Cryopreservation of Red-Blood-Cell-Derived Extracellular Vesicles.

Area of Science:

• Extracellular vesicle (EV) research
• Biotechnology and biomaterials

Background:

• Extracellular vesicles (EVs) are vital in biological processes and explored for diagnostics and therapeutics.
• Red-blood-cell-derived EVs (REVs) show promise for drug delivery but face storage challenges due to their composition.
• Existing EV cryopreservation methods need optimization for REVs.

Purpose of the Study:

• To identify optimal cryopreservation conditions for REVs.
• To evaluate the impact of vesicle concentration and buffer composition on REV stability.

Main Methods:

• Investigated cryopreservation of REVs under varying buffer compositions and initial concentrations.
• Analyzed changes in protein/lipid content, hemoglobin retention, particle recovery, size, and optical properties.
• Compared standard phosphate-buffered saline (PBS) with a novel HEPES, HSA, and trehalose (HAT) buffer system (PBS-HAT).

Main Results:

• Phosphate-buffered saline (PBS) resulted in significant REV loss and compositional changes.
• The PBS-HAT buffer effectively preserved REV biochemical and biophysical integrity.
• Initial REV concentration impacted particle recovery, with losses observed at 1.2 × 10¹¹ particles/mL.

Conclusions:

• Phosphate-buffered saline (PBS) is unsuitable for REV cryopreservation.
• The PBS-HAT buffer system provides effective short- to midterm cryopreservation for REVs.
• Optimal cryopreservation of REVs requires initial concentrations above 10¹² particles/mL.