This work was supported by an AOARD grant (No. FA2386-18-1-4059), the Netherlands Organization for Scientific Research (NWO/OCW) as part of the Frontiers of Nanoscience program, and the Advanced Materials NWO-NSFC program (No. 729.001.016). The authors acknowledge laboratory assistance of Ramon van der Valk and Roland Kieffer.

1. Conversion of a commercial 3D printer into a 3D bioprinter
<ol>
	<li>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.</li>
	<li>Connect a pipette tip (200 &#181;L 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).</li>
	<li>If more than one type of bio-ink will be used, mount additional tubing system(s) and pipette tip(s) to the printer.</li>
</ol>
2. Substrate preparation for 3D printing
<ol>
	<li>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 &#181;g/mL chloramphenicol and 0.5% rhamnose).</li>
	<li>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 &#176;C for up to several days.</li>
</ol>
3. Bio-ink preparation
<ol>
	<li>Prepare a sodium alginate solution (3% w/v), and heat to the boiling point three times to sterilize the solution. Store at 4 &#176;C until used.</li>
	<li>Grow E. coli MG1655 PRO &#916;csgA ompR234 (E. coli &#916;csgA) 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 &#176;C with shaking at 250 rpm in 50 mL of LB medium containing 34 &#181;g/mL chloramphenicol and 0.5% rhamnose.</li>
	<li>Centrifuge the cell culture for 5 min at 3,220 x g to pellet the bacteria. Remove the supernatant.</li>
	<li>Re-suspend the bacteria pellet in 10 mL of LB medium and add 10 mL of sodium alginate (3% w/v).</li>
</ol>
4. 3D printing process
<ol>
	<li>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&#160;by clicking the home button for the X, Y, and Z axes.</li>
	<li>For each print, place a prepared printing substrate onto a particular location on the printing bed.</li>
	<li>Calibrate the height of the printhead in the Z axis.
	<ol>
		<li>Raise the printhead to a height of 22&#160;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).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Program the printing shape by a self-developed point-by-point coordinate-determined method according to the desired trajectory.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>Load the liquid bio-ink into syringe(s), and mount them in the syringe pump(s) of the 3D bioprinter.</li>
	<li>Print the bio-ink onto the printing substrate by clicking the Print button.</li>
	<li>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&#8722;3 s to start the syringe pump after the printing is started.</li>
	<li>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.</li>
	<li>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.</li>
	<li>To measure the width and height of the printed hydrogel, use a steel ruler placed underneath or alongside the sample.</li>
</ol>
5. Growing and testing the effectiveness of biofilm production by E. coli
<ol>
	<li>Incubate the printed samples at room temperature for 3&#8722;6 days to allow the production of biofilm components (curli fibers). Image the plates using a camera or fluorescent scanner.</li>
	<li>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.</li>
</ol>

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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+nacre+by+predesigned+matrix-directed+mineralization.">Synthetic nacre by predesigned matrix-directed mineralization.</a> Science. 354 (6308), 107-110 (2016).</li><li>Gao, H. 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The authors have nothing to disclose.

