Effects of Non-Toxic Cryoprotective Agents on the Viability of Cord Blood Derived MNCs

The present work investigates the effects of a variety of natural cryoprotectants in combination on post-thaw viability and apoptosis of cryopreserved mononuclear cells (MNCs) derived from umbilical cord blood. The extracellular cryoprotectants (10mM) namely trehalose, hydroxyl ethyl starch, polyvinyl pyrrolidine and intracellular CPAs (5mM) like erythritol, taurine and ectoine were used to prepare different combinations of freezing medium following L9 (3⁴) Taguchi orthogonal array. Catalase, coenzyme Q10 and n-acetyl cystine (100 μg/m) were added as antioxidants. Among various combinations, freezing medium consisting of hydroxyl ethyl starch, ectoin and co-enzyme Q10 with 10% FBS is found to be most effective combination achieving maximum cell viability of 93%, 5.6% early apoptotic, 0.7% late apoptotic and 0.1% necrotic cells. SEM and phase contrast microscopy confirmed the normal cell morphology of the post-thaw cultured cells with retaining their membrane integrity. The survival rate of MNCs is higher than the rate achieved using conventional Me₂SO.