Tissue Engineering and Regenerative Medicine

Mammary acini organization plays a fundamental role in tissue development and physiology, but also could offervaluable insights in cancer onset and progression. 3D in vitro models represent a new paradigm in biological sciences.Reconstituted Matrigel® Matrix was successfully used to model 3D tissues; however, Matrigel® encompasses a wide range of different factors, presents large batch-to-batch variation, is expensive and difficult to isolate. Here we designed tunable alginatebased substrates and assessed the assembly of MCF10A human breast-epithelial cells with a focus on the role of laminin-1 in acini maturation and cavitation. Collagen and laminin-1 were used to increase cell adhesive properties of the alginate matrix and to modulate cell activity. We exploited an overlay model consisting of an alginate-based hydrogel substrate and the addition of laminin-1 in the cell culture medium and followed mammary acini morphogenesis. The evolution of cell spheroids was followed for 21 days; immunofluorescence was performed to investigate acini maturation, cavitation and cell polarization were also analyzed in terms of area and roundness. Image analysis was performed to analyze spheroids/acini for area and roundness. We developed an efficient method for the development of 3D cell spheroids/acini in vitro and studied the effect of laminin-1 on spheroids formation (added to medium). This work established tunable, instructive and medium-throughput in vitro culture systems consisting of alginate-based hydrogel substrates and enriched culture media for the development of mammary acini. The modulation of culture substrates and signaling molecules could represent a platform to study the effects of cellular microenvironment on acinar morphogenesis.