Through Vial Impedance Spectroscopy (TVIS): A novel process analytical technology for investigating the thawing of frozen cell suspensions.

Background: Cryopreservation is often used to maintain cell functionality in a state of suspended animation by freezing the cells and holding at low temperatures (–40 °C to –196 °C) for long periods of time. However, many cell types die when they freeze because of intracellular ice formation causing cell disruption from within (Inc. gene and protein expression, etc.) and extracellular ice formation causing concentration of solutes, and osmotic shrinkage, with the potential to damage cell membranes (which then don’t recover on thawing). An effective cryopreservation protocol therefore often relies on the use of chemical agents (cryoprotectants) which impact the (a) the rate of water transport through the cell membrane; (b) ice nucleation temperatures, and (c) the formation of both extracellular and intracellular ice. Despite the recognition of the importance of ice formation (and in particular its suppression) in the role of cryoprotectants there has been no work to date that has attempted to explore the application of dielectric spectroscopy to an understanding of cryopreservation mechanisms; which is surprising given that the dielectric relaxation of ice may be measured in relatively simply experimental studies of bulk suspensions of cells. Such studies have been undertaken recently using a technique known as through-vial impedance spectroscopy, with a view to understanding the lyophilization of simple solutions. From these studies, a number of useful relaxation parameters have been identified, such as the relaxation frequency and increment in dielectric permittivity, that results from the contribution of the polarization of ice to overall measured permittivity. However, an application to the more challenging format of a cell suspension has yet to be approached. Scope, Aim and Methods: In this first ever attempt to explore the use of through vial impedance spectroscopy (TVIS) as a non-invasive PAT for characterizing the freeze-thawing living cells, two cell suspensions were chosen: An example prokaryotic cell (E. coli) and example eukaryotic cell (S. cerevisiae cell). These two cell types were suspended in two diverse types of cryoprotectant solutions: (i) 20 % sucrose with 0.15 M NaCl and (ii) peptone water. Yeast and bacterial strains were cultivated, harvested, and then re-suspended in the cryoprotectant solutions. The cell suspension was then centrifuged and resuspended in the cryoprotectant solution another 2 times (in order to wash away the culture medium and replace it with the cryoprotectant solution). Cell suspension concentrations were defined in terms of the dilution of the centrifuged cell pellet. To this end, the term % RPCS is taken to mean a % dilution by weight relative to the weight of the pelleted cell suspension. For example, 100 % RPCS means that the pellet was used without any dilution, 50 % RPCS was a cell pellet that was diluted using a weight of cryoprotectant solution equal to the weight of the pellet. After an assessment of the viability of the cells, following a freeze-thaw cycle, using a high sensitivity microplate fluorescence assay to measure cytochrome P450 activities before and after the freeze-thawing process, the primary aim of this project is to establish the methods of analysis of the impedance spectra of these systems, and what parameters might be extracted, with a future view to deploy this technique in the evaluation of cryopreservation mechanisms. 2.5 g of each cell suspension were then transferred to a set of TVIS vials, which are 10 mL glass tubing vials that have been modified with a pair of copper foil electrodes attached to the outside. The vials were then frozen to −40 °C, held for 120 min and then reheated to + 20 °C at a rate of 0.5 °C min−1. TVIS has only been applied to the study of relatively easy systems, such as pure water and solutions containing a single solute, such as sucrose, mannitol, and maltodextrin. As a result, a large portion of this thesis is devoted to the development of the techniques for analysing and presenting data from the TVIS spectra. It was anticipated that the spectra acquired for cell suspensions in more complex media would present a more challenging environment for analysis and interpretation. The final goal of the TVIS spectrum analysis was to determine whether it was possible to distinguish between intercellular processes and critical events like (i) the melting of NaCl eutectic, (ii) the glass transition temperature during the thermal cycling stage, and whether it is possible to differentiate between intercellular and extracellular glass transitions. Results: First, by using TVIS, this project looked at how low concentrations of bacteria affected electrical impedance. 