Abstract
One of the beneficial methods being widely used to preserve genetic resources of imperiled animals or improved agricultural animals is cryopreservation of germplasm. During this process, the biological structure of the cells remains relatively intact by freezing and storing them at very low temperatures. Freezing condition plays an essential role in cryopreservation procedure since the exposure of the cells to low temperature can cause damage to the cells [1]. Therefore, accurate monitoring of the freezing process can improve the recovery rate of the cells after thawing. During the phase change phenomena, the dielectric and conduction properties of ice changes [2]. Herein we present, a portable platform for monitoring of the freezing and thawing processes based on the electrical impedance of the samples during cryopreservation.
The system is composed of a portable temperature and impedance measurement system in conjunction with an impedance sensing probe fabricated with printed circuit board (PCB) technology. The impedance measurement circuit, temperature measurement circuit, control unit for data acquisition and wireless communication and touch screen LCD with a graphical user interface are the building blocks of the measurement system that enables users to perform impedance measurement at single or multiple frequencies with various excitation signals (Figure 1A). Utilizing impedance measurement instead of DC resistance measurement enable us to monitor both dielectric properties and resistance changes during phase change and avoids the polarization of the electrodes. In addition, an impedance sensing probe was developed by using PCB technology, which provides accurate electrodes fabrication at low cost. The probe has been designed to be used within 0.5 ml French straw which is a conventional container in cryopreservation application. Pair of parallel electrodes were fabricated on the probe at three detection zones (Figure 1B).
The system was evaluated at different cryopreservation conditions including various sample solutions and cooling rates. Hank's Balanced Salt Solution (HBSS) is being widely used in cryopreservation to maintain cells in a viable state. The system was characterized by measuring the impedance in HBSS and DI water solutions solution. The 0.5 ml French straws were filled sample solution along with the probe and a T-type thermocouple. Next, the samples were placed on top of the liquid nitrogen with a 2 cm gap to freeze, and after the sample temperature reached lower than -40 oC, the samples were left at room temperature to thaw. During this process, the impedance and temperature were measured inside the samples. Figures 1C and 1D show the magnitude and phase of the measured impedance for HBSS and DI water sample respectively. When the HBSS sample starts to freeze, the impedance magnitude does not change before the latent heat removal stage. However, the impedance phase starts to increase after the sample cools down. It reaches a minimum value during nucleation and increases during latent heat removal. Therefore, the phase change phenomena can be detected with the imaginary part of the impedance. In the DI water case, the impedance starts to increase by cooling down the solution when the nucleation happens there it remains relatively constant, and it keeps increasing after that. This shows that the solution containing various ions has different impedance characteristics, which can be used as a parameter to monitor the cryopreservation phenomena. The system was also characterized by different solutions containing salts and cryoprotectants, frozen at various cooling rates, to further investigate the potential of impedimetric sensors in quality control for cell freezing protocols.
Figure 1