Osmometric and permeability characteristics of human placental/umbilical cord blood CD34+ cells and their application to cryopreservation

The transplantation of placental/cord blood-derived HPC (e.g., CD34(+) cells) has become a useful treatment for a broad spectrum of malignant and nonmalignant diseases. The ability to cryopreserve this cell type with high efficiency adds considerable flexibility to cord blood transplantation. The purpose of this study was to develop an understanding of the fundamental cryobiologic factors of these cells, including the osmotic/permeability characteristics, and to use a theoretical approach to optimize freezing procedures. To that end, biophysical parameters, including the osmotically inactive cell volume (V-b), hydraulic conductivity (L-p), and cryoprotectant permeability coefficient (P-CPA) for DMSO and propylene glycol were measured using a modified Coulter Counter (Coulter Electronics, Inc., Hialeah, FL) at 22 degrees C. In addition, the osmotic tolerance of PCB CD34(+) cells was assessed using a colony-forming assay. These experimentally determined parameters were used in a mathematical model to predict optimal cryoprotectant addition and removal procedures. The results demonstrate a V-b, of 0.32 x V-iso, an average L-p of 0.17 +/- 0.03 (mu m/min/atm +/- SD), and a P-CPA of 0.94 +/- 0.004 or 1.0 +/- 0.004 cm/min (x10(-3)) for DMSO or propylene glycol, respectively. No significant difference was determined between the two cryoprotectants used. The osmotic tolerance limits were determined to be 200 and 600 mOsm/kg (1.29 and 0.62 x V-iso, respectively). These results indicate potential benefits of modifications to the widely used method of Rubinstein et al. Proc Natl Acad Sci USA 92:10119-10122, 1995) for cord blood CD34(+) cell cryopreservation. As opposed to Rubinstein's method in which DMSO is added to cooled cell suspensions over a 15-min interval, our data indicate that better results may be obtained by introducing and removing the cryoprotectant at ambient temperature over 5 min both to increase viability by avoiding unnecessary risks from osmotic shock and to simplify the protocol. In addition, substitution of propylene glycol for DMSO may be of benefit during the actual freezing and thawing process.