This research was funded by NIH 1R01HL140239-01 to AMC. We would like to thank the Cell Imaging Center at Drexel University.

1. Breast Epithelial Cell Growth and Assay Media
<ol>
	<li>MCF10A breast epithelial cells 
	NOTE: The non-tumorigenic immortalized breast epithelial cell line is derived from a patient with fibrocystic disease43. Cells do not express estrogen receptor.
	<ol>
		<li>To prepare 20 &#956;g/mL epidermal growth factor (EGF), dissolve 100 &#956;g of lyophilized EGF in 500 &#181;L of sterile dH2O to make 200 &#956;g/mL EGF. Add 500 &#956;L of 200 &#956;g/mL EGF into 4.5 mL of sterile 0.1% BSA in dH2O to make a 20 &#956;g/mL EGF stock solution. Store prepared 20 &#956;g/mL EGF aliquots at -20 &#176;C for up to 12 months.</li>
		<li>To prepare 500 &#956;g/mL hydrocortisone, dilute 1 mg of hydrocortisone in 1 mL of absolute ethanol (200 proof). Add 1 mL of sterile DMEM F:12 to this mixture to make a 500 &#956;g/mL hydrocortisone stock solution. Store hydrocortisone stock aliquots at -80 &#176;C for up to 12 months.</li>
		<li>To prepare 10 &#956;g/mL cholera toxin, dissolve 1 mg of cholera toxin lyophilized powder in 1 mL of sterile dH2O to make a 1 mg/mL cholera toxin stock solution. Store cholera toxin stock aliquots at -80 &#176;C for up to 12 months.</li>
		<li>To prepare Growth Medium, add 500 &#181;L of EGF (20 &#956;g/mL), 500 &#181;L of hydrocortisone (500 &#956;g/mL), 5 &#956;L of cholera toxin (1 mg/mL), 500 &#956;L of bovine insulin (10 mg/mL), 10 mL of penicillin and streptomycin, and 25 mL of horse serum to 500 mL of DMEM F:12 for a final concentration of 20 ng/mL EGF, 500 ng/mL hydrocortisone, 10 ng/mL cholera toxin, 10 ng/mL bovine insulin, 2% v/v penicillin and streptomycin, and 5% v/v horse serum. Antibiotic concentration was increased to 2% to account for decreased sterility in the bioprinting process; however, the antibiotic concentration can be lowered to 1%.</li>
		<li>To prepare Assay Medium, add 500 &#181;L of hydrocortisone (500 &#956;g/mL), 5 &#956;L of cholera toxin (1 mg/mL), 500 &#956;L of bovine insulin (10 mg/mL), 10 mL of penicillin and streptomycin, and 25 mL of horse serum to 500 mL of DMEM F:12 for a final concentration of 500 ng/mL hydrocortisone, 10 ng/mL cholera toxin, 10 ng/mL bovine insulin, 2% v/v penicillin and streptomycin, and 5% v/v horse serum.</li>
	</ol>
	</li>
	<li>MDA-MB-231 
	NOTE: The triple-negative (lacking estrogen receptor, progesterone receptor, and epidermal growth factor receptor 2) breast cancer epithelial cell line is created from pleural effusion of a female patient with metastatic mammary adenocarcinoma44. Cells represent an aggressive, invasive, and poorly differentiated breast cancer.
	<ol>
		<li>To prepare Growth and Assay Medium, add 50 mL of fetal bovine serum, 10 mL of penicillin and streptomycin, and 25 mL of horse serum to 500 mL of DMEM for a final concentration 10% v/v fetal bovine serum, 2% v/v penicillin and streptomycin, and 5% v/v horse serum.</li>
	</ol>
	</li>
</ol>
2. Breast Epithelial Cell Culture
<ol>
	<li>Seed 500,000 MCF10A or MDA-MB-231 cells in a 10 cm (P100) tissue culture dish with 10 mL of MCF10A or MDA-MB-231 growth media. MCF10A and MDA-MB-231 cells reach &#62;90% confluence in 48 hours.</li>
	<li>To passage breast epithelial cells, first wash cells with 10 mL of warm PBS.</li>
	<li>Detach breast epithelial cells by adding 2 mL of 0.05% Trypsin-EDTA to the dish. Place the dish in a 37 &#176;C, 5% CO2 incubator for 20-25 minutes.</li>
	<li>Add 5 mL of MCF10A or MDA-MB-231 Growth Medium to the trypsinized cells to neutralize the trypsin. Pipette the cell-media mixture up and down to resuspend cells and break up cell clusters.</li>
	<li>Add the cell suspension to a 15 mL conical tube and centrifuge at 1,200 x g for 3 minutes. Carefully aspirate the supernatant and resuspend the cell pellet in 5 mL of MCF10A or MDA-MB-231 Growth Medium.</li>
