Small Extracellular Vesicles/Exosome Separation & Purification

Small Extracellular Vesicles/Exosome Separation & Purification

The technologies for exosomes separation and purification include ultracentrifugation (UC), polymer precipitation, tangential flow filtration (TFF), ultrafiltration recovery (UF), and size exclusion chromatography (SEC), inmmunoaffinity chromatography. UC as a relatively gold-standard method for exosome separation, has the highest number of publications. In practical separation experiments, different treatment schemes can be adopted based on the sample volume, downstream experiments, and hardware configuration to achieve the best separation results and cost control.

At NextGen Exsome, our laboratory is equipped with UC, automatic exosome purification system, tangential flow ultrafiltration system, size exclusion chromatographic column purification system, etc., to achieve the separation and purification of exosomes. It can process single batch ranging from 100 μl to 100 L. In addition, NextGen Exsome also has the ability to complete the subsequent treatment of exosomes, including identification and analysis, purity and pollution quality control, as well as freeze-drying and other processing technologies.

Ultracentrifugation(UC)

General differential centrifugation is one of the most commonly used methods for the isolation and purification of exosomes. Initially, cell culture supernatant is centrifuged to remove cell debris and large particles, followed by high-speed centrifugation to precipitate the exosomes. The precipitate is then washed with PBS or another buffer solution to obtain pure exosomes. To further increase the purity of exosomes, especially for samples with high lipoprotein content such as milk, the exosomes can be passed through a sucrose cushion for an additional UC step. If you want to obtain exosomes with extremely high purity, density gradient ultracentrifugation with iodixanol is recommended. This method can separate the exosomes based on the density difference between the exosomes and impurity particles, and collect the corresponding medium (exosome density between 1.13-1.19g/mL) to achieve the best purity.

Tangential Flow Filtration (TFF) and Size Exclusion Chromatography(SEC)

In traditional filtration methods, the liquid flows in the same direction as the filtration (i.e., dead-end filtration), which leads to the accumulation of particles on the membrane surface, thus reducing the filtration efficiency. In contrast, TFF alters the direction of liquid flow to be parallel to the surface of the filter membrane. The supernatant repeatedly circulates along the surface of the membrane, allowing exosomes retained by the membrane to be continuously removed through the circulation flow, while the permeate is effectively eliminated, achieving the purpose of purifying and concentrating exosomes. SEC, on the other hand, is a method that utilizes columns with smaller pore sizes to separate exosomes. When the sample passes through the column, the small-pored column filters out larger cells and cell debris, while exosomes smaller than the pore size pass through the column. This method can separate exosomes of different sizes simultaneously, but it’s complex and time-consuming compared to other methods. The combination of TFF and SEC has been proven to surpass UC in terms of yield, reproducibility, time, cost, and scalability, making it a relatively mainstream solution for separating large volume samples to obtain high-purity exosomes.

1.Clarification Filtration

Utilizing a 0.22 μm sterile filter membrane, the sample undergoes a filtration process to remove impurities, effectively eliminating a large quantity of cell debris and large particle complexes.

2.Volume Concentration

Samples are concentrated using a high-pressure tangential flow ultrafiltration system to achieve a volume concentration of 10:1 at sterile low temperatures.

3.Purification and Recovery

Exosome isolation and subsequent concentration of the concentrated supernatant using size exclusion chromatography can achieve up to 90% separation purity.

Fully Automatic Exosome Purification System (EXODUS)

Based on the principle of negative pressure oscillation system (NPO) combined with double-coupled harmonic oscillation system (HO), acting on a nano ultrafiltration chip, the EXODUS Fully Automatic Exosome Purification System enables the rapid removal of free nucleic acids and proteins and other impurities through nanoscale pores while retaining exosomes, thus purifying and enriching exosomes. Over ten professional papers have been published in prestigious journals such as Nature Methods, PNAS, and ACS Nano.

1.Purification Principle

The Negative Pressure Oscillation (NPO) system, combined with the Dual-Coupled Harmonic Oscillation (HO) system, acts on nano ultrafiltration chips. Free nucleic acids and impurities such as proteins in the sample are rapidly removed and retained through nanopores, thereby purifying and enriching exosomes.

2.Workflow

1) Sample Preprocessing:

Centrifugation or filtration to remove cells and cell debris.

2) Separation and Purification Using EXODUS:

3)Collection of Purified Exosomes for Downstream Applications.