Title : High-throughput in vitro analysis of engineered tumour microenvironments using 3D bioprinting
Traditional 2D flat cell cultures of cancer cells and in vivo animal experiments remain the most commonly used techniques as in vitro and in vivo platforms for cancer research, drug screening and toxicity tests prior to entering human clinical trials. This is due to their low cost, efficient workflows and optimised downstream analysis techniques. Evidence suggests that these traditional cell culture techniques do not accurately replicate the complexity of human tumours. For example, these models have limitations in mimicking tumour stromal heterogeneity and tumour cell-extracellular matrix (ECM) interactions, thus potentially limiting the ability to mimic in vivo tumours realistically. This has led to the development of three-dimensional (3D) in vitro models which have been shown to reflect the cellular responses in vitro.
Synthetic gels have been used extensively over naturally derived biomaterials, such as collagen without the complexity of cell-matrix interactions, as they offer opportunities to control and modulate cells. Herein, we developed an electrostatically crosslinked PEG-based gels system for creating high-throughput 3D in vitro models using a 3D drop-on-demand bioprinter to mimic the extracellular matrix cancer environment. First, the 3-arm PEG-based polymer backbones were conjugated with various degrees of cell adhesive RGD motifs (0%, 25%, 75% and 98%) to study the influences of cell adhesive motifs on breast cancer (MCF-7) spheroid formation. Formation, stability and mechanical properties of gels were tested with and without RGD motifs in different conditions to evaluate cellular response to materials parameters in a 3D environment. Biocompatibility was tested with MCF-7 breast cancer cells by encapsulating cells within a gel for 7 days, where a high cell viability of approximately 99.0 ± 1.4% was observed. The electrostatically crosslinked gels can be degraded in the presence of salts at room temperature by breaking the interaction of oppositely charged polymer chains. As such, both MCF-7 cells and spheroids were released by simply exposing a gel in a 2 M NaCl solution for 5 min. The released MCF-7 breast cancer cells and spheroids remained highly viable with no significant differences before and after release. Finally, the released MCF-7 cells/spheroids were analysed with flow cytometry to characterise cellular responses and behaviours in more detail.