	<li>Plate 1 mL of cell suspension in 5 new 10 cm tissue culture dishes along with MCF10A or MDA-MB-231 Growth Medium. Place the dishes in a 37 &#176;C, 5% CO2 incubator. Cells will be ready to be passaged again in 2-3 days. MCF10A cells can be maintained to passage number 35, after which they show morphological changes. MDA-MB-231 cells can be maintained to passage number 24, after which they show morphological changes.</li>
</ol>
3. Breast Epithelial Spheroid Formation
<ol>
	<li>Freeze 200 &#956;L pipette tips for 30 minutes prior to starting the assay. Keep growth-factor reduced matrix solution (e.g., Matrigel) (10 mg/mL) on ice for the entire process.</li>
	<li>Slowly pipette 30 &#956;L of ice-cold matrix solution using ice-cold 200 &#956;L pipette tips into each well of an 8-well chamber slide. Start from the sides and drag the pipette tip along the corners, adding a final drop to the center of the well to ensure even coating of each well. Avoid air bubbles during this step to ensure a uniform matrix solution layer. Use new pipette tips for each well.</li>
	<li>Incubate chamber slides in a 37 &#176;C, 5% CO2 incubator for 15-20 minutes to polymerize the matrix solution.</li>
	<li>Resuspend trypsinized MCF10A cells in MCF10A Assay Medium at 200,000 cells/mL or resuspend trypsinized MDA-MB-231 cells in MDA-MB-231 Growth Medium at 200,000 cells/mL.</li>
	<li>If using MCF10A cells, prepare fresh MCF10A Spheroid Growth Medium by adding 5 &#956;L of EGF (20 &#956;g/mL) and 100 &#956;L of matrix solution to 5 mL of Assay Medium for a final concentration of 2% matrix solution.</li>
	<li>Remove the chamber slide precoated with matrix solution from the incubator. Add 50 &#956;L of MCF10A or MDA-MB-231 cell suspension (10,000 cells) to each well. If using MCF10A cells, add 450 &#956;L of MCF10A Spheroid Growth Medium. If using MDA-MB-231 cells, add 450 &#956;L of MDA-MB-231 Growth Medium premixed with 2% matrix solution (9 &#956;L). Immediate place the chamber slide in a 37 &#176;C, 5% CO2 incubator.</li>
	<li>Replace the medium every 4 four days. Carefully pipette 200 &#956;L of old media out from one corner of each well using a 200 &#956;L pipette tip. During this process, tilt the chamber slide at 45&#176; to ensure that the spheroids are not disturbed. Add 200 &#956;L of freshly prepared MCF10A Spheroid Growth Medium or MDA-MB-231 Growth Medium previously mixed with 2% matrix solution to each well at the corner in drops to ensure that spheroids remain attached to the matrix layer. Only ~50% of the media is replaced so that all the cytokines produced by the spheroids are not completely depleted. 
	NOTE: MCF10A breast epithelial spheroids take up to 8 days to polarize and form hollow centers. MDA-MB-231 breast epithelial spheroids take up to 5 days to form and have no hollow centers.</li>
</ol>
4. Endothelial Cell Network Formation
<ol>
	<li>Human Umbilical Vein Endothelial Cell (HUVEC) Culture
	<ol>
		<li>Add the contents of a single Endothelial Growth Medium-2 kit containing insulin growth factor, fibroblast growth factor, vascular endothelial growth factor, ascorbic acid, epidermal growth factor, heparin and gentamicin-amphotericin B, along with 50 mL of fetal bovine serum, 5 mL penicillin-streptomycin and 5 mL of 200 mM glutamine to 500 mL of Endothelial Basal Medium-2 (EBM-2) to create complete Endothelial Growth Medium (EGM-2).</li>
		<li>Seed 500,000 endothelial cells in a 10 cm tissue culture dish in 10 mL of EGM-2. Place cells in a 37 &#176;C, 5% CO2 incubator until they reach &#62;80% confluency (around 48 hours).</li>
		<li>To passage cells, wash endothelial cells with 10 mL of warm PBS. Detach HUVEC by adding 2 mL of 0.05% Trypsin-EDTA to the 10 cm tissue culture dish. Closely monitor the cells by phase contrast microscopy. Cells are ready when they are balled up but remain attached to the dish.</li>
		<li>Carefully aspirate the trypsin out of the dish. Add 8 mL of EGM-2 to the dish. Wash cells off the dish by pipetting the EGM-2 up and down over the entire dish surface.</li>
