A Defined Hydrogel-Based Method For Generating Three-Dimensional Human Breast Organoids That Recapitulate Mammary Morphogenesis

Summary

This protocol describes a defined hydrogel-based method to generate human breast organoids that recapitulate key features of mammary morphogenesis in a controlled three-dimensional culture system.

Abstract

The development of physiologically relevant human model systems that recapitulate tissue architecture and cell-state dynamics remains a major challenge in studying breast development and early events in carcinogenesis. Conventional two-dimensional cultures and many three-dimensional systems fail to capture the structural organization and microenvironmental cues that define the human mammary gland. Here, we describe a reproducible method for generating three-dimensional human breast organoids from primary epithelial cells embedded within a defined hydrogel matrix composed of type I collagen, laminin, fibronectin, and hyaluronic acid. This system supports the progression of single cells through key stages of mammary morphogenesis, including progenitor expansion, epithelial patterning, and the formation of terminal ductal lobular unit-like structures, as well as the emergence of a mesenchyme-like compartment, over a 21-day culture period. We provide a step-by-step protocol for hydrogel preparation, cell seeding and culture conditions. The method is compatible with high-content imaging and quantitative analysis of organoid number, size distribution, and architectural complexity. This platform enables mechanistic studies of epithelial plasticity and environmental perturbations, providing a scalable and biologically relevant system for investigating early tissue-level changes associated with breast cancer risk.

Introduction

Understanding human mammary gland development and the early events that predispose tissue to malignant transformation requires experimental systems that faithfully recapitulate tissue architecture, cellular hierarchy, and microenvironmental signaling. While two-dimensional epithelial cultures have provided important mechanistic insights, they lack the structural context necessary to model epithelial organization and morphogenesis¹. Existing three-dimensional culture systems, including those based on basement membrane extracts, have advanced the field but remain limited by variable composition, incomplete control over extracellular matrix compon....

Protocol

Primary tissues that would otherwise have been discarded as medical waste following surgery were obtained in compliance with all relevant laws using protocols approved by the institutional review boards at Maine Medical Center and Tufts Medical Center. All tissues were anonymized prior to transfer and could not be traced to specific patients. For this reason, this research was granted exemption status by the Committee on the Use of Humans as Experimental Subjects at the Massachusetts Institute of Technology and at Tufts University Health Sciences (IRB #13521). All patients enrolled in this study signed an informed consent form agreeing to participate in the study and ....

Results

Successful execution of this protocol results in the formation of three-dimensional organoid structures that exhibit organized epithelial morphology and tissue-specific architectural features. Organoids begin to form within 3–7 days following seeding and continue to develop throughout the culture period. Previous characterization of this hydrogel organoid system demonstrated reproducible organoid formation across multiple independent primary human donors³. In that study, primary epithelial c.......

Discussion

The protocol described here enables the reproducible generation of three-dimensional human breast organoids within a defined hydrogel microenvironment that supports key features of mammary morphogenesis³. Several steps are critical for the success of this method. First, tissue processing and enzymatic dissociation must be carefully controlled to preserve epithelial viability while minimizing overdigestion, which can reduce cell yield and impair subsequent morphogenesis¹⁰. I.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL conical tubes	VWR	89039-664	Sterile tubes for tissue processing and centrifugation
40 μm cell strainer	VWR	732-2757	Filtration device for single-cell suspension preparation
40 μm cell strainer for low volumes	Bel-Art	136800040	Filtration device for single-cell suspension preparation
Automated cell counter	Bio-Rad	1450102	Device used to count cells and determine viability
Bovine pituitary extract	Thermo Scientific	13028014	Supplement for epithelial cell media
Cell counting slides	Bio-Rad	145-0011	Dual-chamber slides used for cell counting
Centrifuge	N/A	N/A	Benchtop centrifuge/s need to have at least 500 x g speed capability and accommodation of 15 mL and 1.5 mL tubes.
Collagen I	Millipore Sigma	08-115	Extracellular matrix protein used for hydrogel formation
Collagenase A	Sigma-Aldrich	11088793001	Enzyme used for tissue dissociation
Cryovials	Corning	976171	Sterile vials for cryogenic storage of samples
Culture vessels (e.g., chamber slides, multiwell plates)	Corning	354104 354108 3603	Platforms for hydrogel deposition and organoid culture.
Dimethyl sulfoxide	Millipore Sigma	317275	Cryoprotectant used in freezing medium
Dispase II	Roche	4942078001	Enzyme used for secondary tissue dissociation
DNase I	Roche	10104159001	Enzyme used during cell dissociation.
Fetal bovine serum	Gibco	10437	Serum supplement used in wash and neutralization media
Fibronectin	Sigma-Aldrich	F2006	Extracellular matrix protein component
GlutaMAX	Thermo Scientific	35050061	Supplement for epithelial cell media
Human epidermal growth factor	Sigma-Aldrich	E9644	Supplement for epithelial cell media
Hydrocortisone	Sigma-Aldrich	H0888	Supplement for epithelial cell media
Hyaluronic acid	Millipore Sigma	385908	Extracellular matrix component for hydrogel formulation
Hyaluronidase	Sigma-Aldrich	H3506	Enzyme used for tissue dissociation
Incubator	Thermo Scientific	3598	Device used for tissue culture incubation
Insulin	Sigma-Aldrich	I9278	Supplement for epithelial cell media
Laminin	Gibco	23017-015	Extracellular matrix protein component
Mammary epithelial basal medium	Thermo Scientific	M171500	Growth medium for epithelial cells
Microcentrifuge tubes (1.5 mL)	Thermo Scientific	3451	Tubes used for small-volume reactions
Orbital rotator	Thermo Scientific	400110	Rotator used for tissue dispersion during enzymatic dissociation
P1000 pipette	Gilson	P1000	Device used to mix and transfer volumes up to 1,000 μL
Penicillin-streptomycin	Thermo Scientific	15140122	Antibiotic supplement for epithelial cell media
Phosphate-buffered saline	Gibco	20012-027	Buffer solution used for washing and rinsing
Precision balance	Mettler Toledo	ML303E	Balance used for tissue weighing
Serological pipette	Nunc	170356N	Pipette used for harvesting non-adherent epithelial cells
Sodium hydroxide (1 N)	Fisher Chemical	SS261	Reagent used for collagen neutralization
Sterile scalpels	Bard-Parker	372615	Tools used for mechanical tissue mincing
Trypan blue solution	Gibco	15250061	Dye used for cell viability assessment
Trypsin (0.25%)	Gibco	25200056	Enzymatic reagent used for cell dissociation
Water bath (37 °C)	VWR	10LA	Device used for controlled thawing of samples