High cell plating density initiates rapid self‐assembly of multipotent mammary cells in three‐dimensional biomatrix

Abstract

Multipotent mammary cells isolated from breastmilk have the potential to differentiate into mammary secretory epithelial phenotypes in culture. The objective of the present study was to investigate how tissue architecture and differentiation of multipotent mammary cells is influenced by cell plating density in three‐dimensional (3D) culture. The total cell population isolated from a human milk sample was cultivated in monolayer to expand and purify the target population. The monolayer was subcultured into 3D biomatrix (Matrigel) at cell densities ranging from 1×104 to 1×107 cells/mL. Architecture and differentiation was tracked using the markers CD49f (progenitor), Cytokeratin 18 (CK18; luminal) and beta‐catenin (epithelial cytoplasm). At low density, single cells underwent division to form alveolus‐like structures with a basal CD49f+ layer and a luminal CK18+ layer after several weeks of growth. At high density, cells formed extensive ductal‐alveolar networks in less than 12 hours that maintained a basal CD49f+ population but did not divide or differentiate. Cellular alignment with matrix fibres preceded visible intercellular interaction, and predigesting the matrix abrogated assembly but not cell viability. This implies that tissue architecture and differentiation are regulated by cell‐extrinsic cues and depend on close intercellular proximity, but not physical interaction.