		<li>Alternatively, add 5 mL of EGM-2 to the trypsinized cells to neutralize the trypsin. Pipette the cell-media mixture up and down to resuspend cells and break up cell clusters. Add the cell suspension to a 15 mL conical tube and centrifuge at 1,200 x g for 3 minutes. Carefully aspirate the supernatant and resuspend the cell pellet in 10 mL of EGM-2.</li>
		<li>Add 1 mL of cell suspension to 9 mL of EGM-2 in a 10 cm tissue culture dish. Place the dishes in a 37 &#176;C, 5% CO2 incubator. Exchange 2/3 of the medium with fresh EGM-2 every 2 days. Cells will be ready to passage in 3-5 days. HUVEC can be maintained up to passage 8 after which they fail to form networks.</li>
	</ol>
	</li>
	<li>Endothelial Cell (HUVEC) Network Formation</li>
	<li>Freeze 200 &#956;L pipette tips for 30 minutes prior to starting the assay. Keep growth-factor reduced matrix solution (10 mg/mL) on ice for the entire process.</li>
	<li>Slowly pipette 30 &#956;L of ice-cold matrix solution using ice-cold 200 &#956;L pipette tips into each well of an 8-well chamber slide. Start from the sides and drag the pipette tip along the corners, adding a final drop to the center of the well to ensure even coating of each well. Avoid air bubbles during this step to ensure a uniform matrix solution layer. Use new pipette tips for each well.</li>
	<li>Incubate chamber slides in a 37 &#176;C, 5% CO2 incubator for 15-20 minutes to polymerize Matrigel.
	<ol>
		<li>Pre-stain a 10 cm dish of HUVEC with 10 &#956;L of red cell tracker (1:1000) for 30 minutes in 37 &#176;C, 5% CO2 incubator.</li>
		<li>Wash endothelial cells with 10 mL of warm PBS. Detach HUVEC by adding 2 mL of 0.05% Trypsin-EDTA to the 10 cm tissue culture dish. Place the dish in a 37 &#176;C, 5% CO2 incubator for 5 minutes to fully detach the cells.</li>
		<li>Add 5 mL of EGM-2 to the dish to neutralize the trypsin. Transfer the cell suspension to a 25 mL conical tube. Centrifuge HUVEC at 1,200 x g for 5 minutes. Aspirate the supernatant and resuspend the cell pellet in 5 mL of serum-free EBM-2.</li>
		<li>Count cells using Trypan blue to determine viable cells. Create a 1 x 106 cell/mL solution.</li>
		<li>Add 100,000 HUVEC to each well with 200 &#956;L of serum free EBM-2. If more cells are added, they will create a surface monolayer rather than a tube network.</li>
		<li>Incubate HUVEC in a 37 &#176;C, 5% CO2 incubator. After 6 hours, image HUVEC networks by phase contrast microscopy. Networks are now ready for coculture with breast spheroids.</li>
	</ol>
	</li>
</ol>
5. Bioprinting Breast epithelial spheroids on pre-formed HUVEC Networks
NOTE: A dual nozzle bio-deposition system should be used for the biofabrication process. In this case, the system had three motion arms to allow micron-scale spatial control of material deposition as well as two screw driven motors to deposit bioink from 10 mL syringes. The system should be functionalized with a high efficiency particulate air filtration system as well as UV-sterilization capabilities to maintain a sterile environment during bioprinting. The bioprinter is UV sterilized for an hour before the printing process.
<ol>
	<li>Create breast epithelial spheroids and HUVEC networks as previously described. Estimate the number of breast epithelial spheroids in each well by counting spheroids in representative phase contrast microscopy images.</li>
	<li>Cut 0.5 cm off the end of a 1000 &#181;L pipette tip. Use the cut pipette tip to carefully pipette all spheroids out of the 8-well chamber slide and into a 50 mL tube. Resuspend spheroids in the selected bioink at 100 spheroids/100 &#181;L. 
	NOTE: Higher spheroid concentrations may result in spheroid clustering and prevent visualization of interactions between the spheroids and the endothelial networks.</li>
	<li>Pass the spheroid mixture through a 70 &#181;m cell strainer to remove any large or clustered spheroids.</li>
	<li>Load the pooled spheroids into a 10 mL sterile syringe and cap it with a 25-gauge sterile needle. Attach the syringe to the bio-deposition system.</li>
	<li>Aspirate the medium off the HUVEC networks in the 8-well chamber slide.</li>
