# Mammary Epithelial Cell Spheroid: Stabilization Through Vascular-Wall Mesenchymal Stem Cells and Endothelial Cells Co-Culture

## TL;DR

This study creates a 3D cell model of the mammary gland using pig cells, showing that combining different cell types leads to more stable and organized structures.

## Contribution

The study introduces a novel 3D co-culture model for domestic species using primary cells from Göttingen Minipigs to mimic mammary gland structure.

## Key findings

- Co-culturing mammary epithelial cells with vascular-wall cells led to more compact and viable spheroids.
- The model showed well-organized cell distribution confirmed by immunofluorescence analysis.
- The 3D model is a promising tool for studying mammary gland physiology and lactation biology.

## Abstract

It has been widely demonstrated that vascular-wall multipotent cells are particularly involved in vascular-remodeling processes, especially when interacting with endothelial cells, by contributing to blood vessel stabilization and maturation. In the mammary alveolus, the vascular wall cells, including endothelial cells, are crucial for maintaining the blood–milk barrier by supporting the function of mammary epithelial cells. Considering that, to date, there is a shortage of 3D co-culture models that mimic in vivo mammary glandular structure for domestic species, in the present research, we described a 3D co-culture model where primary mammary epithelial cells from Göttingen Minipigs were co-cultured with the two vascular-wall cellular populations: the mesenchymal stem cells and endothelial cells. The results showed that co-culturing the mammary epithelial cells with the two vascular wall cell populations led to the formation of more stable mammary gland multicellular spheroids with a well-organized cell structure, offering a strong potential for various applications in veterinary research, including lactation biology and regenerative approaches.

Over the past decade, the interest in using 3D cell culture models for studying the mammary gland in biomedical and veterinary fields has increased, but a fully functional in vitro model for domestic species is still lacking. Multiple cellular components, including epithelial cells, vascular endothelial cells, and stromal/stem cells, sustain the secretory mammary gland tissue in a well-organized 3D architecture. Considering the Göttingen Minipigs widely used for translational lactation studies, this work aimed to establish a 3D culture protocol to generate mammary heterogeneous multicellular spheroids composed of three different Göttingen Minipigs primary cells: mammary epithelial cells (mpMECs), aortic endothelial cells (mpAECs), and vascular-wall mesenchymal stem cells (mpVW-MSCs). Cells were cultured with hanging-drop (HD) and ultra-low-adherence plate (ULA) methods, evaluating aggregate formation in both monocultures and co/triple co-cultures. Brightfield area, eccentricity, viability, and cell distribution were analyzed. Results showed mpMECs formed irregular aggregates in both HD and ULA, while more compact and viable spheroids were formed when co-cultured with mpVW-MSCs and mpAECs by ULA. A well-organized cellular distribution was demonstrated by cytokeratin-18, vimentin, and e-NOS immunofluorescence analysis. In conclusion, this study established a stable 3D mammary multicellular spheroid model, representing a promising tool for future studies on hormonal modulation and mammary gland physiology.