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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<li>Wu, H., Moser, C., Wang, H. Z., Hoiby, N., Song, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strategies+for+combating+bacterial+biofilm+infections%5BTitle%5D)+AND+%22International+Journal+of+Oral+Science%22%5BJournal%5D)">Strategies for combating bacterial biofilm infections.</a> International Journal of Oral Science. 7 (1), 1-7 (2015).</li>
<li>Stewart, P. S., Franklin, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physiological+heterogeneity+in+biofilms%5BTitle%5D)+AND+%22Nature+Reviews+Microbiology%22%5BJournal%5D)">Physiological heterogeneity in biofilms.</a> Nature Reviews Microbiology. 6 (3), 199-210 (2008).</li>
<li>Kikuchi, T., Mizunoe, Y., Takade, A., Naito, S., Yoshida, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Curli+Fibers+Are+Required+for+Development+of+Biofilm+Architecture+in+Escherichia+coli+K-12+and+Enhance+Bacterial+Adherence+to+Human+Uroepithelial+Cells%5BTitle%5D)+AND+%22Microbiology+and+Immunology%22%5BJournal%5D)">Curli Fibers Are Required for Development of Biofilm Architecture in Escherichia coli K-12 and Enhance Bacterial Adherence to Human Uroepithelial Cells.</a> Microbiology and Immunology. 49 (9), 875-884 (2005).</li>
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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 CaCl<s...

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>CaCl&lt;sub&gt;2&lt;/sub&gt; 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&amp;nbsp;</td><td>DENE 3100103/25</td><td></td></tr><tr><td>Syringe pump</td><td>ProSense B.V.&amp;nbsp;</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...

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 ...

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9.6K 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

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15

A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

Immunology and Infection

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

High-resolution Patterned Biofilm Deposition Using pDawn-Ag43

Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms into arbitrary spatial patterns at high spatial resolution. The technique uses a genetically encoded optogenetic construct&#8212;pDawn-Ag43&#8212;that couples biofilm formation in E. coli to optical stimulation by blue light. We detail the process for transforming E. coli with&#160;pDawn-Ag43, preparing the required optical set-up, and the protocol for culturing patterned biofilms using pDawn-Ag43 bacteria. Using this protocol, biofilms with a spatial resolution below 25 &#956;m can be patterned on various surfaces and environments, including enclosed chambers, without requiring microfabrication, clean-room facilities, or surface pretreatment. The technique is convenient and appropriate for use in applications that investigate the effect of biofilm structure, providing tunable control over biofilm patterning. More broadly, it also has potential applications in biomaterials, education, and bio-art.

We demonstrate a method for depositing Escherichia coli bacterial biofilms in arbitrary spatial patterns with a high resolution using optical stimulation of a genetically encoded surface-adhesion construct.

Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create tissues by adding layer upon layer of printable biomaterials, termed bioinks, and cells in an orderly and automatic manner, which allows for creating highly intricate structures that traditional tissue engineering techniques cannot achieve. Thus, 3D bioprinting is an appealing in vitro approach to mimic the native vasculature complex structure, ranging from millimetric vessels to microvascular networks. 
Advances in 3D bioprinting in granular hydrogels enabled the high-resolution extrusion of low-viscosity extracellular matrix-based bioinks. This work presents a combined 3D bioprinting and sacrificial mold-based 3D printing approach for fabricating engineered vascularized tissue flaps. 3D bioprinting of endothelial and support cells using recombinant collagen-methacrylate bioink within a gelatin support bath is utilized for the fabrication of a self-assembled capillary network. This printed microvasculature is assembled around a mesoscale vessel-like porous scaffold, fabricated using a sacrificial 3D printed mold, and is seeded with endothelial cells. 
This assembly induces the endothelium of the mesoscale vessel to anastomose with the surrounding capillary network, establishing a hierarchical vascular network within an engineered tissue flap. The engineered flap is then directly implanted by surgical anastomosis to a rat femoral artery using a cuff technique. The described methods can be expanded for the fabrication of various vascularized tissue flaps for use in reconstruction surgery and vascularization studies.

Engineered flaps require an incorporated functional vascular network. In this protocol, we present a method of fabricating a 3D printed tissue flap containing a hierarchical vascular network and its direct microsurgical anastomoses to rat femoral artery.

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create ...

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

Author Spotlight: Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...

Bioprinting Bacteria in Hydrogel Matrices for the Study of Motility and Growth

3D Printing Bacteria to Study Motility and Growth in Complex 3D Porous Media

Bacteria are ubiquitous in complex three-dimensional (3D) porous environments, such as biological tissues and gels, and subsurface soils and sediments. However, the majority of previous work has focused on studies of cells in bulk liquids or at flat surfaces, which do not fully recapitulate the complexity of many natural bacterial habitats. Here, this gap in knowledge is addressed by describing the development of a method to 3D-print dense colonies of bacteria into jammed granular hydrogel matrices. These matrices have tunable pore sizes and mechanical properties; they physically confine the cells, thus supporting them in 3D. They are optically transparent, allowing for direct visualization of bacterial spreading through their surroundings using imaging. As a proof of this principle, here, the capability of this protocol is demonstrated by 3D printing and imaging non-motile and motile Vibro cholerae, as well as non-motile Escherichia coli, in jammed granular hydrogel matrices with varying interstitial pore sizes.

Author Spotlight: Studying Bacterial Growth in 3D Hydrogel Matrices

This protocol describes a procedure for three-dimensional (3D) printing of bacterial colonies to study their motility and growth in complex 3D porous hydrogel matrices that are more akin to their natural habitats than conventional liquid cultures or Petri dishes.

Bacteria are ubiquitous in complex three-dimensional (3D) porous environments, such as biological tissues and gels, and subsurface soils and sediments. ...