	<li>Extrude 100 &#181;L of breast epithelial spheroids onto 6 wells of HUVEC networks at a flow rate of 1 mL/min. Maintain 2 wells of HUVEC networks as controls.</li>
	<li>Add 400 &#181;L of MCF10A Spheroid Growth Medium if using MCF10A spheroids printed on HUVEC networks or add 400 &#181;L MDA-MB-231 Growth Medium previously mixed with 2% matrix solution if using MDA-MB-231 spheroids printed on HUVEC networks. The respective spheroid growth medium should be used in HUVEC control wells.</li>
	<li>Incubate co-cultures in a 37 &#176;C, 5% CO2 incubator for 24 &#8211; 96 hours without a media change. Co-cultures will remain viable in the original medium for up to four days.</li>
</ol>
6. Confocal Microscopy
<ol>
	<li>Immunofluorescence and PBS-Glycine Wash Buffer Preparation
	<ol>
		<li>Prepare Immunofluorescence (IF) buffer 10x stock solution by adding 2.5 g of sodium azide, 5 g of bovine serum albumin, 10 mL of Triton X-100, and 2.05 mL of Tween-20 in 500 mL of 10x PBS. Adjust pH to 7.4. Store the stock solution for up to a year at 4 &#176;C to avoid sedimentation.</li>
		<li>Create a working IF buffer by diluting 50 mL of 10x IF buffer in 450 mL of sterile deionized water. Store the working solution at room temperature for up to one week.</li>
		<li>Prepare 10x PBS-Glycine buffer stock solution by adding 37.5 g of glycine to 500 mL of 10x PBS. Adjust pH to 7.4. Store the stock solution for up to 6 months at 4 &#176;C to avoid sedimentation.</li>
		<li>Create a working PBS-Glycine solution by diluting 50 mL of 10x PBS-Glycine buffer in 450 mL of sterile deionized water. Store the working solution at 4 &#176;C for up to one week.</li>
	</ol>
	</li>
	<li>Label Bioprinted Samples and Image by Confocal Microscopy
	<ol>
		<li>Aspirate medium from bioprinted 3D co-cultures and rinse 3 times with warm PBS. Fix bioprinted 3D co-cultures with 4% paraformaldehyde for 1 h at room temperature. Rinse samples 3 times for 20 minutes with 1x PBS-Glycine.</li>
		<li>Block samples with IF buffer mixed with 10% goat serum for 90 minutes (primary block), followed by 40 minutes with IF buffer plus 10% goat serum and Af&#64257;nipure Fab fragment (1:100, secondary block).</li>
		<li>If using MCF10A spheroids, label samples with a primary antibody for integrin &#945;6 (1:100) in secondary blocking buffer overnight at 4 &#176;C, followed by an Alexa Fluor 488 secondary antibody (1:200) and Hoescht 33342 (1:1000) for 1 h at room temperature protected from light. MCF10A cell lines express high levels of integrin &#945;6, which is essential for showing spheroid polarization and morphology.</li>
		<li>If using MDA-MB-231 spheroids, which express low levels of integrin &#945;6, label samples with Alexa Fluor 488 phalloidin (1:100) and Hoescht 33342 (1:1000) in secondary blocking buffer for 4 hours at room temperature protected from light. Phalloidin enables actin &#64257;lament visualization so spheroid amorphous and invasive morphology can be assessed.</li>
		<li>Wash samples with 1X PBS-glycine 3 times for 20 minutes.</li>
		<li>Prepare samples for mounting by removing the chamber slide using the manufacturer&#8217;s tool. Add a small drop of antifade solution to each well. Place a 22 mm x 60 mm coverslip on each chamber slide and carefully seal the edges with clear nail polish.</li>
		<li>Image samples using a confocal microscope as Z stacks of ~10 slices in 5 &#956;m steps. If desired, compress Z planes into a single plane using the Extended Focus command in cell imaging software.</li>
		<li>Quantify spheroid adhesion to endothelial networks using Image J. The number of adhered spheroids can be quantified using the analyze plugin with the appropriate particle size and circularity. Normalize the number of attached spheroids to the image area.</li>
		<li>To ensure reproducibility, quantify the number of adhered spheroids in 4 x 4 tiled images of co-cultures. If the spheroid number is statistically significantly lower than other experiments, the experiment should be repeated.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

