This work was supported by the Beijing Natural Science Foundation (3212007), Tsinghua University Initiative Scientific Research Program (20197050024), Tsinghua University Spring Breeze Fund (20201080760), the National Natural Science Foundation of China (51805294), National Key Research and Development Program of China (2018YFA0703004), and the 111 Project (B17026).

1. Cell culture
<ol>
	<li>Supplement high-glucose Dulbecco&#39;s modified Minimum Essential Medium (H-DMEM) with 10% fetal bovine serum (FBS), 1% nonessential amino acid solution (NEAA), 1% penicillin G and streptomycin, and 1% Glutamine supplement as culture media for A549 cells.</li>
	<li>Culture A549 cells in a CO2 incubator at 37 &#176;C and with 5% CO2</li>
	<li>Dissociate cells for subculture using trypsin at approximately 80% confluence.
	<ol>
		<li>Use 3 mL of trypsin to treat the cells in the T75 culture flask at 37 &#176;C for 3 min, and then add 6 mL of culture medium to stop the trypsinization.</li>
		<li>Pipette the culture medium to harvest loosely adhered cells.</li>
		<li>Centrifuge at 72.5 x g for 3 min. Remove the supernatant and resuspend with 3 mL of fresh medium.</li>
		<li>Transfer 1 mL of cell suspension to a new T75 culture flask containing 10 mL of&#160;fresh medium. Change the culture medium every 2 days.</li>
	</ol>
	</li>
</ol>
2. Preparation of nozzles
<ol>
	<li>Load glass micropipettes onto the puller following the manufacturer&#39;s instructions. The outer diameter, inner diameter, and length of the micro-jet pipe are 1.5 mm, 1.1 mm, and 100 mm, respectively.</li>
	<li>Set the pulling parameters for the puller. Specifically, the values of HEAT, PULL, VELOCITY, TIME, and PRESSURE are set to 560, 255, 255, 150, and 500 by default, respectively. Pull off the nozzle under these parameters. 
	CAUTION: The nozzle obtained after pulling off is very sharp, and careless operation may cause injuries.</li>
	<li>Cut off the resulting nozzle (step 2.2) at the designated diameter by a micro-forge device following the manufacturer&#39;s instructions to obtain a specific tip diameter. Specifically, locate the specified diameter of the nozzle onto the heated platinum wire. Heat the wire up to 60 &#176;C for about 5 s and pull the nozzle apart.</li>
	<li>Immerse the printhead of the nozzle in a hydrophobic agent for 30 min, followed by three cycles of rinsing with sterilized water.</li>
	<li>Before printing, sterilize the nozzle in alcohol for 5 min and rinse it with sterilized water three times to remove the residual alcohol.</li>
</ol>
3. Preparation of hydrogel bioink
<ol>
	<li>Dissolve NaCl (0.45 g) into 50 mL of&#160;sterile water to obtain a 0.9% (w/v) NaCl solution. Vibrate the solution to promote dissolution. After being completely dissolved, filter the solution through a 0.45 &#181;m polyethylene terephthalate filter membrane.</li>
	<li>Weigh low-viscosity sodium alginate powder (1 g) and dissolve it in 25 mL of NaCl solution (step 3.1) at a high temperature of 60-80 &#176;C overnight to obtain a 4% (w/v) sodium alginate stock solution. The stock solution can be stored at 4 &#176;C for 1 month.</li>
	<li>Sterilize the sodium alginate stock solution (step 3.2) by heating it three times in an oven (70 &#176;C) for 30 min.</li>
