This research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors. Special thanks to Florian Schmittner, Sandro Gorka, Gurinder Singh, Vincent Tran, and Dominik Vu for their contribution on an earlier prototype of the design.

<ol>
	<li>Brettel, M., Friederichsen, N., Keller, M., Rosenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Virtualization,+Decentralization+and+Network+Building+Change+the+Manufacturing+Landscape:+An+Industry+4.0+Perspective.">How Virtualization, Decentralization and Network Building Change the Manufacturing Landscape: An Industry 4.0 Perspective.</a> World Academy of Science, Engineering and Technology International Journal of Information and Communication Engineering. 8 (1), (2014).</li><li>Gilchrist, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introducing+Industry+4.0.">Introducing Industry 4.0.</a> Industry 4.0. , 195-215 (2016).</li><li>Petrick, I. J., Simpson, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Printing+Disrupts+Manufacturing:+How+Economies+of+One+Create+New+Rules+of+Competition.">3D Printing Disrupts Manufacturing: How Economies of One Create New Rules of Competition.</a> Research-Technology Management. 56 (6), 12-16 (2013).</li><li>Wong, K., Hernandez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review+of+Additive+Manufacturing.">A Review of Additive Manufacturing.</a> ISRN Mechanical Engineering. 10, (2012).</li><li>Lanaro, M., Desselle, M. R., Woodruff, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Printing+Chocolate:+Properties+of+Formulations+for+Extrusion,+Sintering,+Binding+and+Ink+Jetting.">3D Printing Chocolate: Properties of Formulations for Extrusion, Sintering, Binding and Ink Jetting.</a> Fundamentals of 3D Food printing and Applications. , (2018).</li><li>Godoi, F. C., Prakash, S., Bhandari, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3d+printing+technologies+applied+for+food+design:+Status+and+prospects.">3d printing technologies applied for food design: Status and prospects.</a> Journal of Food Engineering. 179, 44-54 (2016).</li><li>Stansbury, J. W., Idacavage, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+printing+with+polymers:+Challenges+among+expanding+options+and+opportunities.">3D printing with polymers: Challenges among expanding options and opportunities.</a> Dental Materials. 32 (1), 54-64 (2016).</li><li>Zhu, W., Ma, X., Gou, M., Mei, D., Zhang, K., Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+printing+of+functional+biomaterials+for+tissue+engineering.">3D printing of functional biomaterials for tissue engineering.</a> Current Opinion in Biotechnology. 40, 103-112 (2016).</li><li>Lanaro, M., Booth, L., Powell, S. K., Woodruff, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrofluidodynamic+technologies+for+biomaterials+and+medical+devices:+melt+electrospinning.">Electrofluidodynamic technologies for biomaterials and medical devices: melt electrospinning.</a> Electrofluidodynamic Technologies (EFDTs) for Biomaterials and Medical Devices. , 37-69 (2018).</li><li>Malone, E., Lipson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fab@Home:+the+personal+desktop+fabricator+kit+Article+information.">Fab@Home: the personal desktop fabricator kit Article information.</a> Rapid Prototyping Journal. 13 (4), 245-255 (2007).</li><li>Vilbrandt, T., Malone, E., Lipson, H., Pasko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+Desktop+Fabrication.">Universal Desktop Fabrication.</a> Heterogeneous Objects Modelling and Applications. , 259-284 (2008).</li><li>Jones, R., 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=RepRap-the+replicating+rapid+prototyper.">RepRap-the replicating rapid prototyper.</a> Robotica. 29, 177-191 (2011).</li><li>Lanaro, M., 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=3D+printing+complex+chocolate+objects:+Platform+design,+optimization+and+evaluation.">3D printing complex chocolate objects: Platform design, optimization and evaluation.</a> Journal of Food Engineering. , (2017).</li><li>Wu, W., DeConinck, A., Lewis, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Omnidirectional+Printing+of+3D+Microvascular+Networks.">Omnidirectional Printing of 3D Microvascular Networks.</a> Advanced Materials. 23 (24), H178-H183 (2011).</li><li>Paxton, N., Smolan, W., B&#246;ck, T., Melchels, F., Groll, J., Jungst, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proposal+to+assess+printability+of+bioinks+for+extrusion-based+bioprinting+and+evaluation+of+rheological+properties+governing+bioprintability.">Proposal to assess printability of bioinks for extrusion-based bioprinting and evaluation of rheological properties governing bioprintability.</a> Biofabrication. 9 (4), 044107 (2017).</li></ol>

The authors have nothing to disclose.