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 CaCl2 solution could enter inside the pipette tip and cause hydrogel formation, causing the pipette tip to become clogged. If the working distance is more than 0.3 mm, the gel cannot be printed continuously. The optimal working distance in this study is 0.2 mm. Good approaches for preparing flat agar printing surfaces are to use larger-diameter Petri dishes (150-mm-diameter Petri dish rather than a 90-mm-diameter plate), place the plates on a flat table, pour the agar solution with fast and even speed, and avoid moving the agar plate during its solidification.
The second critical step is the selection of desired printing parameters including pumping speed, viscosity of the bio-ink used, and printhead speed, which determine the resulting printing resolution. To select these parameters in an efficient manner, the user can sample several extreme values for printhead speed with a constant extrusion rate, noting the width of the printed hydrogel for each set of conditions. Then, repeat this experiment with 4 other extrusion rates. Next, take the five combinations that produced the best printing resolution for the application, and vary both printing parameters (pumping and printhead speeds) in smaller and smaller steps until the desired resolution is obtained.
The thickness of the printed lines has an impact on the ability of the printed engineered bacteria to form stable biofilms. Under optimal printing conditions (pumping speed 0.3 mL/h, printhead speed 300 mm/min, and working distance 0.2 mm), printed lines of bio-ink will produce stable biofilms after 3-6 days of incubation at room temperature. If the lines become thicker, such as by increasing the pumping speed, the middle regions of each line may not be induced sufficiently to produce citrate-stable biofilms.
When printing a multi-layer bio-ink hydrogel, each printed layer is solidified upon contacting the calcium ions that have diffused into the previous printed layer. Since the Ca2+ concentration in the printing substrate is high, Ca2+ ions can rapidly diffuse up through the lower layers. Therefore, the upper layers can be printed immediately after the previous layers have been printed, simply by adjusting the print height in the G-code editor. Additionally, the printing distance of the upper layer should be restricted to only 0.1&#8722;0.2 mm higher than the printing distance of the previous layer. If the added printing distance is less than 0.1 mm, the tip will drag across the first layer and reduce the resolution of the printed hydrogel. If the added printing distance is larger than 0.2 mm, the bio-ink will form drops of liquid during extrusion, causing the printed hydrogel to become discontinuous.
The current bioprinting approach enables the production of reproducible, spatially-controlled engineered biofilms, suitable for use in the study of biofilm mechanical properties or biological resistance of biofilm bacteria to various factors including antibiotics, surfactants, etc. This capability ensures a direct usability of the proposed method. The development of higher-precision do-it-yourself (DIY) bioprinters will likely be possible by maintaining the printing working distance but lowering the pumping speed and the moving speed of the printhead, or by sampling different extruder geometries and bio-ink chemistries. With future improvements to the printing resolution, additional applications can be enabled such as tissue engineering or drug delivery. The 3D bioprinting approach described here should also be able to be expanded to printing additional types of bacteria species that are biocompatible with our alginate-based bio-ink. The current protocol provides sufficient sterility by repeatedly boiling the bio-ink during preparation, using sterile syringes and printing tips, and utilizing antibiotics in both the bio-ink and printing plate. Future experiments using wild-type bacteria may require additional sterilization measures such as replacing or disinfecting the tubing system between prints.
To the authors&#39; best knowledge, the presented method (originally developed in Lehner et al.20) is the first published example of an additive manufacturing style for 3D printing of bacteria. In the first part of this protocol, this general method is described in detail for the 3D printing of bacteria, which is applied to the production of engineered biofilms2. Multiple future applications of 3D-printed biofilms are possible using this method. In nature, multiple bacterial systems have evolved that create various types of biofilms, of which in this article a single system was explored. Multiple other systems can be easily examined by creating 3D-printed biofilms with other bacterial systems, such as Bacillus subtilis or Acetobacter xylinum. Alternative methods23,24 have also been developed for spatial patterning of bacteria at high resolution using optical signals. These approaches require more expensive, complicated equipment to achieve them in comparison to this printer, and are only suitable for patterning of genetically engineered bacteria.
The ability to spatially pattern 3D-printed biofilms with this method can allow for the creation of engineered biofilms that reproduce the spatial heterogeneity of natural biofilms25. Because of the highly detailed arrangement of protein and polymeric fibers within a biofilm, bacteria in a biofilm state achieve a much higher resistance to chemical and physical stimuli, such as an increased resistance to antibiotics as compared to the same bacteria in a planktonic state. Moreover, bacteria within a biofilm show an increased resistance to fluid flow, making the maintenance and sterility of implantable medical devices much more difficult26. Printed engineered biofilms that attempt to reproduce the specific spatial distributions of biofilm components are powerful tools for studying the mechanisms by which bacteria within a biofilm achieve resistance phenotypes.

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 utilization3,4. 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 emerged12,13, 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 biofilms15,16,17. 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.

<table>
	<tbody>
		<tr>
			<td>3D printer</td>
			<td>CoLiDo</td>
			<td>3D-P Kit</td>
			<td></td>
		</tr>
		<tr>
			<td>3D printing software</td>
			<td>CoLiDo</td>
			<td>Print-Rite ColiDo Repetier-Host v2.0.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>05040</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>3886.1</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth powder</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>VWR</td>
			<td>89032-092</td>
			<td>Model 3500</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>VWR</td>
			<td>25384-326</td>
			<td>150 x 15 mm</td>
		</tr>
		<tr>
			<td>Rhamnose</td>
			<td>Sigma-Aldrich</td>
			<td>83650</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon tubing</td>
			<td>VWR&#160;</td>
			<td>DENE 3100103/25</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>ProSense B.V.&#160;</td>
			<td>NE-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium alginate</td>
			<td>Sigma-Aldrich</td>
			<td>W201502</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>71498</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydrooxide</td>
			<td>VWR</td>
			<td>28244.295</td>
			<td></td>
		</tr>
	</tbody>
</table>

three-dimensional patterning of engineered biofilms with a do-it-yourself bioprinter

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.

