miniPURE-EVs Spin Column: A New Platform for a Fast and Convenient Purification of EVs and Nanoparticles from Small Volume Amount

Introduction

High-throughput sample preparation for downstream Extracellular Vesicle (EV) biomarker analysis requires scalable, afford- able, fast, and easy-to-use solutions. Ultracentrifugation, density gradient centrifugation, and dead-end filtration are common- ly used in the upstream processing of complex biofluids [1]. However, these methods are tedious and time-consuming, with often low sample recovery rates [2,3]. Additional pre-analytical purification steps to remove excess dye or antibodies further contribute to sample loss [4].

In this application note, we demonstrate the utility of the new MiniPURE-EVs Spin Size Exclusion Chromatography column for the quick and reproducible purification of EVs from complex samples such as plasma. Furthermore, we showcase the high efficiency of the MiniPURE-EVs Spin column in removing excess dye and antibodies prior to fluorescence analyses using Nanoparticle Tracking Analysis (NTA).

miniPURE-EVs Spin: Workflow

miniPURE-EVs Spin: Applications

1) Purification of EVs from Small Volume Amount of Biofluids

The MiniPURE-EVs Spin column was filled with 100 μL of plasma. EVs were eluted following a two-step centrifugation at 200 xg for 3 minutes each. In the first step, 100 μL of sample was processed, followed by the addition of 50 μL of PBS in the second step. The total elution volume was 150 μL, with a turnaround time of approximately 6 minutes.. After the washing steps, each filtrate was evaluated for particle number and protein amount to confirm reusability.

2) EV Labeling with Membrane Dye: Removal of Dye Excess with miniPURE-EVs Spin

U87-derived EVs were incubated with membrane dye at 37°C for 1 hour. After incubation, a 100 μL sample was loaded onto the Mini- PURE-EVs Spin column and centrifuged for 3 minutes at 200 xg. The total eluate of 100 μL, containing pure labelled EVs, was collected and analyzed with NTA in both scatter and fluorescence modes. A comparative study was performed in parallel using a competitor's spin column.

3) EV Labeling with Fluorophore-Conjugated Antibody: Removal of Dye Excess with miniPURE-EVs Spin

COLO-derived EVs were incubated with an- ti-CD9 antibody (Alexa Fluor 488 conjugate) at 37°C, for 1.5h. After the incubation, the sam- ple (100 μL) was loaded onto the MiniPURE-Evs Spin column and centrifuged for 3 minutes at 200 xg. Additional, 100 μL of PBS was loaded and centrifugation was repeated. Total eluate of 200 μL, containing pure labeled Evs, was col- lected and analyzed with NTA.

Conclusion

- Fast Processing: The MiniPURE-EVs Spin column enables rapid purification of extracellular vesicles (EVs).
- High Sample Recovery: It ensures high recovery rates of EVs, maintaining sample integrity.
- Reusability: The column can be reused, offering a cost-effective solution for EV isolation.
- Versatility: Suitable for both purifying EVs from raw biofluids, cell conditioned medium and for removing excess dye.
- Ease of Use: It is significantly easier to use compared to competitor columns and dead-end filters.

References

[1] Konoshenko, M. Yu.; Lekchnov, E. A.; Vlassov, A. V.; Laktionov, P. P. Isolation of Extracellular Vesicles: General Methodologies and Latest Trends. BioMed Res. Int. 2018, 2018, 1–27. https://doi.org/10.1155/2018/8545347.
[2] Brennan, K.; Martin, K.; FitzGerald, S. P.; O’Sullivan, J.; Wu, Y.; Blanco, A.; Richardson, C.; Mc Gee, M. M. A Comparison of Methods for the Isolation and Separation of Extracellular Vesicles from Protein and Lipid Particles in Human Serum. Sci. Rep. 2020, 10 (1), 1039. https://doi. org/10.1038/s41598-020-57497-7.
[3] Clos-Sansalvador, M.; Monguió-Tortajada, M.; Roura, S.; Franquesa, M.; Borràs, F. E. Commonly Used Methods for Extracellular Vesicles’ En- richment: Implications in Downstream Analyses and Use. Eur. J. Cell Biol. 2022, 101 (3), 151227. https://doi.org/10.1016/j.ejcb.2022.151227.
[4] Rautaniemi, K.; Zini, J.; Löfman, E.; Saari, H.; Haapalehto, I.; Laukka, J.; Vesamäki, S.; Efimov, A.; Yliperttula, M.; Laaksonen, T.; Vuorimaa-Lauk- kanen, E.; Lisitsyna, E. S. Addressing Challenges in the Removal of Unbound Dye from Passively Labelled Extracellular Vesicles. Nanoscale Adv. 4 (1), 226–240. https://doi.org/10.1039/d1na00755f.