A study of the separate effects of influence factors and their coupled interactions on cryoinjury of human erythrocytes

The separate effects of five influence factors and their coupled interactions on cryoinjury of human erythrocytes were investigated experimentally and statistically. The five factors, each having three levels, were as follows: (1) cooling rate: −0.5, −140, and −800 °C/min; (2) warming rate: +0.5, +25, and +200 °C/min; (3) hematocrit: 2, 11, and 60%; (4) concentration of cryoprotectant (glycerol): 1, 2, and 4 M in PBS; and (5) holding temperature at which the frozen samples were kept: no hold, −75 °C for 1.5 hr, and −196 °C for 1.5 hr. Twenty-seven special tests, which were chosen from the 243 possible tests by using the Fractional Factorial Design Technique, an optimum seeking technique, were performed. The conclusions are: (1) the cooling rate is the most significant or sensitive factor causing cryoinjury to the cells; (2) the main effects of the hematocrit and the concentration of cryoprotectant, the interaction between the cooling rate and the warming rate, and the interaction between the cooling rate and the concentration of cryoprotectant are next most significant; (3) the main effect of warming rate, and the interaction between the holding temperature and the cooling rate are less significant; (4) the holding temperature below −75 °C, and the remaining interactions between two factors are relatively not significant; and (5) in the present study, the optimal combination of the five factors for the survival of the cells is: cooling at −0.5 °C/min, warming at +0.5 °C/min, hematocrit at 11%, glycerol concentration at 4 M in PBS, and holding temperature below −75 °C.