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

Area of Science:

• Biomedical Engineering
• Cell Biology
• Regenerative Medicine

Background:

• Current cryopreservation methods often rely on dimethyl sulfoxide (DMSO), a toxic cryoprotectant with adverse clinical side effects.
• Developing effective, non-toxic cryoprotective agents and delivery methods is crucial for advancing cell-based therapies.
• Trehalose, a non-toxic disaccharide, shows potential as a cryoprotectant but requires an efficient loading mechanism into mammalian cells.

Purpose of the Study:

• To assess the efficacy of combining reversible electroporation with trehalose for cryopreserving human adipose-derived stem cells (hADSCs).
• To establish optimal electroporation parameters for maximizing cell permeabilization while maintaining high viability.
• To evaluate the functionality of cryopreserved hADSCs post-thaw.

Main Methods:

• Determined reversible electroporation threshold using propidium iodide staining, identifying optimal parameters (1.5 kV/cm, 8 pulses, 100 µs, 1 Hz).
• Incubated hADSCs in trehalose (250 or 400 mM) and applied electroporation prior to cryopreservation.
• Assessed cell recovery, viability, and differentiation potential (adipogenic and osteogenic) after thawing.

Main Results:

• Electroporation at 1.5 kV/cm achieved high cell permeabilization with good viability.
• Cryopreservation with 250 mM trehalose and electroporation yielded 83.8% cell recovery.
• Cryopreservation using standard 10% DMSO resulted in approximately 91.5% cell survival.
• Electroporated and cryopreserved hADSCs retained successful growth and differentiation capabilities.

Conclusions:

• Reversible electroporation is an effective method for loading trehalose into hADSCs.
• This DMSO-free and xeno-free approach enables efficient long-term cryopreservation of hADSCs.
• The method preserves stem cell functionality, supporting potential clinical applications.