Figure 2

Experimental design for determining key variables to optimize the cryopreservation of HUVECs.

The cells were first subjected to graded freezing vs. two-step freezing in the absence or presence of 10% DMSO. Next, the effect of two cooling rates (0.2 °C/min or 1 °C/min) on graded freezing was examined. Then, graded freezing using a 1 °C/min cooling rate was used to compare three DMSO addition procedures (see Fig. 4 caption for details). To investigate the effect of additional cryoprotectants, the DMSO addition procedure and the interrupted cooling protocol that resulted in the highest membrane integrity were used. Four cryoprotectant solutions were compared: (i) 20% DMSO, (ii) 10% DMSO plus 5% HES, (iii) 10% DMSO plus 8% HES and (iv) 10% DMSO plus 10% HES. Finally, the effect of using a lower concentration of DMSO was evaluated by comparing four cryoprotectant solutions: (i) 7% DMSO plus 7% HES, (ii) 7% DMSO plus 6% HES, (iii) 5% DMSO plus 6% HES and (iv) 3% DMSO plus 6% HES.