Krioprezervacija humanih mezenhimskih matičnih celic in tkiva popkovnice v kombinaciji krioprotektanta trehaloze in reverzibilne elektroporacije : doktorska disertacija

Dovgan, Barbara (author), Miklavčič, Damijan (mentor), Barlič, Ariana (comentor)

Trehaloza je nestrupen disaharid, ki velja za zelo obetaven krioprotektant za krioprezervacijo celičnih produktov, namenjenih zdravljenju pacientov. V naši raziskavi smo preverjali učinkovitost kombinacije reverzibilne elektroporacije v prisotnosti različnih koncentracij trehaloze na zamrzovanje dveh tipov humanih matičnih celic: matičnih celic iz maščobnega tkiva (ASC) in matičnih celic iz tkiva popkovnice (UC-MSC). Z določanjem permeabilizacije membrane in viabilnosti celic smo določili vrednost 1,5 kV/cm kot najbolj primerno jakost električnega polja za učinkovit vnos trehaloze. Celice smo nato elektroporirali v prisotnosti različnih koncentracij trehaloze ter ugotovili, da je za uspešno krioprezervacijo, ki je primerljiva s standardnim postopkom z 10 % dimetil sulfoksidom (DMSO), potrebna vsaj 250 mM trehaloza. Pri zamrzovanju celic brez elektroporacije v prisotnosti 250 mM trehaloze smo dosegli le 10 % nižje vrednosti viabilnosti, ki pa niso bile statistično različne. Rezultati določanja znotrajcelične koncentracije trehaloze kažejo, da je za uspešno krioprezervacijo dovolj okrog 30–40 mM znotrajcelične koncentracije trehaloze. S preverjanjem celične morfologije, celične diferenciacije, izražanja stresnih genov ter izražanja genov povezanih z imuno-aktivacijo smo ocenili ali elektroporacija in krioprezervacija vplivata na celice. Ugotovili smo, da elektroporacija in krioprezervacija ne vplivata na celično morfologijo in sposobnost diferenciacije, saj so se vse testirane celice diferencirale tako v adipogeno kot osteogeno linijo. Pri testiranju celic na izražanje stresnega odziva smo ugotovili, da se celice na elektroporacijo in krioprezervacijo odzovejo s povišanjem izražanja nekaterih stresnih genov sod2 in hspa1a. Kljub temu pa so še vedno sposobne izražati imunomodulacijske gene, ido1, tsg6 in il6, ki jih povzroči simulirano vnetno okolje. Celice ASC in UC-MSC zamrznjeni v prisotnosti trehaloze tako izražajo podobne lastnosti kot celice zamrznjene z DMSO, trehaloza pa ne predstavlja dodatnega tveganja za paciente pri uporabi zamrznjenega celičnega produkta. Poskus zamrznitve tkiva popkovnice v prisotnosti 250 mM trehaloze in elektroporacije je bil neuspešen, medtem ko je bila zamrznitev tkiva popkovnice z 10 % DMSO uspešna.

• Language: Slovenian
• Keywords: trehaloza, krioprezervacija, elektroporacija, DMSO, humane mezenhimske matične celice, ASC, UC-MSC
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Year: 2020
• PID: 20.500.12556/RUL-124425
• COBISS.SI-ID: 49749763
• Publication date in RUL: 21.01.2021

Secondary language

• Language: English
• Title: Cryopreservation of human mesenchymal stem cells and umbilical cord tissues with combination of cryoprotectant trehalose and reversibile electroporation : doctoral dissertation

Abstract

Trehalose, a nontoxic disaccharide, is a promising cryoprotective agent for medically applicable cells. In our study, the efficiency of combining reversible electroporation and trehalose for cryopreservation of two human mesenchymal stem cell types; adipose-derived stem cells (ASC) and umbilical cord-derived stem cells (UC-MSC) was assessed. By optimization of cell permeabilization and viability parameters, 1.5 kV/cm of electric field was determined to successfully introduced trehalose into the cells. Cells were then electroporated in the presence of different extracellular trehalose concentrations. 250 mM extracellular trehalose was determined to be needed for successful cell cryopreservation that is comparable to results obtained by standard dimethyl sulfoxide (DMSO) cryopreservation protocol. Surprisingly, comparable viabilities were obtained in samples treated with or without electroporation in the presence of high extracellular trehalose concentration, where only 10 % lower viability was obtained in non-electroporated cells. At 250 mM extracellular trehalose concentration of about 30–40 mM intracellular trehalose concnetration was shown to be sufficient for successful cryopreservation of the cells. To evaluate the impact of electroporation and cryopreservation on cells, cell morphology, cell differentiation, stress response and immune-activation related gene expressions were analyzed. Electroporation and cryopreservation did not affect cell morphology or cell differentiation since all tested samples were capable of adipogenic and osteogenic differentiation. Despite increased stress response, capacity of ASC and UC-MSC to highly up-regulate immunomodulatory genes in simulated inflammatory environment was not affected. ASC and UC-MSC cryopreserved in trehalose exhibited comparable characteristics to DMSO cryopreserved cells. Importantly, in comparison to DMSO, trehalose does not represent an additional risk for the patient upon cryopreserved cell product administration. Cryopreservation of umbilical cord tissue using electroporation in the presence of 250 mM trehalose was not successful. However, umbilical cord tissue was successfully cryopreserved using 10% DMSO.

• Keywords: trehalose, cryopreservation, electroporation, DMSO, human mesenchymal stem cells, ASC, UC-MSC