This protocol is the first of its kind to bioprint spheroids in their 3D architecture for co-culture with endothelial cells also in their 3D architecture. Critical protocol steps include the initial formation of breast epithelial spheroids and HUVEC networks. Extreme caution must be taken in feeding breast epithelial spheroids, as they are easily disrupted from the matrix solution. Similarly, breast epithelial spheroids must be treated with care when they are pipetted off the matrix solution and mixed into the networks. ...

3D in vitro vascularized tumor models are essential tools for mechanistic study of cancer growth and metastasis. For breast cancer in particular, breast epithelial cells cultured in Matrigel organize into polarized spheroids that more closely resemble the in vivo mammary acinus architecture1,2,3,4,5,6,7,8. 3D breast epithelial cell culture also impacts cell function, with 3D cultures showing differences in epidermal growth factor (EGF) receptor modulation8,9; oncogene function, including ErbB210; growth and apoptosis signaling11,12; and chemotherapy resistance13,14. Vascular endothelial cells similarly respond differently to environmental stimuli in 3D vs. traditional 2D culture15,16,17,18. However, much of the understanding of vascular endothelial and breast epithelial interactions comes from 2D culture using conditioned medium or Transwell inserts, or 3D models in which the two cell types are physically separated19,20,21,22,23. These co-culture models provide limited physiological insight, since both 3D culture and cell-cell contact are critical to vascular endothelial &#8211; breast epithelial cell interactions24,25,26.
3D cancer models have been fabricated using a variety of techniques, including hanging drop spheroid formation, bioprinting, magnetic assembly, and culture within hydrogels or on engineered scaffolds5,27,28,29. More recently, 3D tumor models were created with multiple cell types arranged in their respective 3D structures. In one example of a tumor-on-a-chip platform, cancer, endothelial, and stromal cells were mixed into a matrix and then injected into the three central tissue chambers in a polydimethylsiloxane (PDMS) device. The tissue chambers were bordered by two outer channels that represented an artery and venule. After 5-7 days of culture, endothelial cells formed a microvascular network and cancer cells proliferated to form small tumors near the vasculature. This platform was then used to screen drugs and drug combinations30. Additional tumor-on-a-chip platforms have been created to study metastasis and cancer types with specific mechanical stimuli (e.g., mechanical strain in the lung)31,32. However, these platforms generally do not include both vasculature and cancer in their respective 3D structures.
Biofabrication shows great promise in advancing 3D in vitro vascularized tumor models, since it enables tight spatial control over cell location. Despite growth of bioprinting over the past decade, few studies focus specifically on tumors33,34. In one example, 3D printing of HeLa cells in a gelatin/alginate/fibrinogen hydrogel was used to create an in vitro cervical cancer model. Tumor cells were bioprinted as individual cells and then allowed to form spheroids, which showed a higher proliferation rate, increased matrix metalloproteinase expression, and higher chemoresistance than cells in 2D culture35. In these studies, as in many others36,37, dissociated cell suspensions were bioprinted, and then the cell cultures were provided with the required mechanical and biochemical cues to enable the cells to form a 3D structure. However, cellular self-assembly may take days or weeks, may require complex spatial and temporal environmental cues, or may not occur when two cell types are co-cultured. For example, breast epithelial cells induced cell death in endothelial cells in 2D co-culture, and dissociated breast epithelial cells did not form 3D spheroids when bioprinted in alginate/gelatin hydrogels38. Dissociated breast epithelial or cancer cells formed spheroids in alginate based bioinks only when entrapped in circular PDMS molds. In other cases, spheroids were formed using suspended droplets in ultra-low attachment circular well plates and then mixed into alginate based bioinks39,40.
We now describe an alternative 3D tissue biomanufacturing method in this protocol. Rather than seed dissociated cells and wait for these cells to form the 3D structures, we describe how to create and bioprint 3D tumor spheroids on a&#160;vascular tube network to create a tumor co-culture model that can be used almost immediately. Tumor spheroids can be grown in vitro or derived from human tissues (organoids). Similarly, vascular tubes can be grown or can be derived from adipose tissue microvascular fragments. Bioinks can range from biologically inactive alginate to the highly biologically active Matrigel41. Since this 3D tumor co-culture model can be created with a variety of cell structures and bioinks, it can incorporate multiple cell types, extracellular matrices, and chemokine gradients15,42. While in its current formulation, the endothelial networks cannot be perfused, future iterations could integrate this method with microfluids or -on-chip systems. Bioprinting 3D breast epithelial spheroids onto endothelial networks enables rapid biofabrication of human breast models for drug testing and personalized precision medicine27.

