Extracellular Vesicles from Human Cerebrospinal Fluid Are Effectively Separated by Sepharose CL-6B—Comparison of Four Gravity-Flow Size Exclusion Chromatography Methods

Abstract

Extracellular vesicles (EVs) are a versatile group of cell-secreted membranous nanoparticles present in body fluids. They have an exceptional diagnostic potential due to their molecular content matching the originating cells and accessibility from body fluids. However, methods for EV isolation are still in development, with size exclusion chromatography (SEC) emerging as a preferred method. Here we compared four types of SEC to isolate EVs from the CSF of patients with severe traumatic brain injury. A pool of nine CSF samples was separated by SEC columns packed with Sepharose CL-6B, Sephacryl S-400 or Superose 6PG and a ready-to-use qEV10/70 nm column. A total of 46 fractions were collected and analysed by slot-blot followed by Ponceau staining. Immunodetection was performed for albumin, EV markers CD9, CD81, and lipoprotein markers ApoE and ApoAI. The size and concentration of nanoparticles in fractions were determined by tunable resistive pulse sensing and EVs were visualised by transmission electron microscopy. We show that all four SEC techniques enabled separation of CSF into nanoparticle- and free protein-enriched fractions. Sepharose CL-6B resulted in a significantly higher number of separated EVs while lipoproteins were eluted together with free proteins. Our data indicate that Sepharose CL-6B is suitable for isolation of EVs from CSF and their separation from lipoproteins.

Unveiling the Native Morphology of Extracellular Vesicles from Human Cerebrospinal Fluid by Atomic Force and Cryogenic Electron Microscopy

“…If we compare the concentrations of EVs obtained by NTA and TRPS with the usual values when using the most adopted UC method for isolation, they are expectedly lower due to the dilution effect in SEC [ 17 , 29 ]. Nevertheless, the concentration of particles in the EV pool in this study measured by TRPS with NP400 nanopore ((6 ± 2) × 10 8 particles/mL) is in agreement with our previous results using the same quantification method for a fraction positive for EVs obtained after SEC of CSF (6.02 × 10 8 particles/mL) [ 24 ]. Even though it may seem that NP150 is a better choice for the size range of EVs than NP400, we should point out that the analysis by NP150 was extremely difficult due to clogging of the nanopore.…”

“…CSF was pooled from four TBI patients, whose Glasgow coma scale (GCS) score at admission and discharge and Glasgow outcome scale (GOS) score three months after discharge are shown in Supplementary Table S1 . EVs were isolated from the CSF pool using gravity-driven SEC according to the protocols described previously [ 24 ]. In total, 35 fractions of 2 mL each were collected from the 5 mL CSF pool after SEC, and the slot blot was performed.…”

“…SEC dilutes the sample, which can render the concentration of specific proteins in the eluates too much for their detection by chemiluminescence. To get a higher protein concentration in the eluates, we increased the load volume of CSF pool on the SEC column from 2.8 to 5 mL compared to our earlier publication [ 24 ]. Aiming to obtain an EV pool for further investigation, we performed slot blot on all SEC fractions using both total protein staining and specific immunodetection based on transmembrane CD9 and CD81 proteins, which span the phospholipid bilayer and are known markers also for EVs from CSF [ 6 ].…”

“…The gravity-driven SEC was adopted according to the previous optimization [ 24 ], but with an increased load volume. Five milliliters of pooled CSF from 4 TBI patients (CSF pool) was loaded to the glass SEC column 1.5 × 50 cm with flow adaptor and 30 µm bottom frit (Bio-Rad Laboratories, Hercules, CA, USA).…”

Macrophage-Derived Extracellular Vesicles: A Promising Tool for Personalized Cancer Therapy

“…EVs are released from cells, and their presence was found in several biological fluids: human breast milk, saliva, cerebrospinal fluid and urine [ 81 , 82 , 83 , 84 ]. For this reason, different purification and isolation methods have been developed based on the EVs’ starting source ( Table 1 ), as well as for the detection of their surface biomarkers and cargos.…”