Temperature dependence of the protective effect of pressurized dissolution of xenon gas during cold storage of cells

Abstract

Background

The preservation period afforded by cold storage of cells is short. However, the use of rare gases for cold storage as a means of extending the period of preservation would be highly beneficial.

Objective

To examine the effect of temperature on the protective effect of cold storage of cells using pressurized dissolution of xenon gas, with particular focus on the inhibition of substance transport by viscosity.

Methods

Human dermal fibroblast monolayers incubated in a culture dish for 48 h were used as a test sample, with culture medium used as a preservation solution. Samples were placed into a pressure-resistant vessel, which was pressurized with xenon gas at 0 or 0.5 MPa, and cells were stored at 0 to 5°C for 18 h. Cell activity was evaluated by tetrazolium salt assay. The viscosity of the medium under pressurization at each storage temperature was estimated.

Results

The maximum protective effect against cell damage of cold storage with pressurized dissolution of xenon gas was observed at 4°C. An increase in estimated viscosity by pressurization was correlated with increased cell activity at 4°C.

Conclusion

Analysis of the temperature dependence of the protective effect against cell damage of cold storage with pressurized dissolution of xenon gas revealed that the most effective temperature is 4°C. The data also suggest that increased viscosity due to pressurization plays a role in the protective effect.

Keywords

Cold storage of cells pressurized dissolution of xenon gas protective effect

1. Introduction

Currently, semipermanent preservation by freezing is possible for many kinds of cells and some biological tissues, such as blood vessels, corneas, etc. [1–4]. However, the optimal cryopreservation conditions for other cell types and tissues that cannot bear the stress of freezing or toxicity associated with protective agents remain unclear. Therefore, cells, tissues, and organs that cannot be frozen are often stored in the cold at a temperature of 0–4°C. However, the limit for cold storage of many such cell and tissue types ranges from 12 h to 2 weeks, and the limit for cold storage of organs ranges from only 4 h to 2 days. Considering supply and demand balance, longer preservation periods would be more desirable. Therefore, an effective method for extending the period over which samples can be preserved by cold storage is needed.

Ensuring cell survival and function during cold storage requires control of the rate of chemical reactions essential to metabolism and the prevention of imbalances in electrolytes due to interruption of active transport that occurs below ∽5°C. During cold storage, metabolism can be controlled to some extent by the temperature decline. However, although optimal compositions of storage solutions have been reported (e.g., University of Wisconsin Solution, ET-Kyoto Solution, Celsior solution [5,6]), conventional preservation solutions remain insufficient for preventing substance imbalances [7].

We therefore evaluated the effect of pressurized dissolution of a hydrophobic gas in the sample preservation solution as a new method for reducing the likelihood of substance imbalances during cold storage. We hypothesized that the physical transport of substances could be controlled both intra- and extracellularly by temporarily increasing the viscosity only at the time of preservation to restrain water (liquid state), thereby preventing the generation of hydrates (solid state). Using this approach, we explored the possibility of extending the cold storage preservation period for cells. For this purpose, we used xenon (Xe) gas, which has the highest solubility in water among hydrophobic gases and is biologically safe.

In this study, we aimed to clarify the temperature dependence of the protective effect against cell damage from cold storage using pressurized dissolution of Xe gas. We also investigated the mechanism of the effect, with particular focus on the inhibition of substance transport due to increased viscosity.

2. Materials and methods

2.1. Experimental samples

Samples were prepared using human dermal fibroblast (Cell Systems [Kirkland, WA, USA] Fb Cells, CSC-2F0) monolayers. Dulbecco's Modified Eagle Medium (DMEM; Life Technologies [Waltham, MA, USA] Gibco 12320-032) with 10% fetal bovine serum (FBS; Gibco 26140 lot no. 1215196) and 1% antibiotics (Gibco Antibiotic-Anti-mycotic 15240-062) was used as the cell culture medium. A 2-ml suspension containing 4.5 × 10⁴ cells was added to a culture dish (Corning [New York, NY, USA] 3294, 35-mm diameter), and the cells were cultured in an incubator (Panasonic [Tokyo, Japan] MCO-19AIC-PJ; 37°C, 5% CO₂) for 24 h. Duplicate samples were prepared and left untreated as controls.