<table>
	<tbody>
		<tr>
			<td>37&#176;C incubator, 5% CO2 and 95% humidity</td>
			<td>Sanyo</td>
			<td>MCO-20AIC</td>
			<td>Cell incubation</td>
		</tr>
		<tr>
			<td>3D Bio printer</td>
			<td>custom-made</td>
			<td>None</td>
			<td>Used for bioprinting</td>
		</tr>
		<tr>
			<td>8-well chamber slides</td>
			<td>VWR, Radnor, PA</td>
			<td>53106-306</td>
			<td>for seeding spheroids</td>
		</tr>
		<tr>
			<td>25-gauge needle</td>
			<td>Sigma, St. Louis, MO</td>
			<td>Z192406-100EA</td>
			<td>bioprinting syringe needle</td>
		</tr>
		<tr>
			<td>Absolute ethanol (200 proof )</td>
			<td>Sigma, St.Louis, MO</td>
			<td>E7023-500ML</td>
			<td>reconsitution of media components</td>
		</tr>
		<tr>
			<td>Affinipure F(ab&#8242;)2 fragment goat anti-mouse IgG</td>
			<td>Jackson ImmunoResearch, West Grove, PA</td>
			<td>115006020</td>
			<td>secondary block - Immunofluorescence</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 (1:200)</td>
			<td>Thermo Fisher, Waltham, MA</td>
			<td>A-11006</td>
			<td>Seconday antibody-Immunofluorescence</td>
		</tr>
		<tr>
			<td>Bovine insulin</td>
			<td>Sigma, St.Louis, MO</td>
			<td>I-035-0.5ML</td>
			<td>MCF10A Media additive</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma, St.Louis, MO</td>
			<td>A2153-500G</td>
			<td>Blocking agent -Immunofluorescence</td>
		</tr>
		<tr>
			<td>Falcon 70 &#181;m Cell Strainer</td>
			<td>Corning, Corning, NY</td>
			<td>352350</td>
			<td>Remove large or clustered spheroids</td>
		</tr>
		<tr>
			<td>CellTracker&#8482; Red CMTPX Dye</td>
			<td>Thermo Fisher, Waltham, MA</td>
			<td>C34552</td>
			<td>pre-stain for HUVEC tubes</td>
		</tr>
		<tr>
			<td>Compact Centrifuge</td>
			<td>Hermle- Labnet, Edison ,NJ</td>
			<td>Z206A</td>
			<td>For cell centrifugations</td>
		</tr>
		<tr>
			<td>Cholera Toxin</td>
			<td>Sigma, St.Louis, MO</td>
			<td>C8052-.5MG</td>
			<td>MCF10A Media additive</td>
		</tr>
		<tr>
			<td>Conical tubes 15 mL</td>
			<td>VWR, Radnor, PA</td>
			<td>62406-200</td>
			<td>Collecting and resuspending cells</td>
		</tr>
		<tr>
			<td>Countess II-FL Cell counter</td>
			<td>Thermo Fisher, Waltham, MA</td>
			<td>AMQAF1000</td>
			<td>counting cells</td>
		</tr>
		<tr>
			<td>Glass pipettes (10 mL)</td>
			<td>VWR, Radnor, PA</td>
			<td>76184-746</td>
			<td>cell resuspension</td>
		</tr>
		<tr>
			<td>DMEM F:12</td>
			<td>Thermo Fisher, Waltham, MA</td>
			<td>11320033</td>
			<td>MCF10A basal media</td>
		</tr>
		<tr>
			<td>DMEM 1X</td>
			<td>VWR, Radnor, PA</td>
			<td>10-014-CV</td>
			<td>MDA-MB-231 basal media</td>
		</tr>
		<tr>
			<td>Endothelial Basal Medium-2 (EBM-2)</td>
			<td>Lonza, Durham, NC</td>
			<td>CC-3156</td>
			<td>HUVEC basal media</td>
		</tr>
		<tr>
			<td>Endothelial Growth Medium-2 (EGM-2)</td>
			<td>Lonza, Durham, NC</td>
			<td>CC-3162</td>
			<td>Accompanied with a Bulletkit (containing growth factors)</td>
		</tr>
		<tr>
			<td>Alexa Fluor&#8482; 488 Phalloidin</td>
			<td>Thermo Fisher, Waltham, MA</td>
			<td></td>
			<td>Labelling MDA-MB-231 spheroids</td>
		</tr>
		<tr>
			<td>Fetal Bovine serum</td>
			<td>Cytiva, Logan, UT</td>
			<td>SH30071.03</td>
			<td>HUVEC/MDA-MB-231 media additive</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Thermo Fisher, Waltham, MA</td>
			<td>16210064</td>
			<td>Live and dead cell stain assay for cell viability</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma, St.Louis, MO</td>
			<td>G8898-500G</td>
			<td>immunofluorescence buffer component</td>
		</tr>
		<tr>
			<td>Hoescht 33342</td>
			<td>Thermo Fisher, Waltham, MA</td>
