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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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;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&#39;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

2.8K Views.  Tsinghua University. 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 µm can be obtained and collected for further culturing.

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

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Force System with Vertical V-Bends: A 3D In Vitro Assessment of Elastic and Rigid Rectangular Archwires

A proper understanding of the force system created by various orthodontic appliances can make treatment of patients efficient and predictable. Reducing the complicated multi-bracket appliances to a simple two-bracket system for the purpose of force system evaluation will be the first step in this direction. However, much of the orthodontic biomechanics in this regard is confined to 2D experimental studies, computer modeling/analysis or theoretical extrapolation of existing models. The objective of this protocol is to design, construct and validate an in vitro 3D model capable of measuring the forces and moments generated by an archwire with a V-bend placed between two brackets. Additional objectives are to compare the force system generated by different types of archwires among themselves and to previous models. For this purpose, a 2 x 4 appliance representing a molar and an incisor has been simulated. An orthodontic wire tester (OWT) is constructed consisting of two multi-axis force transducers or load cells (nanosensors) to which the orthodontic brackets are attached. The load cells are capable of measuring the force system in all the three planes of space. Two types of archwires, stainless-steel and beta-titanium of three different sizes (0.016 x 0.022 inch, 0.017 x 0.025 inch and 0.019 x 0.025 inch), are tested. Each wire receives a single vertical V-bend systematically placed at a specific position with a predefined angle. Similar V-bends are replicated on different archwires at 11 different locations between the molar and incisor attachments. This is the first time an attempt has been made in vitro to simulate an orthodontic appliance utilizing V-bends on different archwires.

Force System with Vertical V-Bends: A 3D In Vitro Assessment of Elastic and Rigid Rectangular Archwires

The method presented here is designed to construct and validate an in vitro 3D model capable of measuring the force system generated by different archwires with V-bends placed between two brackets. Additional objectives are to compare this force system with different types of archwires and to previous models.

A proper understanding of the force system created by various orthodontic appliances can make treatment of patients efficient and predictable. Reducing ...

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 ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

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 ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...