Mesenchymal stem cells have been widely implicated in tumour development and metastases. Moving from the use of two-dimensional (2D) models to three-dimensional (3D) to investigate this relationship is critical to facilitate more applicable and relevant research on the tumour microenvironment. We investigated the effects of altering glucose concentration and the source of foetal bovine serum (FBS) on the growth of two breast cancer cell lines (T47D and MDA-MB-231) and human bone marrow-derived mesenchymal stem cells (hBM-MSCs) to determine successful conditions to enable their co-culture in 3D tumour spheroid models. Subsequently, these 3D multi-cellular tumour spheroids were used to investigate the effect of hBM-MSCs on breast cancer cell invasiveness. Findings presented herein show that serum source had a statistically significant effect on two thirds of the growth parameters measured across all three cell lines, whereas glucose only had a statistically significant effect on 6%. It was determined that the optimum growth media composition for the co-culture of 3D hBM-MSCs and breast cancer cell line spheroids was 1 g/L glucose DMEM supplemented with 10% FBS from source A. Subsequent results demonstrated that co-culture of hBM-MSCs and MDA-MB-231 cells dramatically reduced invasiveness of both cell lines (F(1,4) = 71.465, p = 0.001) when embedded into a matrix comprising of growth-factor reduced base membrane extract (BME) and collagen.