This protocol provides detailed instructions for constructing a low-cost melt extrusion-based 3D printer. Construction of the 3D printer can be broken down into subsections including frame, y-axis/bed, x-axis, extruder, electronics, and software. These subsections are presented with detailed diagrams, drawings, files and parts lists. The total price of an ADDME 3D printer comes to $343 AUD ($245 USD as of 01/17/2019), making this the cheapest, reservoir-based melt extrusion 3D printer currently known. It was aimed to mak...

Additive manufacturing is a powerful manufacturing technology that has the potential to provide significant value to the industrial landscape1,2. The attractive features of additive manufacturing involve no tooling costs, high levels of customization, complex geometries, and reduced barriers to entry costs. No retooling costs allow for the rapid manufacturing of prototypes, which is desirable when trying to decrease &#34;time to market&#34;, which is a critical aim of industries in developed nations trying to remain competitive against low-wage competitors1. High levels of customizability allow for a wide variety of products to be fabricated with complex geometries. When these factors are combined with the low costs for set-up, materials, and operator specialization, there is a clear value of additive manufacturing technologies3.
Additive manufacturing, also called 3D printing, involves layer-by-layer fabrication of an object in a computer numerical controlled (CNC) system3. Unlike traditional CNC processes such as milling, in which material is removed from a sheet or block of material, a 3D printing system adds material into the desired structure layer-by-layer.
3D printing can be facilitated through a range of methods including laser, flash, extrusion, or jetting technologies4. The specific technology employed determines the form of the raw material (i.e., powder or melt), as well as the rheological and thermal properties required for processing5. The extrusion-based 3D printing market is dominated by filament-based systems, which is due to filaments being easy to handle, process, and continuously supply large volumes of material to the extrusion head. However, this process is limited by the type of material able to be formed into filaments (mainly thermoplastics). Most materials do not exist in filament form, and the lack of modern low-cost platforms in the market represents a notable gap.
This protocol shows the construction of a reservoir-based extrusion system that allows materials to be stored in a syringe and extruded through a needle. This system is ideally suited to manufacture a wide range of materials including foods6, polymers7, and biomaterials8,9. Furthermore, reservoir-based extrusion techniques are typically less hazardous, lower in cost, and easier to operate than other 3D printing methods.
There is a growing number of university-led teams designing and releasing open-source 3D printing systems to the public. Beginning with the Fab@Home extrusion-based printer in 200710,11, researchers aimed to create a simple and cheap platform to drive rapid expansion in 3D printing technology and applications. Later in 2011, the RepRap project aimed to create a filament-based 3D printing platform designed with parts made by 3D printing, with the goal to create a self-replicating machine12. The cost of 3D printers has been dropping over the years, from $2300 USD for a Fab@Home (2006), $573 USD for a RepRap v1 (2005), and $400 USD for v2 (2011).
In previous work, we demonstrated how an off-the-self 3D printing system could be combined with a custom reservoir-based extrusion system to create complex 3D objects from chocolate13. Further design investigation has shown that considerable cost savings can be achieved compared to this prototype design.
The aim of this protocol is to provide instructions for the construction of a low-cost reservoir-based melt extrusion 3D printer. Presented here are detailed diagrams, drawings, files, and component lists to allow successfully construction and operation of a 3D printer. All components are hosted on the open-source (creative commons noncommercial) platform <a href="https://www.thingiverse.com/Addme/collections" target="_blank">https://www.thingiverse.com/Addme/collections</a>, which allows users to change or add additional features as desired. Viscous cream, chocolate, and Pluronic F-127 (a model for bioinks) are used to evaluate the performance of ADDME and demonstrate application of the ADDME 3D printer to the biomedical and food printing industries.
A laser cutter capable of cutting acrylic and a desktop 3D printer capable of printing PLA or ABS filaments are required for this protocol. A machined heating jacket and heater cartridge or silicone heater can be used to heat the material, depending on which equipment the operator has access to. All CAD files can be found at <a href="https://www.thingiverse.com/Addme/designs" target="_blank">https://www.thingiverse.com/Addme/designs</a>. For firmware and software to control the 3D printer, <a href="http://marlinfw.org/meta/download/" target="_blank">http://marlinfw.org/meta/download/</a> and <a href="https://www.repetier.com/" target="_blank">https://www.repetier.com/</a> are provided resources, respectively. For detailed instructions about the control board, see <a href="https://reprap.org/wiki/RAMPS_1.4" target="_blank">https://reprap.org/wiki/RAMPS_1.4</a>.