	<li>Dilute proper volume of sodium alginate stock solution (step 3.3) in 0.9% NaCl solution (step 3.1) at concentrations of 2%, 1.5%, and 0.5%&#160;(w/v). 
	NOTE: In addition to heating in the oven, other preparation processes of sodium alginate solution should be carried out in the hood.</li>
	<li>Dissolve 1 g of&#160;gelatin powder in 25 mL of NaCl solution (step 3.1) at a high temperature of 60-80 &#176;C overnight to obtain a 4% (w/v) gelatin stock solution. The stock solution can be stored at 4 &#176;C for 1 month.</li>
	<li>Sterilize the gelatin stock solution (step 3.5) by heating it three times in an oven (70 &#176;C) for 30 min.</li>
	<li>Dilute the proper volume of gelatin stock solution (step 3.6) in 0.9% NaCl solution (step 3.1) at a concentration of 1.5% (w/v). 
	NOTE: In addition to heating in the oven, other preparation processes of gelatin solution should be carried out in the hood.</li>
	<li>To promote cell binding on the microcarriers, prepare the alginate-collagen solution by mixing the 2% (w/v) sodium alginate solution and the Type I collagen solution from rat tail (4 mg/mL) at a ratio of 3:1. Adjust the solution to pH 7.2 with 0.1 M NaOH solution and use&#160;immediately after preparation.</li>
	<li>Dissolve the tyrosine-modified HA flocculent solid in phosphate buffer saline (PBS) solution at 60-80 &#176;C overnight to obtain a 0.8% w/w tyrosine-modified hyaluronic acid PBS solution. The stock solution can be stored at 4 &#176;C for 1 month.</li>
	<li>Sterilize the tyrosine-modified HA PBS solution by heating it three times in an oven (70 &#176;C) for 30 min.</li>
	<li>Dissolve horseradish peroxidase powder (500 enzyme activity unit/mg) in PBS solution to obtain 8 enzyme activity unit/mg horseradish peroxidase PBS solution.</li>
	<li>Dilute hydrogen peroxide solution (30%, w/w) by DI water to 4 mM.</li>
	<li>Prepare the modified HA-horseradish peroxidase solution by mixing tyrosine-modified hyaluronic acid PBS solution and horseradish peroxidase PBS solution at a ratio of 1:1. 
	&#8203;NOTE: In addition to heating in the oven, other preparation processes of HA-horseradish peroxidase solution should be carried out in the hood.</li>
</ol>
4. Microdroplets formation based on AVIFJ
<ol>
	<li>Use a home-established cell printing system as previously reported (Figure 1A)26. The electrical signal output by the waveform generator is first amplified and then drives piezoelectric ceramics to generate controllable deformation. The nozzle fixed on the piezoelectric ceramic thus produces controllable vibrations.</li>
	<li>Sterilize the printing system by wiping with 75% (v/v) alcohol and ultraviolet (UV) exposure overnight.</li>
	<li>Load 5 mL of bioink (step 3.1) into a disposable sterile syringe. Install the syringe on the syringe pump.</li>
	<li>Connect the syringe (step 4.3) and nozzle (step 2.5) with a silicone hose of 1 mm inner diameter.</li>
	<li>Fix the nozzle (step 4.4) to be used for printing by adjusting the tightness of the clamping screw. Keep the tip of the nozzle away from the substrate during installation to avoid damage and contamination. 
	&#8203;CAUTION: If the nozzle is broken during installation, please wear thicker gloves and carefully clean up glass fragments and residues.</li>
