26. Optimizing tissue cryopreservation techniques for cryopreservation solution modifications

Abstract

The purpose of this study was to observe how minor changes to cryopreservation solutions affect the success of cryopreservation controlled-rate freeze cycles. Cryopreservation is an important technique utilized in multiple industries. In the medical industry, specifically when used in tissue preservation, there are several components critical to the success of the cryopreservation process including maintaining a constant freeze rate. A typical cooling rate appropriate for vascular tissue, as evaluated in this study, is 1 °C/min. Due to exothermic reactions occurring during phase change, a constant freeze rate is not able to be achieved unless accounted for in a controlled-rate cryopreservation program. Different additives included in cryopreservation media may drastically alter the time and temperature at which these exothermic reactions occur. Unless modifications are made to the cryopreservation freeze program, a constant freeze rate is not achievable. This study evaluated controlled-rate cryopreservation responses to cryoprotectant modifications. Minor changes to a vascular tissue control cryoprotectant containing DMSO were made to determine the effects on the controlled freezing rate. A total of six solutions were evaluated using a controlled-rate freeze program developed to maintain a 1 °C/min freeze rate for vascular tissues cryopreserved in the control cryoprotectant. Solution compositions tested included: (1) control cryoprotectant (CCP); (2) CCP+ 15 g Dextran 40; (3) CCP+15 g Dextran 40 and 1.0 g Mannitol; (4) CCP+ 9.5 g Trehalose Dihydrate; (5) CCP+ 7.3 g trehalose Duhydrate; (6) DMEM with DMSO. The freeze program utilized accommodates for the exothermic phase change occurring when the control cryoprotectant gives off heat as it changes from a liquid to a solid. Cryopreservation reference pouches were filled with each cryoprotectant. Thermocouples were inserted in each of the pouches to monitor the temperature of the solution throughout the controlled-rate freeze. During this process, graphical data reported the temperature inside the cryoprotectant pouches and cryopreservation chamber. Observations were recorded for all solutions to determine if the freeze program was able to accommodate the exothermic phase change. The two parameters assessed were linearity over the entire controlled-rate freeze duration and cryoprotectant temperature increases during the exothermic phase change. All additives resulted in a deviation from the programmed freeze rate of 1 °C/min. The control solution, Solution 1, behaved as expected having a linear, constant freeze rate of 1 °C/min with no temperature increases during the phase change. The results for Solutions 2 and 3 demonstrated that they were unable to maintain a 1 °C/min controlled-rate freeze and had visual temperature increases during the exothermic phase change. Solutions 4, 5 and 6 did not have visual temperature increases during the phase change period, but were not able to maintain a constant controlled-rate freeze of 1 °C/min. For all test solutions evaluated, minor changes to the cryoprotectant resulted in cryopreservation runs unable to maintain a constant 1 °C/min freeze. In conclusion, controlled-rate freezing programs are sensitive to changes in the cryopreservation solution. It is important that new cryopreservation freeze programs be developed to accommodate solution changes thereby ensuring optimal cryopreservation technique.