A mouse mammary gland organoid protocol to mimic breast morphology in vitro

Scope of the method

The Method relates to
  • Animal health
The Method is situated in
  • Basic Research
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Animal derived cells / tissues / organs
Species from which cells/tissues/organs are derived
Type of cells/tissues/organs
mammary gland


Method keywords
  • Mouse mammary gland organoids
  • primary material
  • branched morphology
  • ECM composition
  • growth factor supplementation
  • tumor initiation
  • pubertal development
Scientific area keywords
  • Developmental biology
  • Oncology
  • 3D organoid models
  • stem cell biology
Method description

The protocol is aimed at developing primary mammary gland organoids that have a morphology similar to the one of the in vivo breast, which is organized as a complex network of interconnected branches. The organoids are derived from the mouse mammary gland by mechanical dissociation and enzymatic digestion of the tissue to obtain small mammary tissue fragments that spontaneously organize in sphere-shaped organoids. The sphere-shaped organoids are then transferred in mixed Basement Membrane Extract (BME) and collagen gels, supplemented with growth factors to induce an elongated and branched morphology. By combination of the right ECM stiffness (mixed collagen: Matrigel gels) and growth factor supplementation, we managed to obtain complex organoids, up to 1.2 mm in length and with branches up to the 5th level. This will allow to study how the branching process works in branched organs in our body with reduced use of animal models. Also, mutations can be introduced in the model, which can be used to study tumor initiation and progression.

Method status
  • History of use
  • Published in peer reviewed journal

Pros, cons & Future potential

  • - Organoids are derived from primary material so they are not transformed,
  • - They show a complex morphology similar to the in vivo gland,
  • - Relatively fast procedures,
  • - No need of special equipment.
  • - Relatively low throughput,
  • - Technically challenging,
  • - Long culture time (15-20 days),
  • - Primary material cannot be expanded indefinitely so it still requires use of animals.

We are currently testing additional factors on the organoids to mimic in vitro the remodeling stages of the adult breast during pregnancy, lactation and involution upon weaning to have a model that can reduce the use of animals to study these developmental processes.

Future & Other applications

The model can be used to study the impact of breast remodeling on tumor predisposition. The concept of modulating the matrix stiffness and providing a growth factor alternation may apply to induce branching also in organoid models of other branched organs.

References, associated documents and other information


Caruso M, Huang S, Mourao L, Scheele CLGJ. A Mammary Organoid Model to Study Branching Morphogenesis. Front Physiol. 2022 Mar 16;13:826107. doi: 10.3389/fphys.2022.826107. PMID: 35399282; PMCID: PMC8988230.

Caruso M, Saberiseyedabad K., Mourao L, Scheele CLGJ. A guide towards 3D mammary and breast organotypic cultures. Methods Mol Biol. 2023, Springer Nature (submitted).

Associated documents
A Mammary Organoid Model to Study Branching Morphogenesis.

Contact person

Beaunelle de Bruijn


Oncology - KU Leuven