Advancing humanized 3D tumor modeling using an open access xeno-free medium

Despite limitations like poor mimicry of the human cell microenvironment, contamination risks, and batch-to-batch variation, cell culture media with animal-derived components such as fetal bovine serum (FBS) have been used in vitro for decades. Moreover, a few reports have used animal-product-free media in advanced high throughput three-dimensional (3D) models that closely mimic in vivo conditions. To address these challenges, we combined a high throughput 3D model with an open access, FBS-free chemically-defined medium, Oredsson Universal Replacement (OUR) medium, to create a more realistic 3D in vitro drug screening system. To reach this goal, we report the gradual adaptation procedure of three cell lines: human HeLa cervical cancer cells, human MCF-7 breast cancer cells, and cancer-associated fibroblasts (CAFs) from FBS-supplemented medium to OUR medium, while closely monitoring cell attachment, proliferation, and morphology. Our data based on cell morphology studies with phase contrast and real-time live imaging demonstrates a successful adaptation of cells to proliferate in OUR medium showing sustained growth kinetics and maintaining population doubling time. The morphological analysis demonstrates that HeLa and MCF-7 cells displayed altered cell morphology, with a more spread-out cytoplasm and significantly lower circularity index, while CAFs remained unaffected when grown in OUR medium. 3D fiber scaffolds facilitated efficient cell distribution and ingrowth when grown in OUR medium, where cells expand and infiltrate into the depths of 3D scaffolds. Drug toxicity evaluation of the widely used anti-cancer drug paclitaxel (PTX) revealed that cells grown in 3D cultures with OUR medium showed significantly lower sensitivity to PTX, which was consistent with the FBS-supplemented medium. We believe this study opens the way and encourages the scientific community to use animal product-free cell culture medium formulations for research and toxicity testing.