<table><tbody><tr><td>15 W 12V DC 50x100mm Flexible Silicon Heater</td><td>Banggood</td><td>1280175</td><td>Optional; AU$4.46</td></tr><tr><td>3D Printer</td><td>Lulzbot</td><td></td><td>&lt;a href=&quot;https://download.lulzbot.com/TAZ/6.02/documentation/manual/9780989378482_interior_r6.02.pdf&quot; target=&quot;_blank&quot;&gt;https://download.lulzbot.com/&lt;/a&gt;</td></tr><tr><td>3D Printer</td><td>Ultimaker</td><td>Ultimaker 2+</td><td></td></tr><tr><td>AC 100-240V to DC 12V 5A 60W Power Supply</td><td>Banggood</td><td>994870</td><td>AU$12.7</td></tr><tr><td>Acrylic Sheet White Continuous Cast 1200x600mm</td><td>Mulford Plastics</td><td></td><td>AU$36.95</td></tr><tr><td>Allen Keys</td><td>Metric</td><td></td><td></td></tr><tr><td>Arduino MEGA2560 R3 with RAMPS 1.4 Controller</td><td>Geekcreit</td><td>984594</td><td>AU$28.91</td></tr><tr><td>Carbon Steel Linear Shaft 8mm x 350mm</td><td>Banggood</td><td>1119330</td><td>AU$13.44</td></tr><tr><td>Carbon Steel linear Shaft 8mm x 500mm</td><td>Banggood</td><td>1276011</td><td>AU$19.42</td></tr><tr><td>Chocolate</td><td>Cadbury</td><td></td><td></td></tr><tr><td>Computer with internet access</td><td>Dell</td><td></td><td></td></tr><tr><td>Coupler 5-8mm</td><td>Banggood</td><td>1070710</td><td>AU$6.93</td></tr><tr><td>Hand Cream</td><td>Nivea</td><td>80102</td><td></td></tr><tr><td>Heating Cartridge</td><td>Creality 3D</td><td>1192704</td><td>AU$4.75</td></tr><tr><td>K Type Temperature Sensor Thermocouple</td><td>Banggood</td><td>1212169</td><td>AU$2.37</td></tr><tr><td>Laser Cutter</td><td>trotec</td><td>Speedy 300</td><td>&lt;a href=&quot;https://www.troteclaser.com/fileadmin/content/images/Contact_Support/Manuals/8047-speedy-400-redesign-manual-EN.pdf&quot; target=&quot;_blank&quot;&gt;https://www.troteclaser.com/&lt;/a&gt;</td></tr><tr><td>M10 1mm Pitch Thread Metal Hex Nut + Washer</td><td>UXCELL</td><td></td><td>AU$8.84</td></tr><tr><td>M10 1mm Pitch Zinc Plated Pipe 400mm Length</td><td>UXCELL</td><td></td><td>AU$11.62</td></tr><tr><td>M2 - 0.4mm Internal Thread Brass Inserts</td><td>Ebay</td><td></td><td>AU$5.65</td></tr><tr><td>M2 Nuts</td><td>Suleve</td><td>1239291</td><td>AU$9.17</td></tr><tr><td>M2 x 10 mm Button Hex Screws</td><td>Suleve</td><td>1239291</td><td>AU$9.17</td></tr><tr><td>M2 x 5mm Button Hex Screws</td><td>Suleve</td><td>1239291</td><td>AU$9.17</td></tr><tr><td>M3 - 0.5mm Internal Thread Brass Inserts</td><td>Suleve</td><td>1262071</td><td>AU$7.5</td></tr><tr><td>M3 Nuts</td><td>Suleve</td><td>1109208</td><td>AU$7.85</td></tr><tr><td>M3 Washer</td><td>Banggood</td><td>1064061</td><td>AU$3.05</td></tr><tr><td>M3 x 10mm Button Hex Screws</td><td>Suleve</td><td>1109208</td><td>AU$7.85</td></tr><tr><td>M3 x 20mm Button Hex Screws</td><td>Suleve</td><td>1109208</td><td>AU$7.85</td></tr><tr><td>M3 x 6mm Button Hex Screws</td><td>Suleve</td><td>1109208</td><td>AU$7.85</td></tr><tr><td>M3 x 8mm Button Hex Screws</td><td>Suleve</td><td>1109208</td><td>AU$7.85</td></tr><tr><td>M4 x 8mm Button Hex Screws</td><td>Suleve</td><td>1273210</td><td>AU$4.32</td></tr><tr><td>Needle Luer Lock 18 - 27 Gauge</td><td>Terumo</td><td>TGA ARTG ID: 130227</td><td>AU$3.57</td></tr><tr><td>NEMA 17 Stepper Motor</td><td>Casun</td><td>42SHD0001-24B</td><td>AU$54</td></tr><tr><td>NEMA Stepper Motor Mounting Bracket</td><td>Banggood</td><td>ptNema17br90</td><td>AU$4.79</td></tr><tr><td>Pillow Block Flange Bearing 8mm</td><td>Banggood</td><td>KFL08</td><td>AU$5.04</td></tr><tr><td>PLA Filament</td><td>Creality 3D</td><td>1290153</td><td>AU$24.95</td></tr><tr><td>Pluronic F127</td><td>Sigma Aldrich</td><td>P2443-250G</td><td></td></tr><tr><td>SC8UU 8mm Linear Motion Ball Bearing</td><td>Toolcool</td><td>935967</td><td>AU$21.6</td></tr><tr><td>SG-5GL Micro Limit Switch</td><td>Omron</td><td>1225333</td><td>AU$4.5</td></tr><tr><td>Soldering Station</td><td></td><td></td><td>Solder, Wires, Heat shrink e.c.t.</td></tr><tr><td>Spring</td><td>Banggood</td><td>995375</td><td>AU$2.53</td></tr><tr><td>Syringe 3ml Luer Lock Polypropylene</td><td>Brauhn</td><td>9202618N</td><td>AU$3.14</td></tr><tr><td>Timing Pulley GT2 20 Teeth and Belt Set</td><td>Banggood</td><td>10811303</td><td>AU$11.48</td></tr><tr><td>Trapezoidal Lead Screw and Nut 8mm x 400mm</td><td>Banggood</td><td>1095315</td><td>AU$29.02</td></tr><tr><td>Variable Spanner</td><td></td><td></td><td></td></tr></tbody></table>