			<td>62249</td>
			<td>Nuclei stain immunofluorescence</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Thermo Fisher, Waltham, MA</td>
			<td>16050130</td>
			<td>MCF10A Media additive</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma, St.Louis, MO</td>
			<td>H0888-5G</td>
			<td>MCF10A Media additive</td>
		</tr>
		<tr>
			<td>Human Umblical Vein Endothelial cells (HUVECs)</td>
			<td>Cell applications, San Diego , CA</td>
			<td>200-05f</td>
			<td>Endothelial cell lines</td>
		</tr>
		<tr>
			<td>Integrin &#945;6</td>
			<td>Millipore, Billerica, MA</td>
			<td>MAB1378</td>
			<td>Immunofluorescence spheroid labelling component</td>
		</tr>
		<tr>
			<td>Live Dead assay</td>
			<td>Thermo Fisher, Waltham, MA</td>
			<td>L3224</td>
			<td>Live and dead cell stain assay for cell viability</td>
		</tr>
		<tr>
			<td>LSM 700 Confocal microscope</td>
			<td>Zeiss, Thornwood, NY</td>
			<td></td>
			<td>Used to visualize cells</td>
		</tr>
		<tr>
			<td>Matrigel - growth factor reduced 10 mg/ml</td>
			<td>VWR, Radnor, PA</td>
			<td>354230</td>
			<td>Spheroid formation</td>
		</tr>
		<tr>
			<td>MCF10A cells</td>
			<td>ATCC</td>
			<td>CRL-10317</td>
			<td>Breast cell line</td>
		</tr>
		<tr>
			<td>MDA-MB-231 cells</td>
			<td>ATCC</td>
			<td>HTB-26</td>
			<td>Breast cell line</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma, St.Louis, MO</td>
			<td>158127-500G</td>
			<td>cell fixative</td>
		</tr>
		<tr>
			<td>Penicillin and streptomycin</td>
			<td>Thermo Fisher, Waltham, MA</td>
			<td>15140122</td>
			<td>MCF10A / MDA-MB-231/HUVEC Media additive</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline 1X (PBS)</td>
			<td>Thermo Fisher, Waltham, MA</td>
			<td>7001106</td>
			<td>Wash buffer for cells before trypsinization</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline 10X</td>
			<td>Thermo Fisher, Waltham, MA</td>
			<td>AM9625</td>
			<td>immunofluorescence buffer component</td>
		</tr>
		<tr>
			<td>Prolong gold antifade</td>
			<td>Thermo Fisher, Waltham, MA</td>
			<td>P36934</td>
			<td>immunofluorescence mountant medium</td>
		</tr>
		<tr>
			<td>Recombinant Human Epidermal Growth Factor, EGF</td>
			<td>Peprotech, Rocky Hill, NJ</td>
			<td>AF-100-15</td>
			<td>MCF10A/ assay media component</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma, St.Louis, MO</td>
			<td>S2002-25G</td>
			<td>immunofluorescence buffer component</td>
		</tr>
		<tr>
			<td>Sterile syringe (10 mL)</td>
			<td>VWR, Radnor, PA</td>
			<td>75846-757</td>
			<td>bioprinting process</td>
		</tr>
		<tr>
			<td>Tissue culture dish (10cm)</td>
			<td>VWR, Radnor, PA</td>
			<td>25382-166</td>
			<td>monolayer cell culture</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma, St.Louis, MO</td>
			<td>T8787-250ML</td>
			<td>immunofluorescence buffer component</td>
		</tr>
		<tr>
			<td>Trypan blue 0.4%</td>
			<td>Thermo Fisher, Waltham, MA</td>
			<td>15250061</td>
			<td>cell counter additive</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.05%</td>
			<td>Thermo Fisher, Waltham, MA</td>
			<td>25300054</td>
			<td>cell detachment</td>
		</tr>
		<tr>
			<td>Tween -20</td>
			<td>Thermo Fisher, Waltham, MA</td>
			<td>85113</td>
			<td>immunofluorescence buffer component</td>
		</tr>
		<tr>
			<td>&#62;Vascular Endothelial Growth factor (VEGF165)</td>
			<td>Peprotech, Rocky Hill, NJ</td>
			<td>100-20</td>
			<td>HUVEC tube additive</td>
		</tr>
		<tr>
			<td>Volocity 6.3 cell imaging software</td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>Z stack compresser</td>
		</tr>
	</tbody>
</table>