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 to a tubing system connected to a syringe pump (Figure 1B). The successful conversion of the commercial printer into a bioprinter can be assessed based on the ability to transfer desired bio-ink(s) from the syringe pump through the tubing system and pipette tip(s) onto a printing surface without leaking or heating the bio-ink. If the tubing bulges due to the flow of bio-ink during printing, it may be replaced by tubing with thicker walls. It should be noted that this printing technique should be able to work with any type of commercial 3D printer for which tubing can be attached to the printhead.
The 3D bioprinter can create bacteria-encapsulating hydrogels in a variety of two-dimensional (2D) and 3D shapes (Figure 2). Calcium ions in the printing substrate induce solidification (chelation of calcium ions with alginate carboxyl groups) of the bio-ink upon printing, converting the liquid bio-ink into a solid hydrogel. The resolution of bioprinting will depend on the extrusion speed, the size of the pipette tip, the speed of the printhead, the volume and concentration of CaCl2 solution added to the agar solution, the flatness of the printing surface, and the viscosity of the bio-ink used. The concentration of CaCl2 solution has a great influence on hydrogel sharpness. Four different concentrations of CaCl2 (0.1 M, 0.2 M, 1 M, and 5 M) were sampled, and only 5 M CaCl2 solution resulted in hydrogel that did not become blurred after printing. Therefore, 5 M was chosen as the optimal concentration of CaCl2 solution.
In an earlier version of this protocol, the CaCl2 solution was applied onto the surface of the agar plate rather than mixed into the agar solution before pouring the agar plate. When using this version, the volume of the CaCl2 solution has a critical influence over printing quality and resolution. When using a 150 x 15 mm Petri dish, applying a volume of calcium chloride solution of more than 100 &#181;L (or 30 &#181;L for a 90-mm Petri dish) results in too much liquid remaining on the printing surface. This liquid may spread unevenly when the plate is moved, which can change the working distance and cause blockage of the pipette tip. Too much volume of CaCl2 can also cause printed hydrogels to float and slide across the solution, changing the shape and position of the printed hydrogel. If the volume of calcium chloride solution is too small, some regions of the plate may not receive CaCl2 solution and will have poor hydrogel solidification. In this improved protocol, adding the CaCl2 solution directly into the agar solution prior to pouring the agar plate resulted in substantially less moisture on the surface of the printing substrate compared to the surface-applied method, resulting in dramatically improved printing resolution.
The extrusion speed and printhead movement are interdependent and can be tuned in a coordinated manner to alter the printing resolution. For example, if the printer is operated with extrusion speed between 0.1 mL/h and 0.5 mL/h with a constant printhead movement speed of 300 mm/min, the diameter of the printed hydrogel increases with the increase of extrusion speed2,20. At extrusion speeds over 0.5 mL/h, the outer edges of the printed lines of hydrogel change from straight, parallel lines to wavy lines, and the line width also increases. The velocity of the printhead also has an influence on the printing resolution. With a constant extrusion speed of 0.3 mL/h, increasing the speed of the printhead from 300 mm/min to 500 mm/min results in the width of the printed hydrogel becoming narrower, decreasing from 1.8 mm to 0.9 mm. If the printhead moving speed is over 500 mm/min, the gel line will easily become discontinuous. For a 200 &#181;L pipette tip and the bio-ink used in the current study, several combinations of the printing resolution are considered optimal (Table 2). At pumping speed 0.3 mL/h, printhead movement speed 500 mm/min, and working distance 0.2 mm, printed hydrogel is produced with a width of approximately 0.9 mm.
One crucial achievement of the bacterial 3D printing method is its ability to create engineered biofilms. To create an engineered and spatially-controlled biofilm, the bacteria should not only survive the 3D printing process but should also produce biofilm components while remaining within the printed pattern. The engineered E. coli bacteria used in this protocol, E. coli &#916;csgA bacteria carrying the plasmid pSB1C3-GFP-CsgA, enable controllable expression of curli proteins. The use of a csgA-knockout strain ensures that CsgA protein is only expressed when it is induced from a plasmid with rhamnose. The bacteria export the induced CsgA protein subunits, which then self-assemble21 onto CsgB proteins on the bacterial outer membrane22 to form curli fibers. These amyloid-like fibers are the major proteinaceous components of biofilm extracellular matrix: a connected network of proteins and polymers in which the bacteria are embedded. The printed alginate matrix of the 3D-printing bio-ink lends physical support and structure to the bacteria during the curli production process. The use of constitutive GFP expression allows for visualization and quantification of printed cells via fluorescence imaging.
In order to assess whether the formation of biofilm was successful, the alginate matrix was dissolved using a sodium citrate solution, and the shape of the printed bio-ink was assessed after the citrate treatment (Figure 3). In the case of bio-ink 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 (Figure 3A,B). In the case of bacteria containing the inducible curli production plasmid, the gel was not dissolved after sodium citrate treatment (Figure 3C,D). This result indicates that the printed bacteria were able to form a curli network extensive enough to stabilize the printed pattern of bacteria2.
