Loading trehalose into red blood cells by electroporation and its application in freeze-drying

Freeze-drying is one potentially ideal method for long-term storage of red blood cells (RBCs). Intracellular trehalose is believed to be an effective protectant to stabilize cells during freezing and drying. In this study, we adopted electroporation to load trehalose into human RBCs first. The effects of electroporation parameters (extracellular trehalose concentration, field strength, pulse length and frequency) on loading efficiency were studied. The results show that RBCs can be loaded with 63.7 mM trehalose at 800 mM extracellular trehalose concentration, 1.5 kV/cm,field strength, 1 ms pulse length, 4 pulses in one minute. Then, RBCs loaded with different amount of trehalose by electroporation were freeze-dried and rehydrated. Recovery rates of trehalose-loaded and freeze-dried RBCs increased with intracellular trehalose concentration. The recovery rate of RBCs loaded with 63.7 mM trehalose reached 70.9%. In conclusion, electroporation is an effective method for loading of nonpermeating trehalose into RBCs and therefore benefit freeze-drying of RBCs.