Analysis of the osmotic behaviour for optimal cryopreservation of hMSCs from UCB

In this work, the osmotic behavior for optimal cryopreservation of the human Mesenchymal Stem Cells (hMSCs) from Umbilical Cord Blood (UCB) of three different donors, after isolation and characterization, was investigated. The osmotic properties, i.e., namely inactive cell volume, water and cryo-protectant dimethyl sulfoxide (Me2SO) permeabilities, were evaluated by direct comparison to experimental measurements obtained at three different temperatures under hypertonic conditions (obtained adding sucrose or Me2SO to PBS solutions), and returning back to isotonic conditions. Cell size was determined using an impedance measurement device (Coulter Counter), under both equilibrium and dynamic conditions, while the classic 2-parameters bi-compartimental model was adopted for the linear and non-linear regression analyses. It was found that the inactive volume fraction of hMSCs from UCB apparently increase during swelling, thus limiting the positive excursions of cell volume. This happens either during the swelling phase of the shrink-swell dynamics during Me2SO addition, or during the return back to isotonic conditions after equilibration with a hypertonic solution of the impermeant or permeant solute. Therefore, this cell lineage does not behave as a perfect osmometer and a peculiar cell volume control system needs to be considered to model the osmotic phenomena taking place during its cryopreservation. Source of Funding (if any) Regione Autonoma della Sardegna (RAS) - Progetto L.7/2007 RAS, Bando 2012, CUP F71J12000900002