Comparative Evaluation of EV Isolation Methods: Tangential Flow Filtration vs Ultracentrifugation

Introduction

Extracellular vesicles (EVs) are nanosized membrane-bound particles released by cells and present in a wide variety of biofluids. The purity, yield, and integrity of isolated EVs are critically dependent on the isolation method employed. In this application note, we present a comparative analysis of two widely used EV isolation strategies—Tangential Flow Filtration (TFF) and Ultracentrifugation (UC)—utilizing HansaBioMed’s validated TFF tools and EXONOID’s expertise in mammalian and non-mammalian EV isolation protocols.

In this study, TFF-EVs by HansaBioMed Life Sciences (Product code: HBM-TFF-EVs-S) was used to compare against the gold-standard ultracentrifugation. This filter has 50nm pore size, separating EVs >50nm from proteins and lipoproteins <50nm. The filter is sterile, enabling working directly under the hood. Its re-usability increases the cost efficiency of the method even further.

Methods

TFF is based on filtrating the sample in the perpendicular direction to a lateral flow. Developed initially for bio-pharmaceutical or food industries, the method was well-adapted to EV field in the last decade [1]. Its versatility allows separation, isolation and concentration of EVs, buffer exchange, EV depletion from FBS, and elimination of unwanted proteins (e.g. Tamm Horsfall protein from urine.)

TFF cartridges consist of submillimeter diameter fibers made of non-binding materials (i.e. PES) with set pore size. When the sample is subjected to a laminar flow inside fibers, particles smaller than the pore size are filtrated whereas the larger ones remain. Since the lateral flow creates a cleaning effect on the filter surface, this prevents cake formation and ensures steady filtration.

Results

• 1. EV isolations were performed from each source using TFF and UC with 3 repetitions. With UC, the isolation took 2.5 hours after the pre-processing stage, while with TFF, it took approximately 10 minutes after pre-processing.
• 2. In EV isolations, UC generally offers an advantage in terms of particle number; however, this advantage depends on the source used. For example, there is no significant difference between methods in rosemary EV isolation.
• 3. Importantly and strikingly, despite obtaining more particles with the UC technique, these samples show potential protein contamination, and TFF provides a significant advantage in terms of purity.
• 4. The high purity ratio for UC isolation of milk EVs can be because of high level of lipid particles (or fat content) in the milk, which co-sediments in UC. In TFF, on the other hand, sharp cut-off by 50nm pore size minimizes fat contamination. Further analysis might actually demonstrate TFF to be providing higher purity for milk EVs as well.

Conclusion

In conclusion, HansaBioMed's TFF system offers various advantages over the conventional UC method. These advantages are presented and discussed in Table 1.

Table 1: Comparison of TFF and UC in EV isolation