(F-209) Microbioreactors for Facile Extracellular Vesicle Harvesting and Quantification of Vesicular Heterogeneity in Breast Cancer Tumor at Single-EV Resolution

Introduction:: Currently, well established microgel techniques have been employed for the investigation of tumor spheroid formation. However, these techniques are limited by inadequate fluid exchange. Our novel three-dimensional extracellular vesicle (EV) microbioreactors (^EVµBRs) enable precise control on micro-metastatic EV release. To further enhance the utility, we engineered micro-channels to immobilize the ^EVµBRs for facile harvesting of EVs on a microfluidic device. Our work brings up a significant advancement in the field of cancer research, and this rapid investigating tool could ultimately lead to the development of synthetic EV nanoparticle (EVNP) encapsulation-based therapies to treat cancer.

Materials and Methods:: The flow dynamics on a droplet generator device were applied to produce highly homogeneous calcium-alginate ^EVµBRs. A digital-micromirror device (DMD)-based UV projection system was used for the elaboration of hydrodynamic poly-ethylene-glycol (PEG) traps as large-scale arrays. Lastly, immunofluorescence (IF) with total internal reflective fluorescence microscopy (TIRFM) were employed to quantify these harvested single EVs, thus offering a powerful tool for evaluating vesicular heterogeneity and advancing cancer therapy development.

Results, Conclusions, and Discussions:: Results and Discussions: The flow-focusing microfluidic junction enabled the rapid production of monodispersed ^EVµBRs with coefficient of variation of 1.73 ± 0.46 % (Fig. 1a). Various morphological phenotypes were observed during spheroid growth under exact flow and cell concentration conditions, indicating the heterogeneity of the cell line. By repurposing oxygen inhibition in photopolymerization, two-layer hydrodynamic traps were elaborated in large scale with a DMD-based UV projection system in less than 15 min, providing maskless and rapid microfluidic production unmatched by current soft-photolithographic techniques (Fig. 1b). The hydrodynamic traps immobilized the breast cancer spheroids encapsulated in the ^EVµBRs via a locking mechanism at an efficiency of 94.17 ± 6.97 % (Fig. 1c). Spheroid-derived EVs could be harvested with as little as ~20 µL from the microchannel. The majority of the breast cancer spheroid-derived EVs were small EVs (< 200 nm), which demonstrated a heterogenous expression of CD63 and CD81 (Fig. 1d). The ^EVµBRs were loaded with various densities of cells whereby high-density lent the highest phenotypic heterogeneity. The secretion of EVs across the various densities was analyzed via tunable resistive pulse sensing (TRPS) from suspended ^EVµBRs and single-EV IF from the microfluidic system, where both quantification methods coincided in determining a density-dependent increase of EV production (Fig. 1e).

Conclusion: We developed a novel method to harvest EVs released from biomimetic conditions that require a minimal volume for IF analysis at a single-EV resolution. The platform affords a facile, reliable, tunable, and quantifiable method to investigate micro-metastatic EV release. Our future research aims to utilize novel micro-mixing chip to synthesize highly controllable hollow recombinant EV nanoparticles (rEVNPs) of size 80-120nm, capable of carrying therapeutic agents like small interfering RNA (siRNA) or chemotherapeutic drugs. These rEVNPs can be surface-modified with antibodies that target specific receptors such as Epidermal Growth Factor Receptor (EGFR) and Human Epidermal Growth Factor Receptor 2 (HER2).


Acknowledgements (Optional): :

References (Optional): : Reference: Rima et al., Lab Chip, 2022,22, 2502-2518, HOT Article