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...

The authors have nothing to disclose.

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

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

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

direct bioprinting of 3d multicellular breast spheroids onto endothelial networks

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

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

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

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

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

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

Our protocol facilitates the printing of preformed 3D cellular structures such as breast epithelial spheroids enabling the rapid formation of biologically-relevant models and a variety of bio-inks. The main advantage of this technique is that it facilitates the bioprinting of 3D tumor spheroids onto a vascular network that can be used almost immediately for any experimental study. These 3D bioprinted tissue models provide a rapid in vitro platform that can be expanded for personalized precision medicine.This highly versatile 3D bioprinting method can be applied to other physiological systems in which 3D interactions with the vasculature are important. For breast epithelial spheroid formation, first freeze 200 microliter pipette tips for 30 minutes and place growth factor reduced matrix solution on ice. When the tips have frozen, using one new ice cold pipette tip per well, add 30 microliters of the ice cold matrix solution onto the side and along the corners of each well of an eight-well chamber slide, adding a final drop to the center to ensure an even coating.When all of the wells have been coated, incubate the slide at 37 degrees Celsius and 5%carbon dioxide for 15 to 20 minutes. While the matrix is polymerizing, resuspend MCF 10A cells in MCF 10A assay medium at a two times 10 to the five cells per milliliter concentration. At the end of the incubation, add 50 microliters of cells and 450 microliters of MCF 10A spheroid growth medium to each well and immediately return the slide to the incubator.Every four days, tilt the chamber slide to a 45 degree angle to allow 200 microliters of supernatants to be replaced with fresh spheroid growth medium per well. For HUVEC network formation, prepare a matrix solution coated eight-well chamber slide as demonstrated and pre-stain a 10 centimeter dish of HUVECs with 10 microliters of red cell tracker die for 30 minutes in the cell culture incubator. At the end of the incubation, wash the endothelial cells with 10 milliliters of warm PBS before treating them with two milliliters of 0.05%trypsin EDTA.After five minutes at 37 degrees Celsius and 5%carbon dioxide, neutralize the trypsin with five milliliters of endothelial growth medium two and transfer the cells to a 25 milliliter conical tube for centrifugation. Resuspend the pellet in five milliliters of serum-free endothelial basal medium two for counting and dilute the cells to a one times 10 to the six cells per milliliter concentration. Then add one times 10 to the five HUVECs to each well and place the plate in the cell culture incubator for six hours.After five to eight days of culture, count the number of spheroids in each well in representative phase contrast microscopy images and use a 1, 000 microliter pipette tip with the last 0.5 centimeters of the tip removed to carefully collect all of the spheroids from each well of the eight-well chamber slide into a 50 microliter tube. Resuspend spheroids in an appropriate bio-ink at a 100 spheroids per 100 microliters of ink concentration and load the pooled spheroids into a 10 milliliter sterile syringe. Equip the syringe with a 25 gauge sterile needle and attach the syringe to the biodeposition system.Remove the medium from each well of HUVEC network and extrude 100 microliters of breast epithelial spheroids into each of six wells of the HUVEC network chamber slide at a flow rate of one milliliter of spheroids per minute. Then add 400 microliters of MCF 10A spheroid growth medium to each well and place the co-cultures in the cell culture incubator for 24 to 96 hours. After five to eight days of culture as demonstrated, non-tumorigenic MCF 10A breast epithelial spheroids should appear round with a hollow center with Integrin alpha-6 polarized to the outer edge of the spheroid.Highly invasive MDA-MB-231 breast cancer epithelial cells form irregular spheroids that may show cells migrating out of the spheroids if maintained in the Matrigel culture for too long. After six to eight hours of sparse serum-free culture as demonstrated, HUVEC networks can be imaged by phase contrast or confocal microscopy. After breast epithelial spheroid bioprinting onto the HUVEC networks, both the spheroids and the networks should maintain their original morphology for at least 24 hours.The main thing to remember while attempting this protocol is to handle the spheroids carefully before printing to avoid clustering. Following bioprinting and co-culture, tumor cell viability, proliferation, migration, and other biochemical interactions with the endothelial network can be measured with and without a pharmacological treatment. we have demonstrated that drug testing can be initiated as early as two hours after co-culture bioprinting and have tested spheroid addition to endothelial networks with the anti-cancer drug paclitaxel.

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JoVE Journal

Bioengineering

4.8K vistas.  Drexel University. Our protocol facilitates the printing of preformed 3D cellular structures such as breast epithelial spheroids enabling the rapid formation of biologically-relevant models and a variety of bio-inks. The main advantage of this technique is that it facilitates the bioprinting of 3D tumor spheroids onto a vascular network that can be used almost immediately for any experimental study. These 3D bioprinted tissue models provide a rapid in vitro platform that can be expanded for personalized precisi...