	<li>Rapidly push the syringe pump and load the bioink (step 4.3) to the nozzle (step 4.5). Set the flow rate at 30 &#181;L/min. Wipe off the excess material at the tip.</li>
	<li>Set signal generator parameters. Import self-designed waveform containing 500,000 sampling points. Set the sampling rate and peak-to-peak voltage (Vpp) as 5 million samples/s and 10 V, respectively.</li>
	<li>Clean slides or Petri dishes with deionized water. Place them under the nozzle as the printing substrate.</li>
	<li>Preset the motion path and trigger mode of vibration as drop-on-demand.</li>
	<li>Print the droplets following the pre-designed patterns.</li>
</ol>
5. Microcarriers formation based on AVIFJ
<ol>
	<li>Repeat steps 4.1-4.4, except change the bioink&#160;to 2% (w/v) alginate solution (step 3.4), alginate-collagen solution (step 3.8), or modified HA-horseradish peroxidase solution (step 3.13).</li>
	<li>Dissolve 3 g of calcium chloride into 100 mL of&#160;sterile water to obtain 3% (w/v) calcium chloride solution. After being completely dissolved, filter the solution through a 0.45 &#181;m polyethylene terephthalate filter membrane.</li>
	<li>Add 5 mL of&#160;cross-linking solution (calcium chloride solution or hydrogen peroxide solution) into a Petri dish. Place the Petri dish under the nozzle to work as a substrate.</li>
	<li>After printing, cross-link the microcarriers for 3 min.</li>
	<li>Collect microcarrier suspension in a centrifuge tube, and enrich microcarriers by centrifugation at 29 x g for 3 min. Resuspend the microcarriers in culture media at approximately 600 microcarriers/mL.</li>
</ol>
6. Inoculating cells on the surface of microcarriers
<ol>
	<li>Stain A549 cells with Cell Tracker Green CMFDA to facilitate the observation of cells. Specifically, remove culture media, add 5 mL of&#160;serum-free medium containing 10 &#181;M Cell Tracker dye, and incubate cells for 30 min in an incubator. Then, replace the dye solution with fresh medium.</li>
	<li>Resuspend the A549 cell suspension at the density of 1.6 x 106 cells/mL.</li>
	<li>Add 1 mL of alginate-collagen microcarrier suspension (step 5.3) and 1 mL of A549 cell suspension (step 6.1) into a low-adherent 6-well culture plate.</li>
	<li>Place the plate onto a shaker at 30 rpm in an incubator at 37 &#176;C and 5% CO2.</li>
	<li>Take the plate off the shaker and wait for 30 min to let the microcarriers settle down. Half change the culture medium every 2 days.</li>
</ol>
7. Analysis of microdroplets/microcarriers formation
<ol>
	<li>Observe and measure the nozzle (step 2.2), the Petri dishes (step 4.10 and step 5.4), and cells with microcarriers (step 6.5) under a bright field microscope or confocal microscope. Specifically, the objective magnification is 4x, 10x, or 20x and the eyepiece magnification is 10x.</li>
	<li>Measure the diameter of the microcarriers by ImageJ software. According to the scale bar that comes with the microscope when shooting in the picture, set the actual size of the scale in ImageJ, and draw 10 lines of the radius or semi-major axis of the microcarriers. ImageJ can get the average size and standard deviation of these line segments.</li>
</ol>

