Unloading of cryoprotectants from cryoprotectant-loaded cells on a microfluidic platform.

In this paper, a multistep dilution-filtration microdevice (MDFD) is developed for unloading cryoprotectants from cryoprotectant-loaded cells. The MDFD contained a diluent producing region, a dilution-filtration execution region, and a filtrate collection region. It was made of two patterned PMMA stamps with four pieces of sandwiched PVDF membranes. Firstly, the performances of the mixers that were used in the diluent producing region and the dilution-filtration execution region were assessed using fluorescence experiments. Then, the effect of the MDFD structure on the loss of cells was investigated by applying the MDFD to unload glycerin from glycerin-loaded porcine red blood cells. Finally, the effects of the cell density, glycerin concentration, and membrane pore size on the clearance efficiency of glycerin (C G ), the survival rate of cells (S C ) and the recovery rate of cells (R C ) have been studied. Under the designed conditions, C G achieved ~80% and S C reached ~90%. However, R C was only ~40%, mainly resulting from the cells detained on the membrane surface and squeezed through the membrane pores into the filtrate. Increasing the membrane pore size caused high C G and S C , but low R C . For a low glycerin concentration, C G , S C , and R C were all high. For a high cell density, C G was high, but both S C and R C were low. This work is of significance to develop a microfluidic chip for unloading cryoprotectants from a small amount of cryopreserved cell samples.