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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 ....
1. Breast Epithelial Cell Growth and Assay Media
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 R.......
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. .......
This research was funded by NIH 1R01HL140239-01 to AMC. We would like to thank the Cell Imaging Center at Drexel University.
....Name | Company | Catalog Number | Comments |
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 |
CellTrackerâ„¢ 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 Fluorâ„¢ 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 |
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