A Cell Culture Insert-Based Protocol for Constructing a Simplified Multicellular In Vitro Blood-Tumor Barrier Model...

Effective treatments for breast cancer brain metastases (BCBM) remain limited, largely because the blood-tumor barrier (BTB) restricts the entry of many chemotherapeutic and targeted agents into metastatic lesions within the central nervous system. To model selected features of this complex microenvironment in vitro, this study presents a standardized protocol for constructing a simplified multicellular three-dimensional BTB model. The method comprises three core steps. First, a blood-brain barrier (BBB) compartment is constructed using cell culture inserts with a 0.4 μm pore size. Human brain microvascular endothelial cells (HBMECs) are seeded on the basal side of the porous membrane, and 1 day later, human astrocytes (HA) and human microglial HMC3 cells (HMC3) are seeded on the apical side.Using SK-BR-3 cells as an example, breast cancer cells are cultured by the hanging drop method to form uniform, regularly shaped 3D tumor spheroids after 3 days of culture. The spheroids are then transferred to the apical side of a basement membrane matrix-coated porous membrane, which provides physical separation while allowing chemical communication between the barrier cells and tumor spheroids during co-culture. This platform enables precise control of the experimental timeline and produces astrocytic morphological and marker changes consistent with a peritumoral reactive response while allowing assessment of endothelial barrier integrity and paracellular permeability.