direct bioprinting of 3d multicellular breast spheroids onto endothelial networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

Breast epithelial cells should self-organize into 3D spheroids after 5-8 days of culture on matrix solution and in culture medium with 2% matrix solution. Non-tumorigenic MCF10A breast epithelial spheroids should appear round and have a hollow center, with integrin &#945;6 polarized to the outer edge of the spheroid (Figure 1, inset shows hollow centers). Highly invasive MDA-MB-231 breast cancer epithelial cells form irregular spheroids. Spheroids should be used when they are around 100 &#82...

Watch this Scientific Journal Video about Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks at JoVE.com

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

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Research

JoVE Journal

Bioengineering

4.8K Views.  Drexel University. The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

Video: Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with poor clinical outcomes and currently lacks effective treatment. Both the three-dimensional (3D) environment and the dynamic mechanical forces are very important factors in this metastatic cascade. However, traditional cell cultures fail to recapitulate this natural tumor microenvironment. Thus, in vivo-like models that can emulate the intraperitoneal environment are of obvious importance. In this study, a new microfluidic platform of the peritoneum was set up to mimic the situation of ovarian cancer spheroids in the peritoneal cavity during metastasis. Ovarian cancer spheroids generated under a non-adherent condition were cultured in microfluidic channels coated with peritoneal mesothelial cells subjected to physiologically relevant shear stress. In summary, this dynamic 3D ovarian cancer-mesothelium microfluidic platform can provide new knowledge on basic cancer biology and serve as a platform for potential drug screening and development.

To study ovarian tumor progression in a physiologically relevant model, multicellular spheroids were cultured in a microdevice under simulated fluid flow. This dynamic 3D model emulates the intraperitoneal environment with the cellular and mechanical components where ovarian cancer metastasis occurs.

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

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

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

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

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

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 ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

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 ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

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 ...