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. HUVEC networks should not be plated at too high of a density or left for longer than 16 hours, as they will form a monolayer or die, respectively. Finally, all bioprinting should occur in a sterile environment at 37 &#176;C to maximize cell viability.
Breast epithelial spheroids can be bioprinted in a variety of bioinks besides Matrigel, including alginate and alginate-collagen blends. We demonstrated that spheroids were viable and maintained their morphology when printed in alginate-based bioinks41. Thus while we present bioprinting here in matrix solution-based bioink, other less expensive and easier to use bioinks are also possible. We additionally used other breast cancer cell lines, including MCF-7 and genetically modified MCF10A-NeuN cells with similar success38,41,45. Alternative means could also be used to create the breast epithelial spheroids. For example, Lee et al. used hydrogel microwell arrays produced using PDMS stamps to create uniformly sized spheroids of controlled size46. Finally, alternative endothelial cells such as tumor-derived endothelial cells could be used, and rather than forming HUVEC networks, adipose-tissue derived microvessels could be directly bioprinted as 3D vascular structures47.
A primary limitation of this method was the challenge in controlling bioprinted spheroid location and number. Spheroids had to be printed with relatively large nozzles and in inviscid fluids to prevent spheroid damage. Rapidly gelling bioinks might better control spheroid location. Spheroids also could not be counted in the bioink, since they were too large for our cell counter. We relied instead on spheroid counts derived from phase contrast images taken prior to pipetting spheroids off the Matrigel surface. Alternative means of forming the spheroids could better control their number and size. We were able to control spheroid number and size with moderate accuracy by using cell strainers and culturing spheroids in the same way each time. A final limitation is that breast epithelial cells migrate out of the spheroids and along the endothelial networks over time. It is possible that alternative bioinks would abrogate this limitation.
Direct bioprinting of breast epithelial spheroids on pre-formed HUVEC networks enables the creation of a 3D in vitro tumor co-culture model in a short time. Researchers can then rapidly examine interactions between spheroids and vasculature with higher throughput. In the future, breast epithelial spheroids could be bioprinted onto perfused vasculatures, which would allow study of flow effects. In addition, tumor-derived organoids and endothelial cells could be bioprinted to enable precision medicine through testing of drug efficacy in a patient-specific model.

