the LB-agar solution into each 150 mm x 15 mm Petri dish. Dry 30 min at room temperature with the lid half-open.
   NOTE: The protocol can be paused here by storing these printing substrates at 4 °C for up to several days.

3. Bio-ink preparation

1. Prepare a sodium alginate solution (3% w/v), and heat to the boiling point three times to sterilize the solution. Store at 4 °C until used.
2. Grow E. coli MG1655 PRO ΔcsgA ompR234 (E. coli ΔcsgA) bacteria carrying plasmids pSB1C3-green fluorescent protein (GFP) (constitutive GFP expression)² or pSB1C3-GFP-CsgA (constitutive GFP expression, rhamnose-inducible CsgA expression) overnight at 37 °C with shaking at 250 rpm in 50 mL of LB medium containing 34 µg/mL chloramphenicol and 0.5% rhamnose.
3. Centrifuge the cell culture for 5 min at 3,220 x g to pellet the bacteria. Remove the supernatant.
4. Re-suspend the bacteria pellet in 10 mL of LB medium and add 10 mL of sodium alginate (3% w/v).

4. 3D printing process

1. Install and open the 3D printing software (Table of Materials) on a computer. Connect the 3D printer to the computer. Move the printhead to its home position by clicking the home button for the X, Y, and Z axes.
2. For each print, place a prepared printing substrate onto a particular location on the printing bed.
3. Calibrate the height of the printhead in the Z axis.
   1. Raise the printhead to a height of 22 mm under manual control, so that it will not collide with the edge of the petri dish when moving to the desired position. Position the printhead overtop 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).
   2. Raise the printhead, and move it outside of the plate area by manual control in the X, Y, and Z axes. If the working distance between the printhead and the plate surface is defined as Z2, enter Z1 + Z2 into the printing program as the Z-value during printing.
4. Program the printing shape by a self-developed point-by-point coordinate-determined method according to the desired trajectory.
   1. If the desired trajectory is a straight line, define only the start and end points. Including additional points on curved lines will result in smoother curves. Move the printhead manually to every point sequentially, and record the coordinates of these points in order. Enter all of these coordinates as well as the printhead moving speed for each printed segment into the G-code editor.
5. Both before and after printing, lift the printhead to a distance higher than the plate edge (20 mm), and move directly out of the plate region. Save this program as a G-code file and load directly for use in subsequent prints, while re-measuring the Z axis height for each new printing substrate.
   NOTE: See Table 1 for an example G-code for printing a square.
6. Load the pre-programmed G-code file. Open the G-code editor in the software, and program in the commands for printing the desired shape. At each command line, the position of the printhead may be changed in the X, Y, and/or Z axis. Input the Z value during all printing steps as Z1 + Z2 (height of printing surface + working distance).
   NOTE: The moving speed is also adjustable; 9,000 mm/min is a suitable value for typical printing rates.
7. Load the liquid bio-ink into syringe(s), and mount them in the syringe pump(s) of the 3D bioprinter.
8. Print the bio-ink onto the printing substrate by clicking the Print button.
9. During printing, control the printhead movement entirely by the software. Manually start the syringe pump before the printhead comes into contact with the printing surface.
   NOTE: The coordination of the syringe pump and the printer is empirically determined depending on the extrusion speed, the speed at which the printhead moves to the first print point, and the initial position of the printhead. If the initial printhead position is 20 mm, with a printhead speed of 9,000 mm/min and an extrusion speed of 0.1 mL/h, start the syringe pump immediately after the printing is started. If the extrusion speed is changed from 0.1 mL/h to 0.3 mL/h, then wait 2−3 s to start the syringe pump after the printing is started.
10. Stop the syringe pump as soon as the printhead arrives at the last point of printing. Halt the syringe pump before the printhead lifts up at the end of the printing process, otherwise excess bio-ink will drop onto the printing substrate and reduce the printing resolution.
11. For the construction of 3D structures, control all movements of the printhead in the G-code editor. Type in the printing height of the first layer. Increase the Z-value in the G-code by 0.2 millimeters for the second layer to increase the printing height. Thereafter, increase the Z-value by 0.1 millimeters when moving to a higher layer. Do not move the plate during the printing process.
12. To measure the width and height of the printed hydrogel, use a steel ruler placed underneath or alongside the sample.

5. Growing and testing the effectiveness of biofilm production by E. coli

1. Incubate the printed samples at room temperature for 3−6 days to allow the production of biofilm components (curli fibers). Image the plates using a camera or fluorescent scanner.
2. To dissolve the alginate matrix, add 20 mL of 0.5 M sodium citrate solution (pH = 7 adjusted with NaOH) to the printing substrates, and incubate for 2 h with 30 rpm shaking at room temperature. Discard the liquid and image the plates again to compare with the images of the plates before citrate treatment.

Results

The first step for successful 3D printing of biofilms is converting a commercial 3D printer into a bioprinter. This conversion is achieved by removing the extruder and heater of the printer, designed for printing with a polymeric ink, and replacing these with components appropriate for printing bio-ink containing living bacteria (Figure 1A). The extruder is replaced by a pipette tip (or tips, if multiple bio-inks will be used in the printing process) attached...

Discussion

The protocol presented here for 3D printing of engineered biofilms has two critical steps. First is the preparation of the agar printing surface, which is the most critical factor to producing a specific printing resolution. It is important to ensure that the printing surface is flat and that the pipette tip on the printhead is positioned at the correct height from the surface. If the surface is not flat, the working distance will change during the printing process. If the working distance is less than 0.1 mm, the CaCl

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3D printer	CoLiDo	3D-P Kit
3D printing software	CoLiDo	Print-Rite ColiDo Repetier-Host v2.0.1
Agar	Sigma-Aldrich	05040
CaCl2 dihydrate	Sigma-Aldrich	C7902
Centrifuge	Eppendorf	5810 R
Chloramphenicol	Sigma-Aldrich	3886.1
LB broth powder	Sigma-Aldrich	L3022
Orbital shaker	VWR	89032-092	Model 3500
Petri dish	VWR	25384-326	150 x 15 mm
Rhamnose	Sigma-Aldrich	83650
Silicon tubing	VWR	DENE 3100103/25
Syringe pump	ProSense B.V.	NE-300
Sodium alginate	Sigma-Aldrich	W201502
Sodium citrate monobasic	Sigma-Aldrich	71498
Sodium hydrooxide	VWR	28244.295