Low-Vacuum Filtration as an Alternative Extracellular Vesicle Concentration Method: A Comparison with Ultracentrifugation and Differential Centrifugation

Abstract

Recent years have brought great focus on the development of drug delivery systems based on extracellular vesicles (EVs). Considering the possible applications of EVs as drug carriers, the isolation process is a crucial step. To solve the problems involved in EV isolation, we developed and validated a new EV isolation method—low-vacuum filtration (LVF)—and compared it with two commonly applied procedures—differential centrifugation (DC) and ultracentrifugation (UC). EVs isolated from endothelial cell culture media were characterized by (a) Transmission Electron Microscopy (TEM), (b) Nanoparticle Tracking Analysis (NTA), (c) Western blot and (d) Attenuated Total Reflection Fourier-Transform Infrared Spectroscopy (ATR-FTIR). Additionally, the membrane surface was imaged with Environmental Scanning Electron Microscopy (ESEM). We found that LVF was a reproducible and efficient method for EV isolation from conditioned media. Additionally, we observed a correlation between ATR-FTIR spectra quality and EV and protein concentration. ESEM imaging confirmed that the actual pore diameter was close to the values calculated theoretically. LVF is an easy, fast and inexpensive EV isolation method that allows for the isolation of both ectosomes and exosomes from high-volume sources with good repeatability. We believe that it could be an efficient alternative to commonly applied methods.

Extracellular vesicles derived from microgreens of Raphanus sativus L. var. caudatus Alef contain bioactive macromolecules and inhibit HCT116 cells proliferation

“…It is the first report of the FTIR peak characteristics of EVs derived from plant material. The peaks in the present study are similar to those found in the EVs derived from mammals in other studies 50 – 54 .

Figure 3 ( a ) Primary (black line) and second derivative (blue line) FTIR spectra of biological compositions of EVs derived from Raphanus sativus L. var.

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“…FTIR was used to study the biological molecular compositions in EVs. In addition to proteins, lipids, and nucleic acids, carbohydrates were also detected in EVs derived from different sources e.g., serum 72 , milk 73 , and cell culture-conditioned media 50 . Carbohydrates were reported to be related to the EVs biogenesis 74 , cellular recognition, and uptake of EVs by recipient cells 75 .…”

“…Dynamic light scattering was employed to confirm the size distribution from the transmission electron microscope and determine the zeta potential (stability representation). The biological macromolecular compositions were studied using FTIR analysis 50 – 54 , while the chemical micromolecular compositions were determined using HPLC analysis. The resultant data indicate that the differential centrifugation technique is capable of isolating EVs from plants.…”

Comparative Proteomic Profiling of Ectosomes Derived from Thyroid Carcinoma and Normal Thyroid Cells Uncovers Multiple Proteins with Functional Implications in Cancer

“…200 mL) were collected and subjected to sequential centrifugation. Cells and cellular debris pelleted after centrifugations at 400× g (5 min, 4 °C) and 4500× g (20 min, 4 °C) were discarded, whereas supernatants were collected and concentrated by a low-vacuum filtration (LVF) procedure, as described in [ 24 ]. LVF was performed on the dialysis membrane (Repligen, Waltham, MA, USA, cat.…”

Design and Optimization of a Biosensor Surface Functionalization to Effectively Capture Urinary Extracellular Vesicles

“…First, urine sample was centrifuged at 2000× g for 30 min at room temperature (RT), in order to remove bacteria, cell debris, and the majority of Tamm–Horsfall protein aggregates [ 35 ]. After the centrifugation step, the supernatant was collected and used for the Low-Vacuum Filtration (L-VF) method [ 36 ]. In this method, the urine sample was purified and concentrated using a 1000 kDa (MWCO) dialysis membrane, and a low vacuum (−0.3 Bar) was applied.…”