2.2. Evaluation of cell activity after cold storage

Cell activity was assessed using a water-soluble tetrazolium salt assay (Dojindo [Kumamoto, Japan] WST-8) by measuring the quantity of formazan produced by mitochondrial enzymatic activity using a spectrophotometer (GE Healthcare [Pittsburgh, PA, USA] Gene Quant 1300). WST-8 was added to the sample, the sample was incubated for 2 h (37°C, 5% CO₂), and then the absorbance of the sample supernatant was measured. Relative cell activity was calculated from the sample absorbance divided by the absorbance of the control sample. It has been reported that mitochondria can be damaged during cold storage, leading to cell death [8]; therefore, to assess both cell viability and function, we used the WST-8 assay that evaluates mitochondrial dehydrogenase activity [9].

2.3. Experimental device

The experimental device for pressurized dissolution of Xe gas is shown in Fig. 1. A pressure-resistant vessel (Taiatsu Techno [Tokyo, Japan] TVS-1; internal diameter of 45 mm, 60 mm high, internal volume of approximately 100 ml; pressure-resistant up to 10 MPa) made of stainless steel (SUS316) was used for experiments involving pressurized Xe gas (Tokyo Rare Gas [Tokyo, Japan]; 99.995% purity). Four experimental samples (in 35-mm culture dishes) could be placed in the vessel simultaneously. The vessel was connected to an extension pipe made of stainless steel, which in turn was connected to a pressure regulator attached to a Xe gas cylinder. The device was immersed in a water-filled reservoir made of polystyrene foam and maintained at 4°C using a temperature control water circulator (Yamato [Tokyo, Japan] CTW-801S). The samples in the vessel were pressurized with Xe gas using the cylinder regulator.

Fig. 1.

Experimental system for pressurized dissolution of Xe gas.

2.4. Effect of pressurized Xe gas and temperature dependence of cell activity after cold storage

Four samples were removed from the incubator (37°C, 5% CO₂), the DMEM was removed, and then the cells were washed with 1 ml of Hanks' solution. Next, 1 ml of DMEM without phenol red was added to each sample. The samples were stacked, placed in the vessel, and exposed to Xe gas by pressurization at 0.5 MPa under air at ambient pressure (i.e., Xe pressurization sample). Samples in ambient air were also prepared to provide cell activity reference samples and placed in another vessel (i.e., 0 MPa, non-pressurization sample). All samples in vessels were stored in a refrigerator (Panasonic [Tokyo, Japan] MIR-254-PJ) at 0, 2, 3, 4, or 5°C for 18 h.

The test and control sample vessels were removed from the refrigerator at the end of the storage period. The Xe gas was gradually decompressed from the test samples, and the samples were removed from the vessel. The DMEM was removed, the samples were washed with Hanks' solution, 1 ml of DMEM was added, and then the samples were incubated for 2 h (37°C, 5% CO₂). Next, the sample medium was removed, 1 ml of DMEM and 100 μl of WST-8 were added, and the samples were further incubated for 1 h. The absorbance of the supernatant of each sample was measured spectrophotometrically (wavelength: 450 nm). Control samples were prepared by adding DMEM to samples that were not subsequently pressurized with Xe gas; the control samples were incubated at 4°C for 15 min, and then cell activity was evaluated in the same manner as for the experimental samples.

A pressure setting of 0.5 MPa was chosen as the optimal pressure based on the results of preliminary experiments [10] (i.e., 0.5 MPa was the maximum pressure at which no xenon hydrate formed). Moreover, a maximum storage time of 18 h was also determined based on a preliminary experiment examining the conditions affecting the temperature dependence of the protective effect of Xe gas. Statistical analysis of relative cell activity at each temperature under Xe pressurization was conducted using the nonparametric multiple comparison test to clarify the difference in the protective effect of different storage temperatures.

