Background <p>One of the most important and devastating side effects of breast cancer is brain metastasis. Our understanding of cancer heterogeneity is revolutionized by tumoral organoids and seems promising for personalized medicine. This study aimed to generate a hydrogel-based brain metastasis organoid.</p> Methods <p>Mouse brain metastatic tumor cells (4T1B) were isolated and cultured from the brain metastasis lesions of mice with breast cancer. Different hydrogels, including alginate, carboxymethylcellulose, gelatin, collagen, and matrigel, were prepared. Pre-coated hydrogels in 96-well plates were treated with 4T1B cells. The morphology and viability of metastatic organoids were analyzed after 7 days.</p> Results <p>According to our results, 4T1B cells formed semi-regular cluster structures in alginate hydrogel. In this group, the cell survival rate and formation of three-dimensional structures were significantly higher than in other groups.</p> Conclusion <p>For organoid cultures, there’s a lot of research on natural and synthetic scaffolds that are chemically or mechanically well-designed. In the present study, we used highly brain metastatic tumor cells and detected that alginate hydrogel is the best choice for organoid formation and breast cancer brain metastasis modeling.</p>