Name: Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks (Video) | JoVE Journal
Uploaded: 2020-11-02T13:30:00.000+00:00
Duration: 6 min 7 s
Description: Discover bioprinting of 3D breast epithelial spheroids onto endothelial networks for rapid drug screening. This versatile method creates biologically-relevant co-culture models for precision medicine applications.

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5 minutes

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&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

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 α6 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 – 300 µm ...

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

Research

JoVE Journal

Bioengineering

This video explains the 3D magnetic bioprinting of co-cultured pancreatic cancer cells and fibroblasts. Pancreatic cancer cells and activated pancreatic fibroblast cells are co-cultured and magnetized using a biocompatible nanoparticle assembly, to generate spheroids, under the influence of magnetic forces.

Magnetically Bioprinted 3D Spheroid Co-cultures: A Method for Co-culturing Cancerous Cells and Fibroblasts

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.

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

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.

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

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.

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

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.

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

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.

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

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.

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

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.

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

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.

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

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.

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

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.

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

Discover bioprinting of 3D breast epithelial spheroids onto endothelial networks for rapid drug screening. This versatile method creates biologically-relevant co-culture models for precision medicine applications.

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

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