2.5. Estimation of viscosity under pressurized dissolution of Xe gas

The viscosity under pressurized dissolution of Xe gas was estimated using the falling ball viscosity method and calculated according to the following expression using Stokes' law: η = K (ρ₀ − ρ)t (K: device constant of the experimental system, t: passage time [at terminal velocity], η : viscosity of the fluid, ρ₀: density of the ball, ρ: density of DMEM). The ball (Ametek Brookfield [Middleboro, MA, USA], KF Ball Kit, borosilicate glass, 15.5994 mm in diameter, 4.4101 g) was allowed to fall freely in a clear cylindrical tube (Taiatsu Techno [Tokyo, Japan] HPG-96-3, clear polycarbonate housing, pressurized-resistant up to 2 MPa; tube portion: clear tempered glass, internal diameter of 26.8 mm, 170 mm in depth, internal volume of approximately 96 ml). If the density of DMEM does not change by pressurization of Xe gas, as indicated in the relationship described above, the ratio of the viscosity at 0.5 MPa to that at 0 MPa is expressed as the ratio of passage time. Therefore, the η of DMEM without pressurization of Xe gas (0 MPa) was measured at each temperature using a cone-plate rotational viscometer (Toki Sangyo [Tokyo, Japan], R-500). Five measurements for viscosity were taken at each temperature and the mean value was adopted and assumed to be the true value. The expression K (ρ₀ − ρ) was replaced with K^′, and the value of K^′ at each temperature was determined, using the following expression, from the mean viscosity and mean passage time using the falling ball viscosity method: [K^′ = K (ρ₀ − ρ) = η _mean/t_mean]. Since it can be considered that the density of DMEM practically does not change in relation to pressurization of Xe gas, as demonstrated in the relationships described earlier, the value of K^′ can be determined to be constant at each temperature. The data of the viscosity at each temperature were calculated by evaluating the passage time for the following expression: [ η = K^′ t]. It was assumed that the error of measurement for each viscosity datapoint was affected only by dispersion due to the passage of time. In addition, the estimated effect of the pressure difference of 0.5 MPa on the density of DMEM was ∽0.03% because the bulk modulus of water is approximately 2 GPa (20°C, 1 atm) [11].

First, the relationship between viscosity and temperature in DMEM with pressurization of Xe gas was examined. The ball was placed in the tube of the vessel, and the tube was filled with DMEM as an experimental sample. Xe pressurization (0.5 MPa) and non-pressurization (0 MPa, ambient air) samples were set up. The samples were then stored in the refrigerator at 0, 2, 3, 4, or 5°C for 6 h. Video images of the falling ball (falling distance between measurement sections at terminal velocity: 50 mm) were recorded at 60 fps using a digital camera when the vessel was turned upside down, and the sample was stored in the refrigerator for 10 min every recording to maintain the measurement temperature. The time that passed between measurement sections was read from the recorded video images. The density of the DMEM at each temperature was assumed to be constant regardless of the presence or absence of Xe pressurization.

To examine the relationship between the viscosity of DMEM and the pressure of Xe gas at 4°C, the viscosity of DMEM was estimated at a pressure of 0, 0.2, 0.3, 0.4, and 0.5 MPa using the same method described above. Statistical analysis of viscosity for Xe pressurization and non-pressurization at each temperature was conducted using the nonparametric paired comparison test to clarify the difference in viscosity at different storage temperatures.

3. Results

The effect of temperature on cell activity after cold storage is shown in Fig. 2 (error bars in the figure show standard deviation [SD]). Under the non-pressurization condition (0 MPa), cell activity was low overall, decreasing with increasing temperature from 0 to 3°C, and cell activity was almost 0% at 3, 4, and 5°C. Under the Xe pressurization condition (0.5 MPa), although cell activity was low at 0 and 2°C, it increased with an increase in temperature from 2 to 4°C; cell activity was high at 4 and 5°C, with a maximum at 4°C (92.2%). The difference in relative cell activity between 3 and 4°C was statistically significant (Kruskal–Wallis H test, *P < 0.01, ^**P < 0.05, n = 12), whereas the difference between 4 and 5°C was not (P > 0.05). With regard to the relationship between pressurization of Xe gas and cell activity, although the difference in cell activity in the presence of Xe gas pressurization was small at 0 and 2°C, it was large at 3, 4, and 5°C, with the maximum difference at 4°C.

Fig. 2.

Temperature dependence of cell activity after cold storage in the presence of pressurized Xe gas at 0 to 5°C. (Kruskal–Wallis H test, *P < 0.01, ^**P < 0.05, n = 12).

The relationship between DMEM viscosity and temperature is shown in Fig. 3 (error bars show SD). The SD of the viscosity data reflects the dispersion in the measured value over time when using the falling ball viscosity assay. With non-pressurization, viscosity decreased with increasing temperature. In the case of Xe pressurization, viscosity decreased as the temperature increased from 0 to 2°C and increased from 2 to 4°C, and the viscosity decreased again as the temperature increased from 4 to 5°C. Although there was almost no difference in viscosity at 0 and 2°C in the presence of pressurization, there was a slight difference at 3, 4, and 5°C, with a significant difference at 4°C (Mann–Whitney U test, *P < 0.01, n = 10).

Fig. 3.

Relationship between viscosity and temperature in DMEM in the presence of pressurized Xe gas. (Mann–Whitney U test, *P < 0.01, n = 10).

