Pairing Tangential Flow Filtration and Size Exclusion Chromatography: The Dream Team for EV Isolation From Cell Culture Media

Extracellular Vesicles
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Tangential flow filtration versus ultracentrifugation… which one wins the prize as the best method for preconcentrating samples prior to SEC.. and how exactly do these methods compare?

Extracellular vesicles (EVs) are readily formed by cells in vitro. The highly controlled cell culture environment allows for the isolation of EVs from cells grown in user defined conditions (e.g., in the presence of specific media additives) and manipulated in user defined ways (e.g., engineered to express specific proteins). This makes cell culture conditioned media an ideal source of EVs for therapeutics. Cell culture conditioned media EVs are also ideal for cosmeceuticals, which bridge the gap between traditional cosmetics and pharmaceuticals. However, large volumes of sample are needed to achieve sufficient EVs for therapeutics and cosmeceuticals. We work with customers across a wide variety of applications and sample volumes for exactly these purposes, giving us a strong appreciation of the methodological requirements and challenges involved. Whilst we offer size exclusion chromatography (SEC) qEV columns for samples up to 100 mL, therapeutic and cosmeceutical EVs can necessitate starting volumes of up to tens or even hundreds of litres, necessitating pre-concentration.

Ultracentrifugation has traditionally been used for pre-concentration prior to SEC. However, ultracentrifugation is a technique littered with disadvantages which are especially dire for therapeutic purposes, where poor reproducibility, high isolation of contaminants, and damage to EV integrity and functionality could be devastating for clinical use.1-7 Visan et al. (2022) aimed to identify an alternative by comparing ultracentrifugation with tangential flow filtration (TFF) to pre-concentrate cell culture conditioned media prior to SEC.8 The results were clear: TFF vastly outperformed ultracentrifugation. But how exactly did TFF compare?

Study methodology

Figure 1. Methodology of EV isolation using pre-concentration with ultracentrifugation (blue) or TFF (purple). Adapted from data presented in Visan et al. (2022).<super-script>8<super-script>

In this article, ultracentrifugation combined with SEC will be referred to as UC-SEC and TFF combined with SEC will be referred to as TFF-SEC. Particle concentrations and size were determined using Tunable Resistive Pulse Sensing using the qNano.

Key Results

Figure 2. Comparison of particles and protein isolated using UC-SEC and TTF-SEC for (A) particle concentration, (B) particle size distribution, (C) protein concentration and (D) isolate purity (particle-to-protein ratio).

  1. Yield: TFF-SEC wins. TFF-SEC excelled at particle isolation, retaining up to 23-fold times more particles than UC-SEC (Figure 2a).
  2. Size: it’s a draw. The isolated particles were determined to be of similar size between the two methodologies (Figure 2b).
  3. Protein isolation: TFF-SEC wins. Protein concentration was significantly higher in TFF-SEC isolates than in US-SEC isolates (Figure 2c).
  4. Purity: another draw. TFF-SEC isolated particles with a similar particle-to-protein ratio, suggesting similar purity to UC-SEC (Figure 2c). Nano-flow cytometry identified equal CD9+ and CD63+ EVs in isolates using both methods.
  5. Morphology: draw. Both showed cup-shaped morphology on transmission electron micrographs.
  6. Cost and time: TFF-SEC wins. TFF-SEC is both quicker (Figure 1) and cheaper (< one tenth of the cost) than UC-SEC, rendering it more scalable.
  7. Scalability: TFF-SEC wins. TFF-SEC can process larger volumes, with concentration time being increased. Additional ultracentrifugation steps are required for UC-SEC to effectively concentrate EVs, thereby likely damaging them and causing a loss of EVs9.

The benefits of size exclusion chromatography as an EV isolation method

The benefits of SEC for the isolation of EVs are many. Like TFF, SEC is a gentle method for isolating EVs10,11, meaning that EVs are not damaged and remain highly functional.5,6 This is essential for EV isolation from cell culture conditioned media in particular as these samples are most often used for applications requiring functionality, such as functional experiments and use as therapeutics. Also essential for these purposes is EV isolate purity. SEC using qEV columns isolates EVs with a high level of purity, providing effective separation from free protein contamination.1,9,12,13 SEC is also highly reproducible, especially when considering the additional benefits of using commercially produced qEV columns which are produced in large batches with defined, optimised protocols, and can be combined with the Automatic Fraction Collector to enhance fraction volume accuracy and reproducibility.