design of an open-source, low-cost bioink and food melt extrusion 3d printer

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling costs. This increasing popularity is partly driven by falling barriers to entry such as system set-up costs and ease of operation. The following protocol presents the design and construction of an Additive Manufacturing Melt Extrusion (ADDME) 3D printer for the fabrication of custom parts and components. ADDME has been designed with a combination of 3D-printed, laser-cut, and online-sourced components. The protocol is arranged into easy-to-follow sections, with detailed diagrams and parts lists under the headings of framing, y-axis and bed, x-axis, extrusion, electronics, and software. The performance of ADDME is evaluated through extrusion testing and 3D printing of complex objects using viscous cream, chocolate, and Pluronic F-127 (a model for bioinks). The results indicate that ADDME is a capable platform for the fabrication of materials and constructs for use in a wide range of industries. The combination of detailed diagrams and video content facilitates access to low-cost, easy-to-operate equipment for individuals interested in 3D printing of complex objects from a wide range of materials.

The performance of ADDME during 3D printing was evaluated using a viscous cream (150 mL, Nivea hand cream), chocolate (Cadbury, plain milk), and Pluronic F-127 (Sigma Aldrich). The viscous cream and chocolate were used as is, and the Pluronic was dissolved into a 20% wt solution with ultrapure water and stored refrigerated at 5 &#176;C until needed14,15.
Line testing inv...

Watch this Scientific Journal Video about Design of an Open-Source, Low-Cost Bioink and Food Melt Extrusion 3D Printer at JoVE.com

Design of an Open-Source, Low-Cost Bioink and Food Melt Extrusion 3D Printer

The aim of this work is to design and construct a reservoir-based melt extrusion three-dimensional printer made from open-source and low-cost components for applications in the biomedical and food printing industries.

