Glycerolization of the human neutrophil for cryopreservation: osmotic response of the cell.

The human neutrophil probably requires a very high intracellular concentration of cryoprotectant to avoid cellular injury during cryopreservation. Accordingly, new techniques were developed so that high concentrations of protectant could be safely used. To accomplish that, it was necessary to overcome the osmotic limitations of the neutrophil as well as problems with cytotoxicity and membrane permeability. To reduce complications from toxicity, glycerol was selected as the protective agent. To minimize osmotic stress, the protectant concentration was changed slowly and gently by a system combining cross-flow filtration and exponential gradient glycerolization. With that system, it was possible to introduce and remove high concentrations of glycerol with little loss of neutrophil viability. For both ascending and descending gradients, calculations were used to follow the changes in intracellular glycerol concentration. Similar calculations were used to determine the relationship between cell volume, gradient slope, and medium osmolality. From those data, guidelines were formulated for the glycerolization and deglycerolization of the human neutrophil.