1. Cryopreservation of Human Adipose-Derived Stem Cells in Combination with Trehalose and Reversible Electroporation.
- Author
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Dovgan, Barbara, Barlič, Ariana, Knežević, Miomir, Miklavčič, Damijan, Barlič, Ariana, Knežević, Miomir, and Miklavčič, Damijan
- Subjects
CRYOPRESERVATION of cells ,STEM cell treatment ,ELECTROPORATION ,TREHALOSE ,CLINICAL medicine ,DIMETHYL sulfoxide ,ADIPOSE tissues ,CELL culture ,CELL differentiation ,CELL physiology ,CRYOPRESERVATION of organs, tissues, etc. ,CYTOLOGICAL techniques ,DISACCHARIDES ,STEM cells ,CRYOPROTECTIVE agents - Abstract
New cryopreservation approaches for medically applicable cells are of great importance in clinical medicine. Current protocols employ the use of dimethyl sulfoxide (DMSO), which is toxic to cells and causes undesirable side effects in patients, such as cardiac arrhythmias, neurological events, and others. Trehalose, a nontoxic disaccharide, has been already studied as a cryoprotectant. However, an efficient approach for loading this impermeable sugar into mammalian cells is missing. In our study, we assessed the efficiency of combining reversible electroporation and trehalose for cryopreservation of human adipose-derived stem cells. First, we determined reversible electroporation threshold by loading of propidium iodide into cells. The highest permeabilization while maintaining high cell viability was reached at 1.5 kV/cm, at 8 pulses, 100 µs, and 1 Hz. Second, cells were incubated in 250 or 400 mM trehalose and electroporated before cryopreservation. After thawing, 83.8 ± 1.8 % (mean ± SE) cell recovery was obtained at 250 mM trehalose. By using a standard freezing protocol (10 % DMSO in 90 % fetal bovine serum), cell survival after thawing was about 91.5 ± 1.6 %. We also evaluated possible effects of electroporation on cells' functionality before and after thawing. Successful cell growth and efficient adipogenic and osteogenic differentiation were achieved. In conclusion, electroporation seems to be an efficient method for loading nonpermeable trehalose into human adipose-derived stem cells, allowing long-term cryopreservation in DMSO-free and xeno-free conditions. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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