Tangential Flow Filtration for Highly Efficient Concentration of Extracellular Vesicles from Large Volumes of Fluid

Abstract

Concentration of extracellular vesicles (EVs) from biological fluids in a scalable and reproducible manner represents a major challenge. This study reports the use of tangential flow filtration (TFF) for the highly efficient isolation of EVs from large volumes of samples. When compared to ultracentrifugation (UC), which is the most widely used method to concentrate EVs, TFF is a more efficient, scalable, and gentler method. Comparative assessment of TFF and UC of conditioned cell culture media revealed that the former concentrates EVs of comparable physicochemical characteristics, but with higher yield, less single macromolecules and aggregates (<15 nm in size), and improved batch-to-batch consistency in half the processing time (1 h). The TFF protocol was then successfully implemented on fluids derived from patient lipoaspirate. EVs from adipose tissue are of high clinical relevance, as they are expected to mirror the regenerative properties of the parent cells.

Adipose‐Derived Biogenic Nanoparticles for Suppression of Inflammation

“…The fluid flows tangentially to the membranes, reducing pore clogging, and leading to more effective separation when compared to conventional (dead‐end) filtration. Notably, TFF is very effective at separating nanosized components from larger (micrometer‐sized and above) and smaller (angstrom‐sized) components,16 but is unable to separate different types of nanoparticles (e.g., EVs and lipoproteins). However, one of the main advantages of TFF is that large volumes (>1 L) can be processed for scalable manufacturing, whereas other EV isolation techniques, such as size‐exclusion chromatography (SEC), are limited by much smaller input volumes 26–28.…”

“…However, one of the main advantages of TFF is that large volumes (>1 L) can be processed for scalable manufacturing, whereas other EV isolation techniques, such as size‐exclusion chromatography (SEC), are limited by much smaller input volumes 26–28. TFF also results in higher batch‐to‐batch consistency compared to conventional methods, such as ultracentrifugation 16. Moreover, standard testing for bacteria, mycoplasma, and endotoxin has previously demonstrated that sterility can be maintained throughout the process (from lipoaspiration to obtaining the final product with TFF),16 which is an important aspect for clinical translation.…”

“…TFF also results in higher batch‐to‐batch consistency compared to conventional methods, such as ultracentrifugation 16. Moreover, standard testing for bacteria, mycoplasma, and endotoxin has previously demonstrated that sterility can be maintained throughout the process (from lipoaspiration to obtaining the final product with TFF),16 which is an important aspect for clinical translation. Therefore, in comparison to other isolation methods TFF is more suitable for clinical use, despite not being able to separate different types of biological nanoparticles, resulting in a heterogeneous product.…”

“…The ADSC culture media or content of the Lipogems waste bag was transferred to sterile tubes and centrifuged (800 × g ; 30 min; Sorvall ST 16R centrifuge, Thermo Scientific) to remove large debris. BiNPs were isolated using a KrosFlo Research 2i Tangential Flow Filtration System (Spectrum Labs, Los Angeles, CA, USA) as previously described 16. Briefly, the cell culture media or waste bag supernatant was filtered through sterile hollow fiber polyethersulfone membranes with 0.65 µm pores.…”

“…The clinical translation of EVs and other BiNPs has also been hindered due to inefficient and nonscalable methods of isolation,12–14 such as ultracentrifugation, which is the most commonly used method 15. To overcome the issues faced by conventional isolation techniques, a highly robust, pure, and scalable tangential flow filtration (TFF)‐based method for BiNP isolation was employed 16. The size, morphology, zeta potential, and protein content of patient‐matched ADSC‐EVs and Lipo‐NPs were assessed.…”