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling ...

design-an-open-source-low-cost-bioink-food-melt-extrusion-3d

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.8K Views.  Queensland University of Technology (QUT). The aim of this work is to design and construct a reservoir-based melt extrusion three-dimensional printer made from open-source and low-cost components for applications in the biomedical and food printing industries.

Video: Design of an Open-Source, Low-Cost Bioink and Food Melt Extrusion 3D Printer

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

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Advancing 3D Bioprinting with &#954;-Carrageenan Sub-Microgel Medium

Embedded Bioprinting of Tissue-like Structures Using &#954;-Carrageenan Sub-Microgel Medium

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic scaffolds. However, engineering a suitable gel suspension medium that balances precise bioink deposition with cell viability and function presents multiple challenges, particularly in achieving the desired viscoelastic properties. Here, a novel &#954;-carrageenan gel supporting bath is fabricated through an easy-to-operate mechanical grinding process, producing homogeneous sub-microscale particles. These sub-microgels exhibit typical Bingham flow behavior with small yield stress and rapid shear-thinning properties, which facilitate the smooth deposition of bioinks. Moreover, the reversible gel-sol transition and self-healing capabilities of the &#954;-carrageenan microgel network ensure the structural integrity of printed constructs, enabling the creation of complex, multi-layered tissue structures with defined architectural features. Post-printing, the &#954;-carrageenan sub-microgels can be easily removed by a simple phosphate-buffered saline wash. Further bioprinting with cell-laden bioinks demonstrates that cells within the biomimetic constructs have a high viability of 92% and quickly extend pseudopodia, as well as maintain robust proliferation, indicating the potential of this bioprinting strategy for tissue and organ fabrication. In summary, this novel &#954;-carrageenan sub-microgel medium emerges as a promising avenue for embedded bioprinting of exceptional quality, bearing profound implications for the in vitro development of engineered tissues and organs.

Author Spotlight: Development of Homogeneous &#954;-Carrageenan Sub-Microgel Baths for High-Resolution 3D Bioprinting

This study introduces a novel &#954;-carrageenan sub-microgel suspension bath, displaying remarkable reversible jamming-unjamming transition properties. These attributes contribute to the construction of biomimetic tissues and organs in embedded 3D bioprinting. The successful printing of heart/esophageal-like tissues with high resolution and cell growth demonstrates high-quality bioprinting and tissue engineering applications.

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic ...

Engineering

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.

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 ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...

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 ...

Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several approaches have been developed for the preparation and use of supporting gel beds in 3D bioprinting. This paper describes a process to manufacture microgel suspensions using agarose (known as fluid gels), wherein particle formation is governed by the application of shear during gelation. Such processing produces carefully defined microstructures, with subsequent material properties that impart distinct advantages as embedding print media, both chemically and mechanically. These include behaving as viscoelastic solid-like materials at zero shear, limiting long-range diffusion, and demonstrating the characteristic shear-thinning behavior of flocculated systems. 
On the removal of shear stress, however, fluid gels have the capacity to rapidly recover their elastic properties. This lack of hysteresis is directly linked to the defined microstructures previously alluded to; because of the processing, reactive, non-gelled polymer chains at the particle interface facilitate interparticle interactions-similar to a Velcro effect. This rapid recovery of elastic properties enables bioprinting high-resolution parts from low-viscosity biomaterials, as rapid reformation of the support bed traps the bioink in situ, maintaining its shape. Furthermore, an advantage of agarose fluid gels is the asymmetric gelling/melting transitions (gelation temperature of ~30 &#176;C and melting temperature of ~90 &#176;C). This thermal hysteresis of agarose makes it possible to print and culture the bioprinted part in situ without the supporting fluid gel melting. This protocol shows how to manufacture agarose fluid gels and demonstrates their use to support the production of a range of complex hydrogel parts within suspended-layer additive manufacture (SLAM).

Shear processing during hydrogel formation results in the production of microgel suspensions that shear-thin but rapidly restructure following the removal of shear forces. Such materials have been used as a supporting matrix for bioprinting complex, cell-laden structures. Here, methods used to manufacture the supporting bed and compatible bioinks are described.

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several ...

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 ...