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</ol>

The authors have nothing to disclose.

The protocol described here provides instructions for the preparation of multi-types of hydrogel microcarriers and subsequent cell seeding. Compared to microfluidic chip and inkjet printing methods, AVIFJ approach to constructing microcarriers offers greater flexibility and biocompatibility. An independent nozzle enables a wide range of lightweight nozzles, including glass micropipettes, to be used in these printing systems. The highly controllable processing enables parameters including the volume of the reservoir, the ...

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area and are commonly used for large-scale culture of cells1,2. Their outer surface provides abundant growth sites for cells, and the interior provides a support structure for spatial proliferation. The spherical structure also provides convenience in monitoring and controlling parameters, including pH, O2, and concentration of nutrients and metabolites. When used in combination with stirred tank bioreactors, microcarriers can achieve higher cell densities in a relatively small volume compared to conventional cultures, thereby providing a cost-effective way to achieve large-scale cultures3. Microcarrier culture technology has become one of the main techniques in cytological research, and much progress has been made in the field of large-scale expansion of stem cells, hepatocytes, chondrocytes, fibroblasts, and other structures4. They have also been found to be ideal drug delivery vehicles and bottom-up units, therefore taking on an increasingly important role in clinical drug screening and in vitro tissue engineering repair5.
To meet mechanical property requirements in different scenarios, multiple types of hydrogel materials have been developed for use in the construction of microcarriers6,7,8,9,10,11. Alginate and hyaluronic acid (HA) hydrogels are two of the most used microcarrier materials owing to their good biocompatibility and crosslinkability12,13. Alginate can be easily cross-linked by calcium chloride, and its mechanical properties can be modulated by changing the cross-linking time. Tyramine-conjugated HA is cross-linked by the oxidative coupling of tyramine moieties catalyzed by hydrogen peroxide and horseradish peroxidase14. Collagen, due to its unique spiral structure and cross-linked fiber network, is often used as an adjuvant to mix into the microcarriers to further promote cell attachment15,16.
Current methods for preparing microcarriers include microfluidic chips, inkjet printing, and electrospray17,18,19,20,21,22,23. Microfluidic chips have been proven to be fast and efficient in producing uniform-sized microcarriers24. However, this technology relies on a complex flow channel design and fabrication process25. High temperature or excessive extrusion forces during inkjet printing, as well as intense electric fields in the electrospray approach, may adversely affect the properties of the material, especially its biological activity19. Besides, when applied to various biomaterials and diameters, the customized nozzles used in these methods result in limited processing complexity, high cost, and low flexibility.
To provide a convenient method for microcarrier preparation, a 3D printing technique called alternating viscous-inertial forces jetting (AVIFJ) has been applied to construct hydrogel microcarriers. The technique utilizes downward driving forces and static pressure generated during vertical vibration to overcome the surface tension of the nozzle tip and thus form droplets. Instead of severe forces and thermal conditions, small rapid displacements act directly on the nozzle during printing, causing a minor effect on the physicochemical properties of the bioink and presenting great attraction for bioactive materials. Utilizing the AVIFJ method, microcarriers of multiple biomaterials with diameters of 100-300 &#181;m were successfully formed. Besides, the microcarriers were further proven to bind cells well and provide a suitable growth environment for adhered cells.

<table><tbody><tr><td>A549 cells</td><td>ATCC</td><td>CCL-185</td><td>Human non-small cell lung cancer cell line</td></tr><tr><td>Bright field microscope</td><td>Olympus</td><td>DP70</td><td></td></tr><tr><td>Confocal microscope</td><td>Nikon</td><td>TI-FL</td><td></td></tr><tr><td>Fetal bovine serum, FBS</td><td>BI</td><td>04-001-1ACS</td><td></td></tr><tr><td>Gelatin</td><td>SIGMA</td><td>G1890</td><td></td></tr><tr><td>Glass micropipettes</td><td>sutter instrument</td><td>b150-110-10</td><td></td></tr><tr><td>GlutaMAX</td><td>GIBCO</td><td>35050-061</td><td></td></tr><tr><td>H-DMEM</td><td>GIBCO</td><td>11960-044</td><td>Dulbecco&#039;s modified eagle medium</td></tr><tr><td>Horseradish peroxidase powder</td><td>SIGMA</td><td>P6782</td><td></td></tr><tr><td>Hydrophobic agent</td><td>3M</td><td>PN7026</td><td>Follow the manufacturer&#039;s instructions and use after dilution</td></tr><tr><td>Micro-forge device</td><td>narishige</td><td>MF-900</td><td></td></tr><tr><td>Non-essential amino acids, NEAA</td><td>GIBCO</td><td>11140-050</td><td>non-essential amino acids</td></tr><tr><td>Penicillin G and streptomycin</td><td>GIBCO</td><td>15140-122</td><td></td></tr><tr><td>Petri dish</td><td>SIGMA</td><td>P5731-500EA</td><td></td></tr><tr><td>Puller</td><td>sutter instrument</td><td>P-1000</td><td></td></tr><tr><td>Sodium alginate</td><td>SIGMA</td><td>A0682</td><td></td></tr><tr><td>Trypsin</td><td>GIBCO</td><td>25200-056</td><td></td></tr><tr><td>Type I collagen solution from rat tail</td><td>SIGMA</td><td>C3867</td><td></td></tr></tbody></table>

3d printing of in vitro hydrogel microcarriers by alternating viscous-inertial force jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 &#181;m. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

Printheads of varied convergence rates and diameters were fabricated to achieve the printing of multiple types of materials. The nozzles obtained with increasing pull strength are shown in Figure 1B. The nozzles were divided into three areas: reservoir (III), contraction (II), and printhead (I). The reservoir was the unprocessed part of the nozzle, in which the liquid provided static pressure and bioink input for printing. The contraction area was the main part for generating downward drivin...

