Cryopreservation of Human Umbilical Stem Cells in Combination with Trehalose and Reversible Electroporation

Abstract

Cryopreservation is the most effective method for long-term storage of cells. Cryoprotectant solution usually contains 10% dimethyl sulfoxide and fetal bovine serum. Both represent a limiting factor for clinical applications, due cytotoxicity and presence of animal proteins. We have developed an improved method for cryopreservation of human umbilical cord-mesenchymal stem cells using trehalose in combination with reversible electroporation. First, we demonstrated efficient loading of propidium iodide into cells by reversible electroporation. Propidium iodide (100 μg/ml) was loaded into cells at 0, 100, 180, 300, 650 V, 8 pulses, 100 μs and 1 Hz in 2 mm cuvettes. The highest permeabilization while maintaining high cell viability was reached at 350 V. Second, cells were treated with a combination of 100 mM trehalose and electroporation at 0, 220, 430, 610 V, 8 pulses, 100 μs and 1 Hz, before cryopreservation. After thawing, the highest viability of 61 % was obtained at 430 V. As a control standard freezing protocol with 10 % dimethyl sulfoxide in 90% fetal bovine serum (v:v) was preformed, where 85 % after thawing recovery was obtained. In conclusion, according to our preliminary study electroporation seems an efficient method for loading nonpermeable trehalose into human cells and enables successful cryopreservation of human umbilical cord-mesenchymal stem cells.