PhD Scientific Days 2025

Poster Session II. - B: Molecular Medicine

Characteristics of extracellular vesicles isolated from protoplast cultures

Name of the presenter

Szász Csenge

Institute/workplace of the presenter

Pediatric Center, MTA Center of Excellence, Semmelweis University

Authors

Csenge Szász¹, Ani Barbulova², Kameswari Priyanka Devarakonda², Immacolata Fiume², Angela Mary Joseph², Veronika Kralj-Iglic³, Anna Romolo³, Lilla Turiák^4,5, Mirjam Balbisi^4,5, Ádám Vannay^1,6, Gabriella Pocsfalvi²

1: Pediatric Center, MTA Center of Excellence, Semmelweis University
2: Institute of Biosciences and BioResources, National Research Council of Italy
3: University of Ljubljana, Faculty of Health Sciences, Laboratory of Clinical Biophysics
4: MTA-TTK Lendület (Momentum) Glycan Biomarker Research Group, Institute of Organic Chemistry
5: HUN-REN Research Centre for Natural Sciences
6: HUN-REN - SU Peditarics and Nephrology Research Group

Text of the abstract

Introduction: Extracellular vesicles (EVs) from plant cell cultures can be advantageous over prokaryotic and mammalian cell lines as therapeutic agent or drug delivery system. The plant cell wall is a firm barrier that restricts transport processes thereby also limiting EVs production. Therefore, the aim of this study was to investigate the effect of cell wall removal on the yield and characteristics of EVs isolated from BY-2 protoplast culture, a model species in plant cell biology.
Methods: Protoplast isolation was optimized, viable protoplasts were purified, cultured, and monitored for three days based on cell counting and fluorescein diacetate viability assay. EVs were isolated from the culture supernatant using differential ultracentrifugation and resuspended in phosphate buffered saline. The protein concentration, protein profile, morphology, particle number and size distribution were determined by Qubit assay, SDS-PAGE, cryo-electron microscopy and interferometric light microscopy.
Results: In our experiment conducted in three biological replicates the average protoplast yield was 10.4% determined by cell counting. The protoplasts showed high viability, round morphology and loss of cell-cell connections during the whole course of the experiment. 10 mL of protoplast culture yielded 5.25 μg of protein and 1.11×109 particles on average with median diameters of 260.2, 272.9 and 340 nm. On the electron microscopic images of the isolated EVs, an intermediate density plasma and the surrounding phospholipid bilayer is visible.
Conclusion: We were able to obtain and culture viable protoplasts from BY-2 cell suspension culture, isolate and characterize EVs from their culture supernatant. The optimization of EVs isolation from plant resources might provide multiple opportunities both for basic and translational research fields.
Funding: European Union's Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie Staff Exchange project “FarmEVs” grant agreement N. 101131175 and the National Research, Development and Innovation Office, K-142728; EKÖP-2024-53, EKÖP-2024-160, EKÖP-2024-162 New National Excellence Program of the Ministry for Culture and Innovation; Semmelweis University, TKP2021-EGA-24; Hungarian Academy of Sciences, János Bolyai Research Scholarship.
szasz.csenge@stud.semmelweis.hu
Semmelweis University
Apor Veres-Székely, Ph.D