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&amp;deg;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&amp;prime;)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 &amp;micro;m Cell Strainer</td><td>Corning, Corning, NY</td><td>352350</td><td>Remove large or clustered spheroids</td></tr><tr><td>CellTracker&amp;trade; 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&amp;trade; 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>Immunofluorescence labelling component</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 &amp;alpha;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>Vascular Endothelial Growth factor (VEGF&lt;sub&gt;165&lt;/sub&gt;)</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 &#8211; 300 &#181;m in diameter. When spheroids become too large and get in close proximity, the spheroids will join together to form megaspheroids. In addition, MDA-MB-231 breast epithelial spheroids may show cells migrating out of the spheroids if maintained in the Matrigel culture for too long.
HUVEC should self-organize into tube-like networks after 6-8 hours of sparse, serum-free culture. Samples will have multicellular nodes with connections that are formed of lines of 1-3 cells in parallel. The HUVEC networks can be imaged by phase contrast microscopy or by confocal microscopy if they are labeled with Cell Tracker and Hoescht (Figure 2). The ImageJ angiogenesis analyzer can be used to quantify network junctions, segments, and branches. HUVEC networks will die if left in serum-free medium for longer than 16 hours.
When breast epithelial spheroids are bioprinted onto the HUVEC networks, both spheroids and networks should maintain their original morphology for at least 24 hours. MCF10A breast epithelial spheroids will appear as round objects that adhere directly to the endothelial networks, while MDA-MB-231 breast epithelial spheroids will appear more amorphous yet still attached or in close proximity to the endothelial networks (Figure 3). HUVEC networks will be maintained when co-cultured with breast epithelial spheroids. For co-cultures longer than 24 hours, the breast epithelial cells may migrate out of the spheroids and along the endothelial networks. In our experience, this happens earlier in tumorigenic rather than non-tumorigenic breast epithelial cells38. We previously demonstrated using bioprinted co-cultures that drug testing can be initiated as early as 2 hours after spheroid bioprinting, for example to test spheroid adhesion onto endothelial networks45. We have also shown that 3D breast spheroids are more resistant to anti-cancer drugs like Paclitaxel than when printed as individual cells or in co-culture41,45. In the absence of bioprinted spheroids, HUVEC network control wells on their own fail to hold their network morphology and die after 16 h.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61791/61791fig01.jpg" /> 
Figure 1: Representative confocal microscopy images of breast epithelial spheroids.&#160;MCF10A spheroids were labeled for integrin &#945;6 (green) and nuclei (blue). Cell phenotype can be confirmed after bioprinting when spheroids appear round with a hollow center (inset) and have integrin &#945;6 polarized at the outer edges. MDA-MB-231 spheroids were labeled for actin (green) and nuclei (blue). Cell phenotype can be confirmed when spheroids are irregularly shaped without hollow centers and have cell processes invading into the surrounding matrix. Scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61791/61791fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61791/61791fig02.jpg" /> 
Figure 2: Representative images of HUVEC networks by phase contrast and confocal microscopy.&#160;HUVEC networks appear as small multicellular nodes with lines of cells connecting the nodes. For confocal microscopy, cells were labeled with Cell Tracker Red and Hoescht for nuclei (blue). Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61791/61791fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61791/61791fig03.jpg" /> 
Figure 3: Representative images of breast epithelial spheroids co-cultured with HUVEC networks.&#160;MCF10A spheroids, labeled for integrin &#945;6 (green) and nuclei (blue), remain round and appear adhered directly to the endothelial networks. MDA-MB-231 spheroids, labeled for actin (green) and nuclei (blue), appear amorphous yet remain near or on endothelial networks. Co-cultures maintain this morphology for at least 24 hours after bioprinting, after which breast cells may migrate out along the endothelial tubes. Scale bar = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61791/61791fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

direct-bioprinting-3d-multicellular-breast-spheroids-onto-endothelial

Nanyang Technological University

Research

JoVE Journal

Bioengineering

4.9K Views.  Drexel University. Het doel van dit protocol is om borsteptheelcellen rechtstreeks bioprinten als meercellige sferoïden op voorgevormde endotheelnetwerken om snel 3D-borst-endotheel co-cultuurmodellen te creëren die kunnen worden gebruikt voor geneesmiddelenscreeningsstudies.

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

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Cancer Research

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

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

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

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

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 ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

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

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

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

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

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 ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

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

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

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

3D Bioprinting for Rapid Vascular Network Fabrication Using Vessels-on-a-Plate

High-Throughput Bioprinting Method for Modeling Vascular Permeability in Standard Six-well Plates with Size and Pattern Flexibility

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in coronary arteries and impaired function of the blood-brain barrier. Existing fabrication techniques do not adequately replicate the geometrical variation in vascular networks in the human body, which substantially influences disease progression; moreover, these techniques often involve multi-step fabrication procedures that hinder the high-throughput production necessary for pharmacological testing. This paper presents a bioprinting protocol for creating multiple vascular tissues with desired patterns and sizes directly on standard six-well plates, overcoming existing resolution and productivity challenges in bioprinting technology. A simplified fabrication approach was established to construct six hollow, perfusable channels within a hydrogel, which were subsequently lined with human umbilical vein endothelial cells to form a functional and mature endothelium. The computer-controlled nature of 3D bioprinting ensures high reproducibility and requires fewer manual fabrication steps than traditional methods. This highlights VOP's potential as an efficient high-throughput platform for modeling vascular permeability and advancing drug discovery.

Author Spotlight: Automated Bioprinting for High-Throughput Vascular Model Fabrication

We present a protocol for high-throughput production of vascular channels with flexible sizes and desired patterns on a standard six-well plate using 3D bioprinting technology, referred to as vessels-on-a-plate (VOP). This platform has the potential to advance the development of therapeutics for the disorders associated with compromised endothelium.

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

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