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

Abstract

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.

Keywords: Electroporation; Human umbilical cord mesenchymal stem cells; Me(2)SO- and serum-free cryopreservation; Permeabilization; Sugars.