Cryopreservation of umbilical cord blood: 1. Osmotically inactive volume, hydraulic conductivity and permeability of CD34 + cells to dimethyl sulphoxide

Abstract

Umbilical cord blood (UCB) is an accepted treatment for the reconstitution of bone marrow function following myeloablative treatment predominantly in children and juveniles. Current cryopreservation protocols use methods established for bone marrow and peripheral blood progenitors cells that have largely been developed empirically. Such protocols can result in losses of up to 50% of the nucleated cell population: losses unacceptable for cord blood. The design of optimal cryopreservation regimes requires the development of addition and elution protocols for the chosen cryoprotectant; protocols that minimise damaging osmotic transients. The biophysical parameters necessary to model the addition and elution of dimethyl sulphoxide to and from cord blood CD34 + cells have been established. An electronic particle counting method was used to establish the volumetric response of CD34 + cells to changes in osmolality of the suspending medium. The non-osmotic volume of the cell was 0.27 of the cells isotonic volume. The permeation kinetics of CD34 + cells to water and dimethyl sulphoxide were investigated at two temperatures, +1.5 and +20 °C. Values for the hydraulic conductivity were 3.2×10 −8 and 2.8×10 −7 cm/atm/s, respectively. Values for the permeability of dimethyl sulphoxide at these temperatures were 4.2×10 −7 and 7.4×10 −6 cm/s, respectively. Clonogenic assays indicated that the ability of CD34 + cells to grow and differentiate was significantly impaired outside the limits 0.6–4× isotonic. Based on the Boyle van’t Hoff plot, the tolerable limits for cell volume excursion were therefore 45–140% of isotonic volume. The addition and elution of cryoprotectant was modelled using a two-parameter model. Current protocols for the addition of cryoprotectant based on exposure at +4 °C would require additional time for complete equilibration of the cryoprotectant. During the elution phase current protocols are likely to cause CD34 + cells to exceed tolerable limits. The addition of a short holding period during elution reduces the likelihood of this occurring.