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 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.
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 – 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 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.
1. Breast Epithelial Cell Growth and Assay Media
2. Breast Epithelial Cell Culture
3. Breast Epithelial Spheroid Formation
4. Endothelial Cell Network Formation
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.
6. Confocal Microscopy
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 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.
Figure 1: Representative confocal microscopy images of breast epithelial spheroids. MCF10A spheroids were labeled for integrin α6 (green) and nuclei (blue). Cell phenotype can be confirmed after bioprinting when spheroids appear round with a hollow center (inset) and have integrin α6 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 µm. Please click here to view a larger version of this figure.
Figure 2: Representative images of HUVEC networks by phase contrast and confocal microscopy. 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 µm. Please click here to view a larger version of this figure.
Figure 3: Representative images of breast epithelial spheroids co-cultured with HUVEC networks. MCF10A spheroids, labeled for integrin α6 (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 µm. Please click here to view a larger version of this figure.
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 °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.
The authors have nothing to disclose.
This research was funded by NIH 1R01HL140239-01 to AMC. We would like to thank the Cell Imaging Center at Drexel University.
37°C incubator, 5% CO2 and 95% humidity | Sanyo | MCO-20AIC | Cell incubation |
3D Bio printer | custom-made | None | Used for bioprinting |
8-well chamber slides | VWR, Radnor, PA | 53106-306 | for seeding spheroids |
25-gauge needle | Sigma, St. Louis, MO | Z192406-100EA | bioprinting syringe needle |
Absolute ethanol (200 proof ) | Sigma, St.Louis, MO | E7023-500ML | reconsitution of media components |
Affinipure F(ab′)2 fragment goat anti-mouse IgG | Jackson ImmunoResearch, West Grove, PA | 115006020 | secondary block – Immunofluorescence |
Alexa Fluor 488 (1:200) | Thermo Fisher, Waltham, MA | A-11006 | Seconday antibody-Immunofluorescence |
Bovine insulin | Sigma, St.Louis, MO | I-035-0.5ML | MCF10A Media additive |
Bovine serum albumin (BSA) | Sigma, St.Louis, MO | A2153-500G | Blocking agent -Immunofluorescence |
Falcon 70 µm Cell Strainer | Corning, Corning, NY | 352350 | Remove large or clustered spheroids |
CellTracker™ Red CMTPX Dye | Thermo Fisher, Waltham, MA | C34552 | pre-stain for HUVEC tubes |
Compact Centrifuge | Hermle- Labnet, Edison ,NJ | Z206A | For cell centrifugations |
Cholera Toxin | Sigma, St.Louis, MO | C8052-.5MG | MCF10A Media additive |
Conical tubes 15 mL | VWR, Radnor, PA | 62406-200 | Collecting and resuspending cells |
Countess II-FL Cell counter | Thermo Fisher, Waltham, MA | AMQAF1000 | counting cells |
Glass pipettes (10 mL) | VWR, Radnor, PA | 76184-746 | cell resuspension |
DMEM F:12 | Thermo Fisher, Waltham, MA | 11320033 | MCF10A basal media |
DMEM 1X | VWR, Radnor, PA | 10-014-CV | MDA-MB-231 basal media |
Endothelial Basal Medium-2 (EBM-2) | Lonza, Durham, NC | CC-3156 | HUVEC basal media |
Endothelial Growth Medium-2 (EGM-2) | Lonza, Durham, NC | CC-3162 | Accompanied with a Bulletkit (containing growth factors) |
Alexa Fluor™ 488 Phalloidin | Thermo Fisher, Waltham, MA | Labelling MDA-MB-231 spheroids | |
Fetal Bovine serum | Cytiva, Logan, UT | SH30071.03 | HUVEC/MDA-MB-231 media additive |
Goat serum | Thermo Fisher, Waltham, MA | 16210064 | Live and dead cell stain assay for cell viability |
Glycine | Sigma, St.Louis, MO | G8898-500G | immunofluorescence buffer component |
Hoescht 33342 | Thermo Fisher, Waltham, MA | 62249 | Nuclei stain immunofluorescence |
Horse Serum | Thermo Fisher, Waltham, MA | 16050130 | MCF10A Media additive |
Hydrocortisone | Sigma, St.Louis, MO | H0888-5G | MCF10A Media additive |
Human Umblical Vein Endothelial cells (HUVECs) | Cell applications, San Diego , CA | 200-05f | Endothelial cell lines |
Integrin α6 | Millipore, Billerica, MA | MAB1378 | Immunofluorescence spheroid labelling component |
Live Dead assay | Thermo Fisher, Waltham, MA | L3224 | Live and dead cell stain assay for cell viability |
LSM 700 Confocal microscope | Zeiss, Thornwood, NY | Used to visualize cells | |
Matrigel – growth factor reduced 10 mg/ml | VWR, Radnor, PA | 354230 | Spheroid formation |
MCF10A cells | ATCC | CRL-10317 | Breast cell line |
MDA-MB-231 cells | ATCC | HTB-26 | Breast cell line |
Paraformaldehyde | Sigma, St.Louis, MO | 158127-500G | cell fixative |
Penicillin and streptomycin | Thermo Fisher, Waltham, MA | 15140122 | MCF10A / MDA-MB-231/HUVEC Media additive |
Phosphate Buffered Saline 1X (PBS) | Thermo Fisher, Waltham, MA | 7001106 | Wash buffer for cells before trypsinization |
Phosphate buffer saline 10X | Thermo Fisher, Waltham, MA | AM9625 | immunofluorescence buffer component |
Prolong gold antifade | Thermo Fisher, Waltham, MA | P36934 | immunofluorescence mountant medium |
Recombinant Human Epidermal Growth Factor, EGF | Peprotech, Rocky Hill, NJ | AF-100-15 | MCF10A/ assay media component |
Sodium Azide | Sigma, St.Louis, MO | S2002-25G | immunofluorescence buffer component |
Sterile syringe (10 mL) | VWR, Radnor, PA | 75846-757 | bioprinting process |
Tissue culture dish (10cm) | VWR, Radnor, PA | 25382-166 | monolayer cell culture |
Triton X-100 | Sigma, St.Louis, MO | T8787-250ML | immunofluorescence buffer component |
Trypan blue 0.4% | Thermo Fisher, Waltham, MA | 15250061 | cell counter additive |
Trypsin-EDTA 0.05% | Thermo Fisher, Waltham, MA | 25300054 | cell detachment |
Tween -20 | Thermo Fisher, Waltham, MA | 85113 | immunofluorescence buffer component |
>Vascular Endothelial Growth factor (VEGF165) | Peprotech, Rocky Hill, NJ | 100-20 | HUVEC tube additive |
Volocity 6.3 cell imaging software | PerkinElmer, Hopkinton, MA | Z stack compresser |