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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

Poster J2 - Using Centrifugal Concentrators to Concentrate and Purify Liposomes as a Model for Extracellular Vesicle Purification

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

John T. Chon, Jr.

Undergraduate Research Assistant
Rochester Institute of Technology
Rochester, New York, United States

Co-Author(s)

EK

Ethan Kishimori

Undergraduate Research Assistant
Rochester Institute of Technology, United States
NL

Nick Luey

Undergraduate Research Assistant
Rochester Institute of Technology, United States
MD

Mehdi Dehghani

Manager of Nanoparticle Technologies
Sartorius Stedim Biotech, United States

Primary Investigator(s)

Tom Gaborski, PhD

Professor
Rochester Institute of Technology
Rochester, New York, United States

Introduction: Extracellular vesicles are of interest to researchers due to their ubiquitous role as biomarkers for a whole host of pathologies and conditions. They are distributed by both bacterial and human cells in the body. Thin porous membranes can be used in centrifugal concentrators in order to concentrate these extracellular vesicles. Currently, purification of extracellular vesicles requires expensive and time-consuming ultracentrifugation at 100,000 x g or more. However, with the use of size-based centrifugal concentrators, benchtop centrifuges could provide the necessary conditions to both concentrate and purify extracellular vesicles. Using liposomes as a surrogate for extracellular vesicles, they were concentrated from lower concentration stock solutions, as well as purified from stock solutions containing extraneous proteins. A variety of membrane materials and molecular weight cut-off sizes were tested to quantify the effectiveness of centrifugal concentrators. The concentration data of the concentrated sample was collected by nanoparticle-tracking light-scattering, fluorescence, and flow cytometry, as well as bicinchoninic assay for the samples tested with proteins.

Materials and

Methods: Unilamellar liposomes were obtained from Cellsome with a L-alpha-Phosphatidylcholine to cholesterol molar mass ratio of 70:30. These liposomes were sized to 100 nm and suspended in a PBS buffer with a pH of 7.4. These liposomes were chosen because of their similarity in size to extracellular vesicles as well as their relative size homogeneity. A feed of 1 x 1010 particles per mL was made from the liposomes by serial dilutions. For samples with proteins, bovine serum albumin was inserted such that the concentration of BSA in the feed was 1 mg/mL. This was used to analyze protein filtration. Vivaspin 20TM centrifugal concentrators with molecular weight cut-off filters ranging from 0.2 um to 10 kDa were used to concentrate 10 mL of feed into 9 mL of filtrate and 1 mL of retentate. Vivaspin 15TM concentrators with a 30 kDa molecular weight cut-off were used to analyze the effect of different membrane materials. The concentrators were centrifuged at 500 x g. The feed, filtrate, and retentate were all analyzed with nanoparticle tracking analysis and flow cytometry using the NanoSight NS300TM light-scattering and fluorescence modes as well as the Sartorius Virus CounterTM 3100 fluorescence flow cytometry mode. Cell Mask Orange was used to stain the liposomes for fluorescence analysis. Protein recovery was analyzed using bicinchoninic assay with the Thermofisher PierceTM Micro BCA kit. The recovery rates for each filter were then calculated using the feed, filtrate, and retentate concentrations.

Results, Conclusions, and Discussions: Centrifugal concentrators from 0.2 um to 300 kDa were ineffective at concentrating the liposomes in the retentate (see Figures 1-3). This was due to the pore size of the membrane causing the liposomes to either pass through or remain inside of the filter. The filter sizes 100 kDa and smaller were found to be effective at concentrating liposomes in the retentate. The 100 kDa filter size was further selected for use with a mixed liposome and bovine serum albumin experiment due to its ability to retain liposomes. Previous research also indicated that this cutoff was effective at passing albumin through the filter (see figure 4). It was found that this molecular weight cut-off was effective at both concentrating liposomes and filtering albumin (see figure 5). Potentially, the 100 kDa cut-off could be effective at purifying liposomes from extraneous proteins (see figure 6). In addition, the effects of membrane material were tested using Vivaspin 15TM concentrators. Two different regenerated cellulose membranes and one polyethersulfone membrane were compared in their ability to recover liposomes in the retentate. Generally, it was found that the membrane materials varied slightly in effectiveness, with the HydrosartTM regenerated cellulose membrane being the worst and the other regenerated cellulose being the most effective at concentrating liposomes (see figure 7).

Future work should be done with actual purified extracellular vesicles. Because extracellular vesicles vary more in size and charge, there may be different results. However, thin porous membranes show potential for size-based purification of extracellular vesicles, as well as purification of extraneous proteins from conditioned media.

Acknowledgements (Optional): This work was completed with support from Sartorius Stedim Biotech.