Extracellular vesicles produced in hollow fiber bioreactor as potential drug delivery systems – cell uptake tracking with label-free optical diffraction tomography

Extracellular vesicles (EVs) are emerging as one of the most promising candidates for novel drug delivery systems (DDS). Unique features, like biocompatibility, encapsulation capability and functionalization for precise targeting make them an attractive candidates for biomedical applications. However, despite the numerous benefits, progress in EVs-based DDS translation from bench to clinics remains highly challenging. One of key limitations that needs to be addressed are low production yield, limited reproducibility and poor understating of their natural, in vitro/in vivo behavior. To address these challenges, we aimed to establish a bioreactor-based system for long-term, high-yield EV production and evaluate their internalization capabilities using label-free tomography. Hollow fiber system was implemented as bioreactor for production and pre-purification of extracellular vesicles in long time manner. The obtained vesicles were characterized by spectral flow cytometry, tunable resistive pulse sensing (tRPR) and transmission electron microscopy (TEM). Subsequently, in order to investigate their biological functions, EVs uptake by target cells was evaluated with label-free techniques, such as Optical Diffraction Tomography (ODT) and Fourier Ptychographic Microscopy (FPM). Bioreactor enabled to performed five week long continuous production of extracellular vesicles with high homogeneity among each collected batch. This include tetraspanins expression, and more importantly, narrow size distribution with low, ~5% differences. Reconstructed tomographic images revealed that obtained extracellular vesicles can effectively internalize into the target, pancreatic, cells and were preferentially located in perinuclear area. This first stage results are highly promising for further development such approaches for high yield EVs production and its label-free in vitro tracking.