As shown in Figure 1, ultracentrifugation is a time-consuming method, but it is also limited by scale. Ultracentrifugation machines can only be so large, necessitating multiple runs to isolate EVs from large volumes of starting material. This is expensive not just in ultracentrifuge tubes and tube cleaning, but in staff time. A tissue culture EV precipitation-based kit from another company takes even longer than ultracentrifugation, with overnight (at least 12h) incubation required. Comparatively, qEV isolation can be completed in as little as 15 minutes. SEC also scales linearly with size, meaning that smaller scale trials can be easily scaled to larger columns capable of isolating EVs from much larger starting volumes.

The future of large-scale EV isolation

The study by Visan et al. (2022) clearly showed that TFF outperforms ultracentrifugation for the concentration of cell culture conditioned media, prior to SEC. Equitable size, purity and morphology was seen for EVs pre-concentrated with TFF or ultracentrifugation prior to qEV-based SEC EV isolation. Given the vastly improved yield of EVs when TFF-SEC was used over UC-SEC and the lower cost and time expenditure, it is clear that TFF-SEC is a preferable workflow for EV isolation. Whilst not explored in this study, TFF is unlikely to cause the same negative impacts to EV integrity and function as ultracentrifugation due to the lack of high gravitational forces in TFF.

Izon Science supports both academic and industry-focused researchers to ensure that they get the best from their samples, including through the customisation of high throughput EV isolation from large starting volumes. This includes expertise in implementing automated pump systems to enable high-throughput EV isolation from large volumes. Our scientists have also optimised TFF for use prior to EV isolation using qEV columns and can provide support in this area. We can also provide technical expertise in media clarification, pre-concentration by depth filtration, and EV isolation by SEC. Here at Izon Science we pride ourselves on producing custom solutions for our customers, helping them to strive for better EV isolation from basic research to therapeutic or cosmeceutical purposes.

Learn more about customised extracellular vesicles separation solutions!


  1. Takov, K., Yellon, D. M. & Davidson, S. M. Comparison of small extracellular vesicles isolated from plasma by ultracentrifugation or size-exclusion chromatography: yield, purity and functional potential. J Extracell Vesicles 8, 1560809 (2019).
  2. Yuana, Y., Levels, J., Grootemaat, A., Sturk, A. & Nieuwland, R. Co-isolation of extracellular vesicles and high-density lipoproteins using density gradient ultracentrifugation. J Extracell Vesicles 3 (2014).
  3. Crigna, A. T. et al. Inter-Laboratory Comparison of Extracellular Vesicle Isolation Based on Ultracentrifugation. Transfusion Medicine and Hemotherapy 48, 48-59 (2021).
  4. Linares, R., Tan, S., Gounou, C., Arraud, N. & Brisson, A. R. High-speed centrifugation induces aggregation of extracellular vesicles. Journal of Extracellular Vesicles 4 (2015).
  5. Mol, E. A., Goumans, M. J., Doevendans, P. A., Sluijter, J. P. G. & Vader, P. Higher functionality of extracellular vesicles isolated using size-exclusion chromatography compared to ultracentrifugation. Nanomedicine-Nanotechnology Biology and Medicine 13, 2061-2065 (2017).
  6. Nordin, J. Z. et al. Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties. Nanomedicine-Nanotechnology Biology and Medicine 11, 879-883 (2015).
  7. Yang, Y. et al. Extracellular vesicles isolated by size-exclusion chromatography present suitability for RNomics analysis in plasma. Journal of Translational Medicine 19 (2021).
  8. Visan, K. S. et al. Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles. J Extracell Vesicles 11, e12266 (2022).
  9. Lobb, R. J. et al. Optimized exosome isolation protocol for cell culture supernatant and human plasma. Journal of Extracellular Vesicles 4 (2015).
  10. Li, J., He, X. Q., Deng, Y. Y. & Yang, C. X. An Update on Isolation Methods for Proteomic Studies of Extracellular Vesicles in Biofluids. Molecules 24 (2019).
  11. Kang, H., Kim, J. & Park, J. Methods to isolate extracellular vesicles for diagnosis. Micro and Nano Systems Letters 5, 15 (2017).
  12. Veerman, R. E. et al. Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin. Journal of Extracellular Vesicles 10 (2021).
  13. Stranska, R. et al. Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma. J Transl Med 16, 1 (2018).

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