An Integrating Microfluidic System for Concentration Gradient Generation of Exosomes and Exosome-Assisted Single-Cell-Derived Tumor-Sphere Formation

To enhance exploration on tumor stem-like cells (TSCs) without altering their cellular biological characteristics, researchers advocate for application of single-cell-derived tumor-spheres (STSs). TSCs are regulated by their surrounding microenvironment, making it crucial to simulate a tumor microenvironment to facilitate STS formation. Recently, exosomes that originated from the tumor microenvironment have emerged as a promising approach for mimicking the tumor microenvironment. In the tumor microenvironment, various associated cells (such as fibroblasts, endothelial cells, and immune cells) play crucial roles. Utilizing exosomes derived from these cells enabled us to simulate the tumor microenvironment and promote STS formation. Herein, we have developed an integrated microfluidic platform to generate serial concentration gradients and evaluate the effects of multiple exosomes on STS formation. To demonstrate the feasibility of our approach, we generated serial concentration gradients of exosomes derived from two different cell types (HUVEC and NIH/3T3 cells) and assessed their effects on STS formation. Subsequently, we investigated the drug resistance of STSs treated with free doxorubicin and doxorubicin-loaded poly(lactic-<i>co</i>-glycolic acid) (PLGA) nanoparticles. Our findings revealed that the serial concentration gradients of mixed exosomes could be successfully generated, leading to an enhanced formation rate and size of STSs. Compared to exosomes derived from one cell type, the mixed exosomes exhibited superior promotion of STS formation. Additionally, nanomedicines demonstrated a reduction in the drug resistance of TSCs compared to free drug treatment, particularly in smaller and/or more deformable TSCs. This platform provides an innovative approach for STS formation enhancement and tumor microenvironment simulation.