Watch this Scientific Journal Video about 3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting at JoVE.com

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 &#181;m can be obtained and collected for further culturing.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...

3d-printing-vitro-hydrogel-microcarriers-alternating-viscous-inertial

Research

JoVE Journal

Bioengineering

3.0K Views.  Tsinghua University. Wij bieden een mild biogeprint proces voor het bereiden van microdragers met een goede beheersbaarheid en biocompatibiliteit. Na inkapseling door cellen kunnen de dragers ook worden gebruikt in in vitro celexpansie en functionele microtissueconstructie. Beperkte verplaatsing, in plaats van zware krachten onder vergelijkbare omstandigheden, werken tijdens het printproces op het mondstuk, waarbij de oorspronkelijke fysische / chemische eigenschappen van de bio-inkt maximaal behouden blijven.

Video: 3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

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

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

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

An Additive Manufacturing Technique for the Facile and Rapid Fabrication of Hydrogel-based Micromachines with Magnetically Responsive Components

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels have simple monolithic architectures and often function as scaffolding materials for tissue engineering applications. More sophisticated structures typically take a long time to fabricate and do not contain moving components. This protocol describes a photolithography method that allows for facile and rapid microfabrication of PEG structures and devices. This strategy involves an in-house developed fabrication stage that allows for the rapid fabrication of 3D structures by building upwards in a layer-by-layer fashion. Independent moving components can also be aligned and assembled onto support structures to form integrated devices. These independent components are doped with superparamagnetic iron oxide nanoparticles that are sensitive to magnetic actuation. In this manner, the fabricated devices can be actuated using external magnets to yield movement of the components within. Hence, this technique allows for the fabrication of sophisticated MEMS-like devices (micromachines) that are composed entirely out of a biocompatible hydrogel, able to function without an onboard power source, and respond to a contact-less method of actuation. This manuscript describes the fabrication of both the fabrication set-up as well as the step-by-step method for the microfabrication of these hydrogels-based MEMS-like devices.

An additive manufacturing strategy for processing UV-crosslinkable hydrogels has been developed. This strategy allows for the layer-by-layer assembly of microfabricated hydrogel structures as well as the assembly of independent components, yielding integrated devices containing moving components that are responsive to magnetic actuation.

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

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 ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication of soft tissue constructs. However, granular gel formulations have been restricted to a limited number of biomaterials that allow for the cost-effective generation of large amounts of hydrogel microparticles. Therefore, granular gel support media have generally lacked the cell-adhesive and cell-instructive functions found in the native extracellular matrix (ECM).
To address this, a methodology has been developed for the generation of self-healing annealable particle-extracellular matrix (SHAPE) composites. SHAPE composites consist of a granular phase (microgels) and a continuous phase (viscous ECM solution) that, together, allow for both programmable high-fidelity printing and an adjustable biofunctional extracellular environment. This work describes how the developed methodology can be utilized for the precise biofabrication of human neural constructs.
First, alginate microparticles, which serve as the granular component in the SHAPE composites, are fabricated and combined with a collagen-based continuous component. Then, human neural stem cells are printed inside the support material, followed by the annealing of the support. The printed constructs can be maintained for weeks to allow the differentiation of the printed cells into neurons. Simultaneously, the collagen continuous phase allows for axonal outgrowth and the interconnection of regions. Finally, this works provides information on how to perform live-cell fluorescence imaging and immunocytochemistry to characterize the 3D-printed human neural constructs.

This work describes a protocol for the freeform embedded 3D printing of neural stem cells inside self-healing annealable particle-extracellular matrix composites. The protocol enables the programmable patterning of interconnected human neural tissue constructs with high fidelity.

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

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