To construct multi-layered structures, additional layers were printed (Figure 4) by adjusting the print height and print trajectory in 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 (Figure 5)2,20, but even 5-layer printed structures could be created with a resolution of millimeters to sub-millimeters. When E. coli engineered to inducibly produce curli proteins were printed into multi-layered structures, sodium citrate treatment did not dissolve the samples, whereas multi-layer structures containing non-curli-producing E. coli were dissolved in sodium citrate solution (Figure 6). This experiment demonstrates that engineered biofilms can be created in multi-layered, three-dimensional printed structures, as well as in single-layer printed structures.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59477/59477fig1.jpg" /> 
Figure 1: Photos showing the conversion of a commercial 3D printer into a 3D bioprinter. (A) The components of the 3D bioprinter after conversion from a commercial 3D printer. (B) The bio-ink extruder formed by a tubing system attached to a pipette tip. Additional printing tips can be added in the second printhead hole or by adding additional holes to the printhead, for use in printing multiple types of bio-ink. <a href="https://www.jove.com/files/ftp_upload/59477/59477fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59477/59477fig2.jpg" /> 
Figure 2: Examples of 3D bioprinted patterns containing E. coli pSB1C3-GFP-CsgA. These images were taken two days after printing. This printing resolution was obtained with pumping speed 0.3 mL/h, printhead movement speed 300 mm/min, and working distance 0.2 mm. The G-codes for printing these shapes may be found in the Supplemental Files. <a href="https://www.jove.com/files/ftp_upload/59477/59477fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59477/59477fig3.jpg" /> 
Figure 3: A method of verifying whether biofilm components have been produced by E. coli bacteria within a printed pattern. When printed E. coli contained a plasmid that did not encode for curli induction, the printed pattern was completely dissolved by sodium citrate treatment (A and B). When E. coli containing a plasmid encoding inducible curli proteins was used, the printed biofilm was resistant to sodium citrate treatment (C and D). The programming process and explanations of the G-code for printing this square pattern are provided in Table 1. <a href="https://www.jove.com/files/ftp_upload/59477/59477fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59477/59477fig4.jpg" /> 
Figure 4: Top view (A) and side view (B) of multi-layered printed structures containing E. coli pSB1C3-GFP-CsgA. This sample was printed with pumping speed 0.3 mL/h, printhead movement speed 200 mm/min, and working distance 0.2 mm. <a href="https://www.jove.com/files/ftp_upload/59477/59477fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59477/59477fig5.jpg" /> 
Figure 5: The line width and height of printed hydrogels containing different numbers of printed layers. The measurements were performed on samples printed with pumping speed 0.3 mL/h, printhead movement speed 500 mm/min, and working distance 0.2 mm. <a href="https://www.jove.com/files/ftp_upload/59477/59477fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59477/59477fig6.jpg" /> 
Figure 6: A method of verifying whether biofilm components have been produced by E. coli bacteria within multi-layer printed structures. Engineered E. coli was printed into 1-, 3-, or 5-layer hydrogels and incubated for 6 days. When the printed E. coli contained a plasmid that did not encode for curli induction, the printed pattern was completely dissolved by sodium citrate treatment (A and B). When the printed E. coli contained a plasmid encoding inducible curli proteins, the printed biofilm was resistant to sodium citrate treatment (C and D). <a href="https://www.jove.com/files/ftp_upload/59477/59477fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>G-code commands</td>
			<td>Tasks</td>
		</tr>
		<tr>
			<td>G1 Z20 F9000</td>
			<td>Lift the Z-axis to a height of 20 mm with a 9,000 mm/min moving speed.</td>
		</tr>
		<tr>
			<td>G1 X95 Y65 F9000</td>
			<td>Move to the starting point of the first line with a 9,000 mm/min moving speed.</td>
		</tr>
		<tr>
			<td>G1 Z6 F9000</td>
			<td>Move downwards in the Z-direction to a proper (here Z = 6 mm) printing distance.</td>
		</tr>
		<tr>
			<td>G1 X95 Y105 F300</td>
			<td>End point of the first line and starting point of the second line.</td>
		</tr>
		<tr>
			<td>G1 X135 Y105&#160;</td>
			<td>End point of the second line and starting point of the third line.</td>
		</tr>
		<tr>
			<td>G1 X135 Y65</td>
			<td>End point of the third line and starting point of the fourth line.</td>
		</tr>
		<tr>
			<td>G1 X95 Y65</td>
			<td>End point of the fourth line and starting point of the first&#160; line; a square is formed.</td>
		</tr>
		<tr>
			<td>G1 Z20 F9000</td>
			<td>Lift the Z-axis to a height of 20 mm at 9,000 mm/min.</td>
		</tr>
		<tr>
			<td>G1 X55 Y40 F9000</td>
			<td>Move to a coordinate (55, 40) outside of the Petri dish range.</td>
		</tr>
	</tbody>
</table>
Table 1: Programming process and explanations of G-code for printing a square.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Extrusion speed (mL/h)</td>
			<td>Printhead moving speed (mm/min)</td>
			<td>Gel width (mm)</td>
		</tr>
		<tr>
			<td>0.1</td>
			<td>100</td>
			<td>1.6 &#177; 0.1</td>
		</tr>
		<tr>
			<td>0.1</td>
			<td>200</td>
			<td>1.1 &#177; 0.1</td>
		</tr>
		<tr>
			<td>0.1</td>
			<td>300</td>
			<td>1.0 &#177; 0.1</td>
		</tr>
		<tr>
			<td>0.3</td>
			<td>300</td>
			<td>1.8 &#177; 0.1</td>
		</tr>
		<tr>
			<td>0.3</td>
			<td>400</td>
			<td>1.2 &#177; 0.1</td>
		</tr>
		<tr>
			<td>0.3</td>
			<td>500</td>
			<td>0.9 &#177; 0.1</td>
		</tr>
		<tr>
			<td>0.5</td>
			<td>200</td>
			<td>2.2 &#177; 0.2</td>
		</tr>
		<tr>
			<td>0.5</td>
			<td>1,200</td>
			<td>1.2 &#177; 0.2</td>
		</tr>
		<tr>
			<td>0.7</td>
			<td>200</td>
			<td>2.8 &#177; 0.1</td>
		</tr>
		<tr>
			<td>0.7</td>
			<td>1,200</td>
			<td>1.3 &#177; 0.1</td>
		</tr>
	</tbody>
</table>
Table 2: The optimal printing parameters for hydrogels with high resolution. Five points were measured for each condition. The average value and standard deviation are shown in the table.

