Izolacija, vrednotenje in ugotavljanje funkcijskega potenciala zunajceličnih veziklov pridobljenih iz kultur človeških mezenhimskih matičnih/stromalnih celic in vitro

Nemčić, Laura (author), Jeras, Matjaž (mentor), Barlič, Ariana (comentor)

Zunajcelični vezikli (EV) so v zadnjih letih postali eno izmed najbolj raziskovanih orodij za uporabo v regenerativni medicini. So pomembni prenašalci signalnih molekul in na ta način sodelujejo kot posredniki medcelične komunikacije. V magistrski nalogi smo preučevali kako sestava različnih gojišč vpliva na količino, sestavo in funkcijski učinek izoliranih zunajceličnih veziklov. Človeške mezenhimske matične/ stromalne celice (MSC) smo gojili v štirih različnih medijih: v DMEM/F12 z 10 % človeškega seruma AB+, v DMEM/F12 z dodanim bFGF, ter v dveh specialnih, RoosterBio Collect in FujiFilm. Zunajcelične vezikle smo izolirali s pomočjo tangencialne pretočne filtracije (TFF) in jih nato dodatno koncentrirali še s centrifugalno filtracijo, da smo dobili še čistejše pripravke. Za analizo zunajceličnih veziklov smo uporabili metodo sledenja nanodelcem (NTA), s katero smo analizirali velikost in koncentracijo delcev v vzorcih pripravkov, vsebnost topnih proteinov smo ovrednotili s testom Bradford, njihov biološki učinek na človeško keratinocitno linijo NCTC 2544 pa smo določali s testom MTS in vitro. Najvišje koncentracije delcev smo določili v primeru uporabe medija DMEM/F12 z 10% serumom AB+, največjo vsebnost proteinov pa v vzorcih, za pripravo katerih smo uporabili DMEM/F12 z 10% serumom AB+ in FujiFilm. Ugotovili smo tudi, da so zunajcelični vezikli, pridobljeni v specialnih gojiščih RoosterBio in FujiFilm, najbolj pospešili presnovno aktivnost keratinocitov NCTC 2544. Naši rezultati kažejo, da sestava uporabljenega gojišča pomembno vpliva na količino, kakovost in funkcijski učinek EV iz MSC. Pokazali smo, da osnovna karakterizacija EV ni dovolj, saj lahko šele z ustreznimi biološkimi testi in vitro ocenimo njihov terapevtski potencial. Ugotovili smo tudi, da so EV, pridobljeni iz MSC gojenih v medijih RoosterBio in FujiFilm gojiščih, izkazali največji funkcijski potencial.

• Language: Slovenian
• Keywords: mezenhimske matične/ stromalne celice (MSC), zunajcelični vezikli (EV), izolacija in karakterizacija, funkcijski potencial
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2025
• PID: 20.500.12556/RUL-174228
• Publication date in RUL: 30.09.2025

Secondary language

• Language: English
• Title: In vitro isolation, characterisation and functional potential assessment of extracellular vesicles obtained from human mesenchymal stem/stromal cell cultures

Abstract

Extracellular vesicles (EVs) are one of the most studied tools in regenerative medicine in recent years. They play an important role as carriers of signaling molecules and act as mediators of intercellular communication. In this master’s thesis, we explored how the composition of different culture media affects the quantity, composition and functional potential of extracellular vesicles (EVs) derived from human mesenchymal stromal/stem cells (MSCs). The cells were cultured in four different media: DMEM/F12 with 10 % human serum AB+, DMEM/F12 with added bFGF, and in two special ones, RoosterBio Collect and FujiFilm. We isolated extracellular vesicles using tangential flow filtration (TFF), followed by their further concentration with centrifugal filtration to obtain purer samples. To analyze the EVs, we used nanoparticle tracking analysis (NTA) to determine the size and concentration of particles in the prepared samples. The soluble protein content was measured using the Bradford assay, and their biological effect on the human keratinocyte cell line NCTC 2544 was tested in vitro using the MTS assay. The highest concentration of particles was found in samples prepared with DMEM/F12 medium containing 10% AB+ serum, while the highest protein content was found in samples prepared using DMEM/F12 with 10% AB+ serum and FujiFilm. We also found that EVs obtained from specialized media, i.e. RoosterBio and FujiFilm, most effectively enhanced the metabolic activity of NCTC 2544 keratinocytes. Our results show that the choice of composition of the culture medium significantly influences the quantity, quality, and functional effects of EVs derived from MSCs. We showed that basic characterization is not enough, as their therapeutic potential can only be properly assessed through relevant in vitro biological assays. EVs from MSCs grown in RoosterBio and FujiFilm media exhibited the highest functional potential.

• Keywords: mesenchymal stem/ stromal cells (MSCs), extracellular vesicles (EVs), isolation and characterisation, functional potential