Kryoprezervácia buniek je jednou z hlavných výziev súčasnej kryobiológie, najmä s ohľadom na progresívne rastúcu oblasť tkanivového inžinierstva. Spôsob kryoprezervácie môže výrazne ovplyvniť kvalitu buniek po rozmrazení. Z tohto dôvodu je potrebné zaviesť protokoly, ktoré zabezpečia dobrý fyziologický a adherentný stav zmrazovaných buniek, pretože ich efektívna regenerácia vedie k vysokej aplikačnej účinnosti v bunkových terapiách. Úspešná kryoprezervácia si vyžaduje starostlivé zváženie niekoľkých kľúčových faktorov vrátane výberu kryoprotektívneho činidla (CPA) a jeho koncentrácie.
Predkladaná práca sa zaoberá štúdiom efektu kryoprezervácie buniek s rôznymi nízkymi koncentráciami kryoprotektívnej látky dimetylsulfoxid (DMSO). Je známe, že DMSO je schopný zmierniť poškodenie buniek súvisiace so zmrazovaním počas pomalého ochladzovania, ale jeho účinky na bunkovú biológiu sú stále predmetom diskusie.
Preskúmali sme kryoprezerváciu ľudských keratinocytov s použitím štandardných a nižších dávok DMSO v zmrazovacej zmesi, ktoré minimalizujú jeho toxické účinky, ale ešte si zachovajú kryoprotektívne účinky. Bunky keratinocytov sme zmrazovali s (1,8 %; 2,2 %; 5 % a 10 %, v/v) koncentráciami DMSO a krátkodobo (počas troch dní) ich uchovávali pri teplote ̶ 80 °C. Po rozmrazení sme ich porovnali s nezmrazenými bunkami. Zamerali sme sa na viabilitu a proliferáciu buniek po rozmrazení a analyzovali sme zmenu ich morfológie a ultraštruktúry spôsobenú vplyvom kryoprezervácie. Skúmali sme vplyv DMSO na vybrané vlastnosti buniek v závislosti od jeho koncentrácie a zvažovali sme postupy na zníženie jeho toxických účinkov. Preukázali sme, že DMSO aj v nižších koncentráciách (do 2,2 %) môže ochrániť keratinocyty počas kryoprezervácie a krátkodobého skladovania pri nízkej teplote. Koncentrácia DMSO nižšia ako 2,2% už nemusí mať dostatočný kryoprotektívny účinok, pretože boli preukázané rozdiely v štrukturálnych a dynamických vlastnostiach zmrazovacej zmesi, ktoré spôsobujú, že bunky po rozmrazení podliehajú apoptóze. Tieto zistenia by mohli mať prínos v optimalizácii metódy kryoprezervácie kožných buniek pre biotechnológie aj základný výskum.
Táto práca bola vypracovaná za podpory projektov APVV-SK-BY-RD-19-0019 a APV-SK-PL-21-0073, NAWA EUROPARTNER, Polish National Agency for Academic Exchange (2019-2022) a KEGA 041UK-4/2020 (2020-2022).
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