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(+) cell s) has become a useful treatment for a broad spectrum of malignant and nonm alignant diseases. The ability to cryopreserve this cell type with high eff iciency adds considerable flexibility to cord blood transplantation. The pu rpose of this study was to develop an understanding of the fundamental cryo biologic factors of these cells, including the osmotic/permeability charact eristics, and to use a theoretical approach to optimize freezing procedures . To that end, biophysical parameters, including the osmotically inactive c ell volume (V-b), hydraulic conductivity (L-p), and cryoprotectant permeabi lity coefficient (P-CPA) for DMSO and propylene glycol were measured using a modified Coulter Counter (Coulter Electronics, Inc., Hialeah, FL) at 22 d egrees C. In addition, the osmotic tolerance of PCB CD34(+) cells was asses sed using a colony-forming assay. These experimentally determined parameter s were used in a mathematical model to predict optimal cryoprotectant addit ion 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 cryo protectants used. The osmotic tolerance limits were determined to be 200 an d 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 Rubinstei n 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 cryopr otectant at ambient temperature over 5 min both to increase viability by av oiding unnecessary risks from osmotic shock and to simplify the protocol. I n addition, substitution of propylene glycol for DMSO may be of benefit dur ing the actual freezing and thawing process.