Monitoring the quality of frozen-thawed venous segments using bioimpedance spectroscopy

In order to preserve the viability and structural integrity of living cells and tissues for a long period, they are stored under very low (cryogenic) temperatures where all the chemical, biological and physical reactions in living cells would be suspended or dramatically reduced. This process is called cryopreservation (Karlsson and Toner 1996, Pegg 2007, Jang et al 2017). Cryopreservation is usually performed at −80 °C and below as traces of unfrozen solution can still be found in tissue above this temperature, and consequently the optimal duration of cryopreservation would play an important role in the prevention of any kind of damage to the cells (Mazur 1984). However, if the cryopreservation is performed at −130 °C and below then all biological activities, including biochemical reactions that can lead to cell death, would stop. Furthermore, at −196 °C (liquid nitrogen temperature) there would not be enough thermal energy to drive any type of biochemical process (Mazur 1984, Gao and Critser 2000, Woods et al 2016). Under cryogenic temperatures, background ionizing radiation (which causes photophysical reactions) could be the only cause of damage to tissues, and this is not considerable (Woods et al 2016). Thus, in temperatures lower than −130 °C the duration of cryopreservation does not play a very important role in the viability of the cells, so the cryopreservation period can range from short (Ku et al 1992, Müller-Schweinitzer 1994a) to undefined periods (Elmore et al 1991, Bujan et al 2000).

The advantage of cryopreserved cells and tissues is their constant availability for both research and clinical application, where their quality can be tested for suitability for transplantation purposes (Ibars et al 2016, Jang et al 2017). However, experimental findings have shown that the cryopreservation process can be damaging to the cells (Mazur 1963, Gao and Critser 2000). One of the methods for cryopreservation of cells and tissues is the freezing-thawing procedure (Karlsson and Toner 1996). Although storage under cryogenic temperatures is beneficial for long-term preservation of cells and tissues, the freezing and thawing processes can be detrimental to their viability (Karlsson and Toner 1996, Bia et al 2006, Müller-Schweinitzer et al 2007). However, the challenge is not only to withstand storage at high subzero temperatures but also to withstand the transition phases and to traverse the temperature range of −15°C to −60°C. By the time the tissue reaches the temperature of −15 °C, ice crystals are already formed in the extracellular matrix but the cell membrane blocks the growth of ice in the intracellular matrix. Consequently, water has started flowing out of the cells osmotically, due to its higher chemical potential in comparison to the water in the extracellular matrix. Therefore, the temperature zone in which both ice crystallization in the extracellular matrix and loss of intracellular water happen is considered a lethal zone for cells, especially when they need to traverse it twice (once during freezing and once during thawing) (Mazur 1963, Gao and Critser 2000, Woods et al 2016).

Both cooling (freezing) and warming (thawing) rates play important roles in determining the viability of cells and tissue (Pegg 2007) and should optimally result in the recovery and survival of cell and tissue structure (Karlsson and Toner 1996). The rate of temperature change controls the transport of water out of the cells during freezing and into the cells during thawing, and thus indirectly influences the concentration of the solution surrounding the cells (Mazur 1963, Pegg 2007). At low temperatures, permeability of the cell membrane to water is higher than to the corresponding solute, which makes the cell membrane semipermeable (Lynch and Diller 1981, Karlsson and Toner 1996). In other words, formation of intracellular ice is influenced by cooling rate and what happens to that ice is controlled by warming rate (Pegg 2007). Freezing rates that are too rapid or too slow can both cause damage to the cells, however the damage mechanisms are different (Gao and Critser 2000).

On the other hand, if cells are cooled too rapidly (figure 1) the intracellular water volume remains almost the same, as water does not have time to leave the cells fast enough by osmosis to maintain equilibrium (Karlsson and Toner 1996). In this way, icy crystals would be formed inside the cell (which is called 'Intracellular Ice Formation' or IIF), which can be lethal to the cells (Mazur 1963, Mazur et al 1972, Gao and Critser 2000). Therefore, damage to the cells during rapid cooling is due to IIF or osmotic rupture (Karlsson and Toner 1996).

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Changes in temperature and concentrations of intra- and extracellular solutes damage the lipid-protein complexes in the cell membrane (Lovelock 1953, Gao and Critser 2000), causing leakage and also changes in membrane permeability (Pegg 2007, Müller-Schweinitzer 2009). Therefore, an optimal cooling rate (which would be high enough to minimize the solution effect and low enough to avoid IIF) is necessary to maintain the viability and integrity of the cells and their membranes (Mazur 1963, Mazur et al 1972, Gao and Critser 2000).

