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Abstract The human mammary gland represents a highly organized and dynamic tissue, uniquely characterized by postnatal developmental cycles. During pregnancy and lactation, it undergoes extensive hormone-stimulated architectural remodeling, culminating in the formation of specialized structures for milk production to nourish offspring. Moreover, it carries significant health implications, due to the high prevalence of breast cancer. Therefore, gaining insight into the unique biology of the mammary gland can have implications for managing breast cancer and promoting the well-being of both women and infants. Tissue engineering techniques hold promise to narrow the translational gap between existing breast models and clinical outcomes. Here, we provide an overview of the current landscape of breast tissue engineering, outline key requirements, and the challenges to overcome for achieving more predictive human breast models. We propose methods to validate breast function and highlight preclinical applications for improved understanding and targeting of breast cancer. Beyond mammary gland physiology, representative human breast models can offer new insight into stem cell biology and developmental processes that could extend to other organs and clinical contexts.

Bioprinting, combined with other tissue engineering techniques, is a promising method to create engineered tissues or standardized 3D cell culturing models. It has potential applications in the development of animal free models for drug toxicity screenings or for personalized medicine approaches. Here we report the conversion of the worldwide used open source 3D printer (RepRap, Prusa i3) to a functional and affordable bioprinter by substituting the common plastic-extruder with the Nydus One Syringe Extruder (NOSE). The NOSE modification enables mechanical hydrogel extrusion as well as a tunable deposition precision and volume by featuring a modular syringe-holder concept. A cost effective and simple hardware design including a custom software (termed ‘composer’) was developed to prove that this technique can be made accessible for a broader public. Furthermore, the printing procedure was optimized for the peer-reviewed FRESH-method by Hinton et al. which allows to print geometrical complex cell-laden constructs. We provide a detailed protocol on the creation of the FRESH-gel to ensure the reproducibility of this state of the art method. As a proof of concept HEK293 cells as well as mouse embryonic stem cells (mESC) were utilized for cell-laden printing. Depending on the cellular source the survival rates range from 60% up to 95%. Automatized quantitative viability analysis was performed using the open source image analysis tool Icy, in particular the “spot detector” plugin. A detailed protocol allows the recreation of the assay. Further data utilizing a support bath printing technique reveal limitations regarding the printing and residential time of cell-laden constructs. Keywords: Open Source Bioprinter, RepRap Modificiation, Mechanical Syringe Extruder, Bioprinting Protocol, Bioprinting Software