27/06/2018

Concentrating exosome samples with a tangential flow filtration system

The two main membrane filtration methods are Direct Flow Filtration (DFF) and Tangential Flow Filtration (TFF). Both methods can be used for the concentration of exosomes

Direct Flow Filtration vs Tangential Flow Filtration

DFF also called “dead-end filtration”, has a feed stream that is perpendicular to the filtration membrane, so the feed must pass straight through the membrane to be filtered.

In DFF the pores of the membrane may become blocked by larger particles making it difficult for the smaller particles to pass through the filter. As a result, this can cause the feed flow to reduce dramatically, requiring increased pressure to be applied to the fluid so that smaller particles can eventually pass through the membrane.

TFF has a feed stream that passes parallel to the filtration membrane, so the feed passes across the surface of the fliter..

In TFF, as the feed passes across the surface of the membrane, this constant agitation in the feed flow prevents blockage of the membrane, making TFF a fast and efficient method for the purification, concentration and buffer exchange of exosome samples.

In TFF what passes through the membrane is called the permeate and what is retained is called the retentate. The retentate can then be recirculated to the feed reservoir until the desired concentration of the sample has been achieved.

Concentrating EV samples with HansaBioMed TFF-easy.
 
HansaBioMed TFF-Easy is a filter cartridge made from polysulfone hollow fibres with 20 nm pores and based on the tangential flow filtration methodology as described above. It allows the concentration and the removal of small proteins and molecules from diluted matrices (cell conditioned media, urine, etc..) prior to the EV purification and EV dialysis for changing buffer conditions. Water and small molecules (<20 nm) pass through the hollow fibre pores and are collected in the collection bag and extracellular vesicles can be easily recovered from the hollow fibre cartridge with a syringe.














The protocol is simple, using a syringe containing the sample at one end and a clean empty syringe at the other. Then to start the concentration process, simply push the two syringes alternately until the desired volume has been obtained.


Comparison between TFF-easy and MWCO spin concentrator.
When compared with the MWCO spin concentrator in an ELISA assay assessing the expression of CD81 in LnCAP CCM, TTF-easy showed better performance and low variability between samples as seen in the graph below.








Combine TFF-easy with the HansaBioMed Size exclusion Chromatography Columns to obtain Pure Exosomes and Extracellular Vesicles.

Ultracentrifugation is currently the gold standard methodology for Extracellular Vesicle isolation from biological fluids or cell conditioned media. However, it does not isolate EVs efficiently, tends to alter the vesicle shape and functionality, requires expensive equipment and is time-consuming. To address these issues, HBM-LS has developed optimised tools for the isolation of total or specific EV populations.

In particular, HansaBioMed-LS has developed different classes of Size Exclusion Chromatography columns from different sample matrices and volumes (from 100ul and up to 20 mL). Size exclusion chromatography is currently one of the best methods to isolate exosomes and EVs. As a result, using TFF-easy to concentrate samples before using the PureEV SEC columns from HansaBioMed allows researchers to isolate easily and efficiently highly pure exosomes and EVs for further studies.




















Written by Magalie Dale
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