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Cryopreservation of Human Adipose-Derived Stem Cells in Combination with Trehalose and Reversible Electroporation

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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.

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Acknowledgments

The authors would like to thank Prof. Dr. Marija Bešter Rogač and Anton Kelbl from the Faculty of Chemistry and Chemical Technology, the University of Ljubljana and Janja Dermol from Faculty of Electrical Engineering, and the University of Ljubljana for their help with the osmolality measurements. Research was supported with Slovenian Research Agency through Research Programme, Electroporation-Based Technologies and Treatments (P2-0249), and Infrastructure Programme, Network of research infrastructure centres at University of Ljubljana (IP-0510). The work was performed in the scope of LEA EBAM.

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Authors and Affiliations

  1. Educell Ltd, Prevale 9, 1236, Trzin, Slovenia

    Barbara Dovgan, Ariana Barlič & Miomir Knežević

  2. Faculty of Electrical Engineering, University of Ljubljana, Tržaška 25, 1000, Ljubljana, Slovenia

    Damijan Miklavčič

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  1. Barbara Dovgan
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Correspondence to Damijan Miklavčič.

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Dovgan, B., Barlič, A., Knežević, M. et al. Cryopreservation of Human Adipose-Derived Stem Cells in Combination with Trehalose and Reversible Electroporation. J Membrane Biol 250, 1–9 (2017). https://doi.org/10.1007/s00232-016-9916-z

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