From Organoids to Bioprinting: Advancing the Building Blocks of Breast Tissue Engineering

Abstract

Breast tissue is essential for milk production and has significant health implications due to the high prevalence of breast cancer. Despite advances in understanding breast biology and cancer treatment, challenges remain. Current treatment development is hindered by inefficiencies, high costs, and ethical concerns over animal models, which poorly mimic human breast biology. Improving and humanizing preclinical models is crucial for better insights into breast function and disease. The mammary gland’s three-dimensional structure is vital to its function, making 3D organoid models central to breast tissue engineering. While organoids improve on traditional 2D cell cultures in complexity, no fully functional, lactating mammary organoid model exists. Key limitations include a lack of spatial control, hormonal cues, stromal tissue, and vasculature. Advances in tissue engineering, biofabrication, and microfluidics offer potential solutions, with 3D bioprinting enabling precise deposition of cells and matrix components. Light-based methods, especially volumetric bioprinting, show promise due to their high resolution and layer-free printing. Additionally, bioreactors and organ-on-a-chip systems provide dynamic control over cell environments, guiding self-organization and maturation. This thesis, From Organoids to Bioprinting: Advancing the Building Blocks of Breast Tissue Engineering, aimed to develop an advanced in vitro mammary gland model bridging gaps between simplified cell cultures, non-human animal models, and complex human biology. Key Findings: Chapter 2: Identified research priorities, including integrating biofabrication with organoid technology, improving multicellularity, scalability, and post-printing maturation. Human breast milk was highlighted as a valuable cell source for tissue engineering. Chapter 3: Established breast milk as a non-invasive source for mammary organoids (MBOs), encompassing all major epithelial cell types and forming TDLU-like structures—the milk-producing units of the breast. Chapter 4: Used MBOs to assess breast cancer treatment safety. Given the abundance of milk, MBOs could serve as a biobank for population-wide tissue variations, identifying rare side effects and screening immunotherapy targets. Chapter 5: Developed a biofabrication pipeline integrating volumetric bioprinting, cell seeding, perfusion, and imaging-based validation. Findings suggest future enhancements should include hormonal stimulation, stromal cells, and MBO integration. Chapter 6: Introduced AnyBio, an affordable (€350) open-source mSLA-based bioprinter, demonstrating its versatility in biomaterial printing and cellular viability. Chapter 7 & Annex 1: Explored two biomaterials—supramolecular GelMA for vascularization, organoid culture, and T-cell migration within a 3D-printed breast cancer model, and gel-norbonene for light-induced porosity, supporting vascularization in a vessel-on-a-chip model. Conclusion: This thesis approached breast tissue engineering from multiple angles, emphasizing the need for improved multicellularity, multi-material composition, and architectural complexity. The integration of diverse fabrication techniques and a deeper understanding of the mammary extracellular matrix and stem cell organization will be crucial for future progress. Dynamic post-fabrication culture conditions have shown promise in functional maturation. Core Principles: Open Science & Accessibility: Open-source equipment and workflows promote democratization of knowledge in biofabrication. Sustainability: Computational data analysis and resource-efficient lab practices minimize waste. Future work could lead to a fully functional human mammary gland model, advancing research on lactation, breast cancer, and treatment development in an animal-free framework.