The relationship between the viscosity of DMEM and pressure of Xe gas at 4°C is shown in Fig. 4. The relationship between cell activity after 24 h of storage at 4°C (right vertical axis) and Xe gas pressure from our previous report [7] is plotted with data from the present study for comparison (error bars show SD; viscosity, n = 10; cell activity, n = 12). The SD reflects the variation over time when using the falling ball viscosity assay. The viscosity increased with increasing Xe gas pressure and reached a maximum at 0.5 MPa. This tendency was the same as that observed between Xe gas pressure and cell activity.

Fig. 4.

Relationship between viscosity of DMEM and pressure of Xe gas at 4°C.

4. Discussion

With regard to the relationship between cell activity and temperature (Fig. 3), cell activity in samples stored at 4°C with Xe pressurization was higher than that of samples stored at other temperatures and without Xe pressurization. These data indicate that the protective effect against cell damage is greatest at a temperature of 4°C under Xe pressurization. That is, the preservation period under cold storage is the longest under these conditions.

Under non-pressurization conditions at temperatures ranging from 0 to 3°C, viscosity decreased monotonically with increasing temperature; therefore, it is possible that cell activity also increases with increasing viscosity (Fig. 3). In contrast, under Xe pressurization, DMEM viscosity was the highest at 4°C. In terms of the relationship between viscosity and pressure (Fig. 4), the maximum viscosity was observed at a pressure of 0.5 MPa. This result was consistent with the previously reported relationship between cell activity and pressure of Xe gas [10]. These data indicate that at pressures ranging from 0 to 0.5 MPa at 4°C, the protective effect increases with increasing viscosity resulting from the increase in Xe gas pressure. However, the low degree of variation in viscosity relative to temperature suggests that viscosity greatly influences the protective effect (Fig. 2).

Because the solubility of Xe gas increases with decreasing temperature, the degree of physical dissolution increased with decreasing temperature at a constant pressure of 0.5 MPa in this study. Thus, the increase in viscosity with increasing Xe gas pressure is not directly associated with the increase in the degree of physical dissolution of Xe gas by pressurization. That is, physical restraint of water molecules by Xe is greatest at a storage temperature of 4°C and Xe gas pressure of 0.5 MPa, which appears to result in an increase in DMEM viscosity [12]. One factor that affects the restraint of water molecules is the density of the solution, as restraint of water molecules becomes stronger as the density increases. Under non-pressurization conditions, the density of culture medium will increase with a decrease in temperature because it is known that the density of a saline solution increases with a decrease in temperature. Its viscosity also increases with a decrease in temperature (Fig. 3), and this is reflected in the increase in the protective effect from 3 to 0°C (Fig. 2). Under Xe pressurization, the density of the culture medium was difficult to measure, and we could not find any reference data, but it may be at a maximum at 4°C, as in the case of water. In addition, the viscosity of the extracellular solution was measured in this study, but it is possible that the intracellular viscosity also increases under pressurized dissolution of Xe gas, which could affect the temperature dependence of the protective effect.

Although the viscosity of DMEM with pressurization of Xe gas was lower at 3 and 5°C than at 0°C, there was a large difference in cell activity with pressurization of Xe gas (Figs 2 and 3). Consequently, it is possible that factors other than viscosity are involved that increase cell activity with pressurized dissolution of Xe gas. Xe exhibits slight chemical activity and is known to have an anesthetic effect [13]. This effect is due to chemical inhibition of NMDA-type glutamate receptor (ion channel) activation in nerve cells by Xe. Abnormal activation of these receptors is a factor in nerve cell damage, and Xe gas administration can protect cells by inhibiting the activation of the receptors [14]. However, whether Xe has any effect on cells other than nerve cells, and the relationship between temperature and its anesthetic effect, are unknown. As cell metabolism is suppressed at lower storage temperatures, it is expected that Xe gas would have less of a chemical effect at 0°C than 5°C. Therefore, any effect due to chemical activity of Xe gas (e.g., anesthetic effect) would not be observed in this study at 0°C or 2°C due to the lack of a difference in cell activity in the presence of pressurized dissolution of Xe gas, but such an effect would be observed at 3°C or higher.

5. Conclusion

Using pressurized dissolution of Xe gas as a new technology for cold storage is an effective method for extending the preservation period under cold storage. The most effective temperature for protecting cells from damage during cold storage with Xe gas pressurization was found to be 4°C. The slight increase in the viscosity of the preservation solution under pressurized dissolution of Xe gas appears to play a role in the temperature dependence of the protective effect.

Footnotes

Acknowledgements

The authors thank Ms. Sayaka Kiyokawa, Ms. Minori Inoue, and Ms. Shiori Matsushita (School of Allied Health Sciences, Kitasato University) for their technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research (19K04222) from the Japan Society for the Promotion of Science.

Conflict of interest

None to report.

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