Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Summary

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.

Abstract

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.

Introduction

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 architecture^{1,2,3,4,5,6,7,8}. 3D breast epithelial cell culture also impacts cell function, with 3D cultures showing differences ....

Protocol

1. Breast Epithelial Cell Growth and Assay Media

1. 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.
   1. To prepare 20 μg/mL epidermal growth factor (EGF), dissolve 100 μg of lyophilized EGF in 500 µL of sterile dH₂O to make 200 μg/mL EGF. Add 500 μL of 200 μg/mL EGF into 4.5 mL of sterile 0.1.......

Discussion

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. .......

Materials

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
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