20.500.12556/RUL-182147 Primerjava učinkovitosti izolacije primarnih celic iz perinatalnih tkiv z uporabo dveh encimskih metod Comparison of primary cell isolation efficiency from perinatal tissues with two enzymatic methods Primarne mezenhimske matične/stromalne celice (MSC) predstavljajo pomemben vir celic, ki imajo velik potencial za uporabo v regenerativni medicini, tkivnem inženirstvu, naprednih celičnih terapijah zaradi svojih proliferativnih, imunomodulatornih lastnosti ter sposobnosti diferenciacije. MSC iz perinatalnih tkiv, natančneje iz amnijske membrane, popkovnice in placente, so priročen vir MSC, saj po porodu predstavljajo biološki odpadek. Njihovo pridobivanje je neinvazivno in etično nesporno, v nasprotju z nekaterimi drugimi viri MSC. Kljub dobremu terapevtskemu potencialu MSC pa ostaja optimizacija postopkov njihove izolacije eden ključnih izzivov. Izbira metode, reagentov in pogojev izolacije ima lahko pomemben vpliv na kakovost izoliranih celic, sposobnost proliferacije in sposobnost tvorbe kolonij. Namen te magistrske naloge je primerjati učinkovitost izolacije primarnih MSC iz različnih perinatalnih tkiv z uporabo dveh encimskih metod, izolacije s kolagenazo in izolacije z mešanico encimov, ki poleg kolagenaze vsebuje tudi DNazo I in dispazo II. V raziskavo smo vključili 15 darovalk iz Kliničnega oddelka za perinatologijo Ginekološke klinike Univerzitetnega kliničnega centra Ljubljana. Od vsake porodnice smo pridobili po tri perinatalna tkiva, ki smo jih uporabili za izolacijo primarnih celic z uporabo obeh metod. Celice izolirane po obeh metodah so bile gojene v standardnih pogojih za primarne humane celične kulture. Učinkovitost izolacije smo ocenili na podlagi več parametrov, in sicer s sposobnostjo celic, da tvorijo kolonije, s časom do tripsinizacije pri pasaži 0 (p0), ter s številom tripsiniziranih celic pri p0. Na podlagi teh rezultatov smo določili število učinkovito in neučinkovito izoliranih vzorcev pri obeh metodah. Testirali smo naslednje hipoteze, 1. da je učinkovitost izolacije primarnih celic enaka pri obeh metodah, 2. da je čas do tripsinizacije pri p0 enak pri obeh metodah, ter 3. da je število tripsiniziranih celic pri p0 enako pri obeh metodah izolacije. S statistično analizo smo potrdili vse tri hipoteze. Rezultati so namreč pokazali, da se uspešnost izolacije, čas do tripsinizacije pri p0 ter število tripsiniziranih celic pri p0 med izbranima metodama statistično ne razlikujejo. Pri obeh metodah pa smo ugotovili relativno nizko učinkovitost izolacije. Na učinkovitost izolacije poleg koncentracije oz. aktivnosti encimov vplivajo še številnih drugi dejavniki kot so čas inkubacije z encimi, , čas od odvzema tkiv do izolacije celic ter drugi dejavniki, ki bi jih bilo potrebno v prihodnosti natančneje preučiti. Primary mesenchymal stromal cells (MSCs) represent an important cell source with great potential for use in regenerative medicine, tissue engineering, and advanced cell therapies due to their proliferative capacity, immunomodulatory properties, and differentiation potential. MSCs derived from perinatal tissues, like the amniotic membrane, umbilical cord, and placenta, represent a convenient source of MSCs, as these tissues are considered biological waste after childbirth. Their procurement is therefore non-invasive and ethically acceptable, in contrast to some of the other sources of MSCs. Despite the considerable therapeutic potential of MSCs, the optimization of isolation procedures remains one of the key challenges. The choice of isolation method, reagents, and conditions can significantly influence the quality of the isolated cells, their proliferative capacity, and their ability to form colonies. The aim of this master’s thesis was to compare the efficiency of primary MSC isolation from different perinatal tissues using two enzymatic methods: isolation with collagenase and isolation with an enzyme mixture containing collagenase as well as DNase I and dispase II. This research includes 15 donors from the Department of Perinatology, Division of Gynaecology and Obstetrics, University Medical Centre Ljubljana. We obtained three perinatal tissues per donor that we used for primal cell isolation using both methods. The isolated cells were cultured under standard conditions for primary human cell cultures. The efficiency of isolation was evaluated based on following three parameters: their colony-forming ability, time until trypsinization at passage 0 (p0) and with cell count of the trypsinizated cells at p0. Based on the collected data, we determined the success rate for the isolation with both methods. We tested the following hypotheses: 1. the efficiency of primary cell isolation is the same for both methods, 2. the time to trypsinization at p0 is the same for both methods and 3. the number of trypsinized cells at p0 is the same for both isolation methods. The results showed no significant differences in isolation efficiency between the two selected methods based on these parameters. For both methods, an overall low efficiency was observed for primal cell isolation. The effectiveness of the isolation is influenced by several other factors besides the enzymes concentrations or activities, including the time of incubation with enzymes, the time from tissue sampling to cell isolation, and others that should be more closely examined in the future. primarne mezenhimske matične/ stromalne celice perinatalna tkiva encimska izolacija kolagenaza DNaza dispaza II primary mesenchymal stem/stromal cells perinatal tissues enzyme isolation collagenase DNase dispase II true false false [N. Cvetko] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-04-25 08:45:05 2026-04-25 08:45:16 2026-05-05 10:55:59 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 576.5:577.15:577.08:615.36(043.2) 125244 276775171 31929.pdf 31929.pdf 1 6F07115AA4AB9DF195313594B17755EB 9c10ffccee9973fba2238d8789fb90262af20869c80917db6f7d745be7c7c078 111857b8-4072-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=233121 Fakulteta za farmacijo 0 0 0