Warming or thawing can be as challenging for the survival of the cells as the cooling or freezing process (Mazur 1984, Gao and Critser 2000). Studies have shown that maximum thermal stress to the cells occurs during the warming or thawing process (Zhang et al 2005, Müller-Schweinitzer 2009) as small intracellular ice crystals show different behaviors when they are treated by slow or rapid warming (Pegg 2007). Therefore, the extent to which the warming rate would affect the tissue under cryopreservation actually depends on whether the prior rate of cooling has caused cellular dehydration or intracellular ice formation (Gao and Critser 2000, Woods et al 2016).

Slow warming gives enough time for the small intracellular ice crystals to recrystallize, join and grow into harmful ice crystals (Pegg 2007). Therefore, although there is no change in the amount of ice in the cells, what causes injury is the size and shape of the ice. In addition, during slow warming the cells would be exposed to concentrated solutes for a longer period of time, which increases the chances of toxicity caused by the solutes (Woods et al 2016).

In contrast, during rapid warming (which is generally applied in a 37 °C water bath) (Bellon et al 1999) there is not enough time for the ice crystals to recrystallize, merge and grow bigger, so the ice crystals just melt (Pegg 2007). In this way, rapid warming prevents the formation of large ice crystals that can be damaging to the cells and thus helps rescue them (Mazur 1984, Gao and Critser 2000).

Previous research has shown that blood vessel cells and tissues can be seriously damaged when they are subjected to cooling and warming (freezing and thawing). This damage can range from loss of contractility in the smooth muscle layer to loss of function in endothelial cells (Müller-Schweinitzer 2009). According to some studies, storage of blood vessels at −80 °C would provide better preservation than lower temperatures such as −160 °C/−190 °C (Ku et al 1994, Müller-Schweinitzer 1994a, 1994b, Bujan et al 2000). Storage of vascular tissues in temperatures of around −80 °C for a short period of time (3–4 weeks) can preserve the structural integrity of the tissues. For example, studies have shown that the viscoelastic properties of human veins would not change during the cryopreservation period (Bia et al 2006, Müller-Schweinitzer et al 2007). However, storage under these temperatures for a long period would not guarantee the survival of mammalian cells (Müller-Schweinitzer et al 1986, Ku et al 1994). According to previous clinical research, slow warming of arteries and animal vessels (with a rate of 1 °C min⁻¹) would cause less damage to the endothelial cell layer in comparison to rapid warming (which can cause fractures in the vascular wall) (Hunt et al 1994, Wassenaar et al 1995, Pegg et al 1997, Bujan et al 2000, Pascual et al 2004, Pasquinelli et al 2006, Müller-Schweinitzer et al 2007).

It is important to evaluate the viability and structural integrity of the cells and tissues after cryopreservation (freezing-thawing cycle) and before their clinical application (such as transplantation). Some of the monitoring methods that are currently employed for this purpose include microcomputed tomography imaging (Bischof et al 2007); magnetic resonance swift imaging (Zhang et al 2014, Manuchehrabadi et al 2017); FT-IR spectroscopy (Giugliarelli et al 2016); label-free fluorescence imaging with optical fiber (Alfonso-Garcia et al 2018); light and cryomicroscopy, scanning and transmission electrode microscopy imaging (Bujan et al 2000); and general histological evaluation methods (Merdassi et al 2011). In addition there are mechanical tests such as tonometry for evaluating the biomechanical properties and functionality of tissues, such as their viscoelasticity (Bia et al 2006). However, these methods are mostly invasive and destructive to the tissues as they require segmenting the tissue into tiny rings or pieces, and also staining. Therefore, there is a need for a non-destructive method which would be able to monitor the tissue quality after its cryopreservation in a non-destructive manner.

Electrical bioimpedance measurements facilitate non-destructive miniaturized methods for real-time evaluation of the state or properties of biological materials. The bioimpedance measurement technique has been used in clinical applications to determine fluid content, electrolytic composition, cellular morphology, organ perfusion, inflammation, body composition, etc. The spatial resolution can range from molecular-size to whole-body, and can be focused by proper development of measurement setup and electrode design (Grimnes and Martinsen 2015). Structural tissue properties are usually highly detectable in a bioimpedance measurement, as are the concentration of cells and their geometry. Destruction of cell membrane and cell death are examples of detectable vital signs in a tissue (Amini et al 2018).

Therefore our hypothesis is that a potential change in the viability of the cells due to freezing/thawing will be detectable with a properly designed bioimpedance setup. Bioimpedance spectroscopy is one of the bioimpedance measurement techniques, which records the electrical impedance of a tissue over a frequency range to detect the frequency-dependent electrical properties of the tissues. As electrical properties of the tissue are related to their physiological and morphological properties, bioimpedance spectroscopy can be employed for evaluation of the tissue of interest (Dean et al 2008, Grimnes and Martinsen 2015).

The aim of the present study was therefore to employ bioimpedance spectroscopy as a non-invasive and non-destructive measurement technique, and to evaluate its ability in monitoring changes in venous segments which have undergone multiple freezing-thawing cycles.