Primerjava učinkovitosti izolacije primarnih celic iz perinatalnih tkiv glede na čas razgradnje tkiv s kolagenazo D

Krajnc, Nika (author), Zupan, Janja (mentor), Trojner Bregar, Andreja (comentor)

Mezenhimske matične/stromalne celice so heterogena populacija multipotentnih celic, ki imajo zaradi svojih proliferacijskih, diferenciacijskih in imunomodulatornih lastnosti velik potencial za uporabo v regenerativni medicini in celičnih terapijah. Pomemben vir teh celic predstavljajo perinatalna tkiva, saj so po porodu enostavno dostopna in se običajno zavržejo kot medicinski odpadek, njihovo pridobivanje pa ne zahteva invazivnih posegov v darovalca. Posledično je njihova uporaba povezana z bistveno manj etičnimi zadržki kot uporaba embrionalnih matičnih celic. Kljub njihovemu terapevtskemu potencialu ostaja optimizacija postopkov izolacije primarnih celic iz teh tkiv pomemben izziv, saj lahko različni pogoji encimske razgradnje vplivajo na uspešnost pridobivanja celic. Namen magistrske naloge je bil primerjati učinkovitost izolacije primarnih celic iz amnijske membrane, placente in popkovnice glede na čas razgradnje tkiva s kolagenazo D. Primerjali smo 90-minutno in 24-urno razgradnjo tkiva pri 49 vzorcih. Po izolaciji smo celice gojili v pogojih in vitro ter s fazno-kontrastnim mikroskopom spremljali njihovo pritrditev na plastično podlago, rast in nastanek kolonij. Učinkovitost izolacije smo ovrednotili glede na uspešnost izolacije, število tripsiniziranih celic in sposobnost tvorbe fibroblastnih kolonijskih enot pri pasaži 0. Rezultati so pokazali, da uspešnost izolacije primarnih celic med 90-minutno in 24-urno razgradnjo tkiva ni statistično različna. Prav tako med skupinama nismo ugotovili statistično značilne razlike v številu tripsiniziranih celic pri pasaži 0, čeprav je bilo njihovo število pri 90-minutni razgradnji večje. Tudi sposobnost tvorbe fibroblastnih kolonijskih enot se med skupinama ni statistično značilno razlikovala, kljub višjim vrednostim pri 24-urni razgradnji. Na podlagi rezultatov ugotavljamo, da čas encimske razgradnje v uporabljenih eksperimentalnih pogojih ni bil povezan s statistično značilnimi razlikami v učinkovitosti izolacije primarnih celic. Ker smo med posameznimi tkivnimi vzorci opazili precejšno variabilnost, bi bilo v prihodnjih raziskavah smiselno vključiti večje število vzorcev ter podrobneje ovrednotiti vpliv vrste perinatalnega tkiva in drugih dejavnikov, ki lahko vplivajo na izolacijski postopek.

• Language: Slovenian
• Keywords: mezenhimske matične/stromalne celice, perinatalna tkiva, kolagenaza D, encimska izolacija, sposobnost tvorbe kolonij
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FFA - Faculty of Pharmacy
• Publisher: [N. Krajnc]
• Year: 2026
• PID: 20.500.12556/RUL-190900
• UDC: 615:57(043.2)
• COBISS.SI-ID: 304797699
• Publication date in RUL: 06.11.2026

Secondary language

• Language: English
• Title: Comparison of the isolation efficiency of perinatal tissues-derived primary cells according to tissue digestion time with collagenase D

Abstract

Mesenchymal stem/stromal cells are a heterogeneous population of multipotent cells with considerable potential for use in regenerative medicine and cell-based therapies due to their proliferative, differentiative and immunomodulatory properties. Perinatal tissues represent an important source of these cells, as they are readily accessible after delivery, usually discarded as medical waste and can be collected without invasive procedures involving the donor. Consequently, their use is associated with substantially fewer ethical concerns than the use of embryonic stem cells. Despite their therapeutic potential, optimization of primary cell isolation procedures from these tissues remains an important challenge, as different conditions of enzymatic tissue digestion may affect the efficiency of cell isolation. The aim of this master's thesis was to compare the efficiency of primary cell isolation from the amniotic membrane, placenta and umbilical cord according to tissue digestion time with collagenase D. We compared 90-minute and 24-hour tissue digestion in 49 samples. Following isolation, the cells were cultured under in vitro conditions, and their plastic attachment, growth and colony formation were monitored by phase-contrast microscopy. Isolation efficiency was evaluated based on isolation success, the number of trypsinized cells and colony-forming unit-fibroblast activity at passage 0. The results showed that primary cell isolation success did not differ significantly between 90-minute and 24-hour tissue digestion periods. Similarly, no statistically significant difference in the number of trypsinized cells at passage 0 was observed between the groups, although the number was higher after the 90-minute digestion period. The colony-forming unit-fibroblast values also did not differ significantly between the groups, despite higher values following the 24-hour digestion period. Based on the obtained results, we conclude that, under the experimental conditions used, the duration of enzymatic digestion was not associated with statistically significant differences in the efficiency of primary cell isolation. Considerable variability was observed among individual tissue samples. Therefore, future studies should include a larger number of samples and evaluate in greater detail the influence of the type of perinatal tissue and other factors that may affect the isolation procedure.

• Keywords: mesenchymal stem/stromal cells, perinatal tissues, collagenase D, enzymatic isolation, colony-forming capacity