50 % E. coli RPCS with 50 % w/w peptone demonstrated three main processes: the high-frequency process indicates the dielectric relaxation of ice that occurs in this process; the intermediate frequency process indicates the Maxwell-Wagner mechanism, where the polarisation of the glass wall through the conductivity of the unfrozen fraction occurs; and the low-frequency process suggests that the Maxwell-Wagner mechanism may also be present but on a smaller scale, i.e., the ice may as opposed to 50 % E. coli RPCS with 50 % sucrose-NaCl solution and 50 % S. cerevisiae RPCS with 50% peptone water and sucrose-NaCl solution, which both demonstrated two processes, high-frequency and low-frequency. Secondly, with the application of TVIS, investigation of changes in the electrical impedance of high concentration of 100 % S. cerevisiae RPCS with peptone and sucrose with NaCl showed two processes including the high-frequency process suggesting that the frozen fraction refers to dielectric relaxation of ice (MW process for the extracellular phase) and the low-frequency process suggesting that unfrozen fraction refers to MW polarization mechanism, the polarization of the interstitial spaces between ice crystals. This process mainly depends on cell type, not on the suspending media. Thirdly, the investigation of low-frequency processes revealed that sucrose-NaCl eutectic crystallisation of frozen yeast S. cerevisiae RPCS occurred between the frequencies of 10 Hz and 12 Hz at −23 °C. The concentration of NaCl inside and outside of the cell was measured as a result of temperature dependence shifting towards the low frequency. Fourthly, S. cerevisiae cell pellets treated with buffer solution before the freeze-thawing process produce high cell functionality, according to research on the effects of suspending media on E. coli and S. cerevisiae cell viability using a microplate fluorescence assay to measure cytochrome P450 activities. The outcomes of this experiment indicated that for yeast S. cerevisiae suspended in peptone water/sucrose-NaCl/buffer solution after freeze-thawing process, all cells were not viable due to osmotic stress. Interestingly, a β-dispersion in the high-frequency range was then observed through the merging of two processes identified in a 50 % S. cerevisiae RPCS with 50 % peptone water study. The critical temperature was found to be −33.3˚C, as indicated by the temperature dependency of the dielectric properties. By using the line-fit method, the extrapolated approach data at −25˚C produced 1.6066 pF (high frequency) and 1.0328 pF (low frequency), respectively. On a log frequency scale, the distance between the two peak frequencies is one unit. The CC model thus confirmed the establishment of the two peaks and their beginning to compensate one another due to a high probability of compensation between the increments in the capacitances of the two processes. Furthermore, TVIS is able to use the LOD method to determine the intracellular and extracellular glass transitions (T_g^') of yeast S. cerevisiae RPCS with sucrose-NaCl and peptone events of the freeze-thaw process inside a glass vial was demonstrated. Conclusions: This research project demonstrated the TVIS technology proved the ability to measure the intracellular and extracellular glass transitions (T_g^') of yeast S. cerevisiae RPCS with sucrose-NaCl and peptone system and failure to determine intracellular and extracellular ice formation. Those results were marginally satisfying. Furthermore, a eutectic behaviour of NaCl of a frozen solution of yeast S. cerevisiae was observed at low-frequency process. This study has exposed the mechanism of two peaks (dielectric relaxation of ice and the Maxwell-Wagner mechanism) in 50 % yeast S. cerevisiae RPCS with 50 % peptone water to determine the glass transitions (T_g^') of peptone water by using the take-off linear approach and LOD method. As such, the development of a methodology for a complex system was demonstrated.

Name dc:type.qualificationname

MPhil

Level dc:type.qualificationlevel

Masters

Grantor dc:publisher.institution

De Montfort University

Year dc:date.issued

2023

Author dc:creator

• Kaasi, Vaithianathan

Licence

• https://dora.dmu.ac.uk/bitstreams/0db35c2d-235e-407b-b22f-3884676e458b/download