Scientific Context of the Project

This project includes three main sections. In the first section, the source of extracellular vesicles will be prepared from breast cancer cell lines MDA MB 231 and MCF7.  Conditioned media will be prepared from the cell lines cultured in serum-free conditions.

In the second part, new high flux ultrafiltration membranes exhibiting a low fouling tendency will be developed to isolate and separate extracellular vesicles. The membrane formulation will be optimized based on the filtration capacity, pore size, and recovery of the vesicles. The relationship between the morphological features of the membrane and its filtration performance will be investigated through detailed characterizations.

In the third part, the isolated high-purity Evs will be characterized. EV markers and cargo will be analyzed by western blot and Real-Time PCR for protein and RNA content. Furthermore, the effect of isolated EVs will be compared to source conditioned medium in terms of cell proliferation and invasion.

Innovative Aspects of the Project

Size exclusion chromatography (SEC) and filtration are commonly used size-based separation techniques for extracellular vesicles (Evs) isolation. While SEC dilutes the isolates, filtration concentrates EVs, making them suitable for large-scale applications. The main challenge in filtration is the membrane fouling due to the deposition of EVs on the surface, causing reduced fluxes. To solve the low-flux problem, the transmembrane pressure is usually increased; however, this strategy may damage EVs. In this study, new high flux ultrafiltration membranes exhibiting a low fouling tendency will be developed to isolate and separate extracellular vesicles from different biofluids. The main motivation is to make the production method standardized, scalable, and cost-effective to isolate high-yield and high-purity EVs from various biological fluids.

Research Environment and Infrastructure

IZTECH has all the facilities for membrane preparation, testing and characterization. These facilities include membrane casting device, filtration set-up, surface charge, contact angle measurement devices, surface roughness and morphological characterization tools.

IZTECH has all the facilities required to perform purity and content analysis of isolated extracellular vesicles. Briefly, basic BSL2 cell culture facility for functional analysis, western blot for protein analysis and real-time PCR for RNA analysis are available.