Watch this Scientific Journal Video about Three-dimensional Patterning of Engineered Biofilms with a Do-it-yourself Bioprinter at JoVE.com

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

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.

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced ...

three-dimensional-patterning-engineered-biofilms-with-do-it-yourself

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.5K Views.  Delft University of Technology. 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.

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

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the living body than two-dimensional cell culture. In this study, we fabricated an electrical motor-driven lab-on-a-CD (compact disc) platform, called a centrifugal microfluidic-based spheroid (CMS) culture system, to create three-dimensional (3D) cell spheroids implementing high centrifugal force. This device can vary rotation speeds to generate gravity conditions from 1 x g to 521 x g. The CMS system is 6 cm in diameter, has one hundred 400 &#956;m microwells, and is made by molding with polydimethylsiloxane in a polycarbonate mold premade by a computer numerical control machine. A barrier wall at the channel entrance of the CMS system uses centrifugal force to spread cells evenly inside the chip. At the end of the channel, there is a slide region that allows the cells to enter the microwells. As a demonstration, spheroids were generated by monoculture and coculture of human adipose-derived stem cells and human lung fibroblasts under high gravity conditions using the system. The CMS system used a simple operation scheme to produce coculture spheroids of various structures of concentric, Janus, and sandwich. The CMS system will be useful in cell biology and tissue engineering studies that require spheroids and organoid culture of single or multiple cell types.

We present a motor-powered centrifugal microfluidic device that can cultivate cell spheroids. Using this device, spheroids of single or multiple cell types could be easily cocultured under high gravity conditions.

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...