TY - JOUR

T1 - Me2SO- and serum-free cryopreservation of human umbilical cord mesenchymal stem cells using electroporation-assisted delivery of sugars

AU - Mutsenko, Vitalii

AU - Barlič, Ariana

AU - Pezić, Tamara

AU - Dermol-Černe, Janja

AU - Dovgan, Barbara

AU - Sydykov, Bulat

AU - Wolkers, Willem F.

AU - Katkov, Igor I.

AU - Glasmacher, Birgit

AU - Miklavčič, Damijan

AU - Gryshkov, Oleksandr

N1 - Funding Information: This work was supported by the German Research Foundation through the Cluster of Excellence REBIRTH ( EXC 62/1 ), DAAD projects Eastern Partnership ( 54364768 ) and IP@Leibniz ( 57156199 ) as well as a PostDoc grant "Ways to Research II" of Leibniz University Hannover ( 60442522 ). This work was partially performed within the network of research and infrastructural centres of University of Ljubljana , which is financially supported by Slovenian Research Agency through infrastructural grant IP-0510 and P2-0249 .

PY - 2019/12

Y1 - 2019/12

N2 - Cryopreservation is the universal technology used to enable long-term storage and continuous availability of cell stocks and tissues for regenerative medicine demands. The main components of standard freezing media are dimethyl sulfoxide (hereinafter Me2SO) and fetal bovine serum (FBS). However, for manufacturing of cells and tissue-engineered products in accordance with the principles of Good Manufacturing Practice (GMP), current considerations in regenerative medicine suggest development of Me2SO- and serum-free biopreservation strategies due to safety concerns over Me2SO-induced side effects and immunogenicity of animal serum. In this work, the effect of electroporation-assisted pre-freeze delivery of sucrose, trehalose and raffinose into human umbilical cord mesenchymal stem cells (hUCMSCs) on their post-thaw survival was investigated. The optimal strength of electric field at 8 pulses with 100 μs duration and 1 Hz pulse repetition frequency was determined to be 1.5 kV/cm from permeabilization (propidium iodide uptake) vs. cell recovery data (resazurin reduction assay). Using sugars as sole cryoprotectants with electroporation, concentration-dependent increase in cell survival was observed. Irrespective of sugar type, the highest cell survival (up to 80%) was achieved at 400 mM extracellular concentration and electroporation. Cell freezing without electroporation yielded significantly lower survival rates. In the optimal scenario, cells were able to attach 24 h after thawing demonstrating characteristic shape and sugar-loaded vacuoles. Application of 10% Me2SO/90% FBS as a positive control provided cell survival exceeding 90%. Next, high glass transition temperatures determined for optimal concentrations of sugars by differential scanning calorimetry (DSC) suggest the possibility to store samples at −80 °C. In summary, using electroporation to incorporate cryoprotective sugars into cells is an effective strategy towards Me2SO- and serum-free cryopreservation and may pave the way for further progress in establishing clinically safe biopreservation strategies for efficient long-term biobanking of cells.

AB - Cryopreservation is the universal technology used to enable long-term storage and continuous availability of cell stocks and tissues for regenerative medicine demands. The main components of standard freezing media are dimethyl sulfoxide (hereinafter Me2SO) and fetal bovine serum (FBS). However, for manufacturing of cells and tissue-engineered products in accordance with the principles of Good Manufacturing Practice (GMP), current considerations in regenerative medicine suggest development of Me2SO- and serum-free biopreservation strategies due to safety concerns over Me2SO-induced side effects and immunogenicity of animal serum. In this work, the effect of electroporation-assisted pre-freeze delivery of sucrose, trehalose and raffinose into human umbilical cord mesenchymal stem cells (hUCMSCs) on their post-thaw survival was investigated. The optimal strength of electric field at 8 pulses with 100 μs duration and 1 Hz pulse repetition frequency was determined to be 1.5 kV/cm from permeabilization (propidium iodide uptake) vs. cell recovery data (resazurin reduction assay). Using sugars as sole cryoprotectants with electroporation, concentration-dependent increase in cell survival was observed. Irrespective of sugar type, the highest cell survival (up to 80%) was achieved at 400 mM extracellular concentration and electroporation. Cell freezing without electroporation yielded significantly lower survival rates. In the optimal scenario, cells were able to attach 24 h after thawing demonstrating characteristic shape and sugar-loaded vacuoles. Application of 10% Me2SO/90% FBS as a positive control provided cell survival exceeding 90%. Next, high glass transition temperatures determined for optimal concentrations of sugars by differential scanning calorimetry (DSC) suggest the possibility to store samples at −80 °C. In summary, using electroporation to incorporate cryoprotective sugars into cells is an effective strategy towards Me2SO- and serum-free cryopreservation and may pave the way for further progress in establishing clinically safe biopreservation strategies for efficient long-term biobanking of cells.

KW - Electroporation

KW - Human umbilical cord mesenchymal stem cells

KW - MeSO- and serum-free cryopreservation

KW - Permeabilization

KW - Sugars

UR - http://www.scopus.com/inward/record.url?scp=85073528948&partnerID=8YFLogxK

U2 - 10.1016/j.cryobiol.2019.10.002

DO - 10.1016/j.cryobiol.2019.10.002

M3 - Article

C2 - 31593692

AN - SCOPUS:85073528948

VL - 91

SP - 104

EP - 114

JO - Cryobiology

JF - Cryobiology

SN - 0011-2240

ER -