Key Topics

Cryopreservation – effect on cell quality

The first practical demonstration of the cryopreservation of living cells (spermatozoa) was performed by Polge et al in 1949. Since then the procedure has become routine in cell biology laboratories for the storage and transport of cells as well as being used in a range of applications from fertility treatment, cell therapies, drug screening and cell banking. In simple terms cryopreservation is the process whereby cells are preserved through cooling to extremely low temperatures, typically -196°C (the boiling point of liquid nitrogen). At this low temperature biological activity within the cell is arrested allowing cells to be held in stasis for long periods of time. Despite the widespread use of cryopreservation the processes involved in cell freezing are often practiced sub-optimally with little control over the “quality” of the cells following recovery. As in-vitro systems are increasingly used as therapies and/or alternatives to animal testing more importance is being directed to the development of robust approaches to cryopreservation to ensure cell viability and function are maintained.

The key to successful cryopreservation is controlling the formation of ice, both intracellular and extracellular, during the freezing process. This can be achieved through several methods the most common of which, controlled rate freezing, can be broken down into several steps (Figure 1) each of which impact cell quality following recovery. During controlled rate freezing cells are removed from cell culture and placed into a cryoprotectant solution usually formed of dimethyl sulfoxide (DMSO) or glycerol with varying levels of animal serum. The cryoprotectants are designed to permeate the cell membrane where their large molar volumes (due to high molecular weight) slow water loss from the cell during freezing as well as lowering the freezing temperature and delaying ice crystal formation. Cells are then frozen to -80^oC at a rate of ~1^oC per minute using an isopropanol jacketed freezing box. This step allows the cells to vitrify while avoiding cell injury due to intracellular and trans-membrane ice formation formed during rapid freezing processes. Next the frozen cells are transferred for storage in liquid nitrogen where the extremely low temperatures hold the cells in stasis. To recover the cells the samples are removed from liquid nitrogen and rapidly warmed to 37^oC in a waterbath; this process melts the ice in the solutions while maintaining the integrity of the cell membrane. The cells are then transferred into growth medium and placed back into cell culture.

Figure 1 – The processes involved in cell cryopreservation via controlled rate freezing. Cells are removed from culture and placed into a cryoprotectant solution; cells are control frozen at ~1^oC per minute down to -80^oC; cells are transferred for storage in liquid nitrogen. When needed cells are removed from nitrogen storage and rapidly warmed to 37^oC before being returned to cell culture.

Through the Chemical and Biological Metrology Programme project LS3 - Authenticated cell lines towards ‘The Controlled Cell’, LGC is measuring which aspects of the cryopreservation process impact most on cell quality by examining the effect of a range of cryoprotectant solutions on the viability, proliferation and function of cells frozen to -80^oC over different time periods and then stored in liquid nitrogen for periods up to 10 weeks (Figure 2). Standard methods for measuring cell “quality” following cryopreservation utilise single end point measurement to assess cell recovery and generate a snapshot of cell behaviour, this ignores many of the early processes involved in cell recovery which ultimately impact on the “fitness for purpose” of cells in downstream applications. To address this problem the LS3 project is using a unique approach to measure cell characteristics in real-time over 7 days using impedance measurements (Figure 3). This cutting edge technology uses an electrical sensor array to allow label free measurements of cell recovery, adherence and division in the cell culture environment. Using this process LGC is able to examine in detail all stages of the cryopreservation processes and assess how each step impacts the cells following liquid nitrogen storage.

This project has shown that the different methods of cryopreservation have little effect on cell viability following recovery, but cell proliferation and function are significantly affected by the time cells spend in the controlled freezing step and the amount of time they are stored in liquid nitrogen. This project is now being incorporated as part of a larger TIERed testing strategy approach to assessing cell quality and fitness for purpose based on cryopreservation, cell age (passage level), genetic stability and cell authenticity (STR fingerprinting).

Figure 3 – Label free cell measurements using impedance. (A) Baseline electronic impedance is determined by applying an electrical field across an electrode array (arrows in A) on the base of the cell culture vessel. (B) When a cell adheres to the electrode array changes in the local ionic environment at the electrode/solution interface create an impedance measurement which is detected by the system. (C) As cells divide the electrode impedance is increased allowing proliferation rates to be measured. (D) Impedance map showing differences in cell proliferation rates for different cryoprotectants.