Optimization of Cryopreservation Technique for Human Cord Blood Nucleated Cells Using Combination of Cryoprotectant DMSO and Antioxidant N-acetyl-L-cysteine

The paper evaluated the efficiency of N-acetyl-L-cysteine (AC) antioxidant during cryopreservation of human cord blood nucleated cells (CBNCs) with various concentrations of endocellular cryoprotectant dimethyl sulfoxide (DMSO). It has been found that rise in DMSO concentration (from 2.5 and 5 up to 7.5% and 10%) and exposure time of the CBNCs suspension with cryoprotectant (from 15 to 30 min and more) resulted in a significant increase in the amount of cells with excess reactive oxygen species (ROS) (from (7.5 ± 0.8)% at 5% DMSO and 15-min incubation to (28.9 ± 3.2)% with 10% DMSO and 60 min incubation), decrease in their viability and preservation rate. Supplementing 10 mM AC to the cryopreservation medium led to in a reduction in the amount of cells with excess ROS and rise of their preservation rate and viability at the stage of equilibration with cryoprotectant, as well as after freeze-thawing of CBNCs suspension. Maximum effect was achieved after AC supplementing to the media with 7.5 and 10% DMSO concentrations. We proved that the use of antioxidant contributed to the rise in preservation rate and viability of CBNCs if cryoprotectant concentration and exposure time with it were optimal.

Recently the application of cord blood (CB) in clinical practice has become widespread.Since 2009, a number of performed allotransplantations of CB hematopoietic stem cells exceeded that of transplantations of the bone marrow cells [5,14].Nowadays about 3 thousand transplantations of CB cells are annually performed around the world to patients in different diseases [20,15].Such an attention of clinicians to CB application and increased number of its transplantations have resulted in a need to establish the network of cryobanks [12], where the samples are stored in a frozen state at -196°C with no loss in their biological properties for unlimited time period.So, the small amounts of each CB dose and impossible re-sampling make it necessary to choose the optimal cryopreservation method for nucleated cells (NCs), comprising hematopoietic stem cells.In this regard, the development of novel methods for CB cryopreservation and optimizing the existing ones have still remained actual.
The cryoprotectant dimethyl sulfoxide (DMSO) at 5-10% concentration is quite often used to freeze the CB NCs [13].During cryopreservation the CB NCs undergo a significant stress effect, which may result in apoptosis, both prior to and after thawing [16].So, it is important to assess the cell survival and viability not only immediately after thawing, but some time after transferring them into the conditions close to physiological ones.It is to be noted that the cell apoptosis may be initiated inter alia by different factors, in particular, reactive oxygen species (ROS).Due to this fact of importance is to develop the methods, which could prevent the ROS accumulation at all the cryopreservation stages, to either avoid or slow down the oxidative stress development.The supplement of cell suspension with the substances possessing pronounced antioxidative and cytoprotective properties may be prospective.Among others one could mention N-acetyl-L-cysteine (AC), not only featured by a direct antioxidant activity, but also participating in glutathione synthesis and providing the cytoskeletal structure with thiol groups, protecting the cells from destruction [10].However, the design of cryopreservation techniques is impossible without determining this antioxidant effect on CB NCs during freeze-thawing.Therefore this work purpose was to determine the effect of different concentrations of DMSO and N-acetyl-L-cysteine on the survival and viability indices and reactive oxygen species content in cord blood nucleated cells prior to and after cryopre-servation.
vein during natural labour after child birth and separation from placenta.
The NCs fraction was isolated from the whole CB by sedimentation in Polyglucinum (6% dextran solution (JSC Biokhimik, Russia) with 60,000 molecular weight).For this purpose the blood was first mixed with Polyglucinum in 1:1 (v/v) ratio, then precipitated up to distinct separation of erythrocytes and nucleated cells appearance.Sedimentation time made from 30 to 50 min, then the supernatant was collected and centrifuged for 5-7 min at 800g to obtain the NCs concentrate.
Cell suspension was supplemented with 25% DMSO solution up to 2.5; 5; 7.5 and 10% final concentrations in a sample.Cell suspensions were treated with cryoprotectant at a low positive temperature 0...4°C.Before mixing the cell suspension and cryoprotectant solutions were brought to the appropriate temperatures; DMSO was dropwise added during a constant stirring.
Here we used the AC (Sigma-Aldrich, USA) in a 10 mmol/L final concentration, supplemented to DMSO containing samples prior to cryopreservation.
Samples were cryopreserved using a programmable freezer (Cryoson, Germany) with 1-3 deg/min rate down to -80°C, followed by immersion into liquid nitrogen (-196°C) [4].Thawing was done in a 37-40°C water bath with constant shaking until a solid phase disappeared.
The absolute cell number was counted in Goryaev's chamber according to the standard procedure [9].The cell survival was determined as the percentage of cell number in the studied sample with respect to their initial one prior to any impact (incubation with cryoprotectant, freeze-thawing).
The viability of CD45 + -cells was assessed by the standard ISHAGE protocol (International Society of Hematotherapy and Graft Engineering) using a monoclonal antibody CD45-FITC and DNA dye 7-aminoactinomycin D (7AAD) [24] by means of flow cytometry.For this purpose the whole blood (50 µl) was supplemented with 10 µl of reagents (FITC-labeled CD45, clone 2D1 and 7AAD), then mixed and incubated for 15 min at room temperature in the dark.Each tube was supplemented with 1 ml ammonium chloride solution (BD, USA), which caused cell lysis.Samples were analyzed with the CellQuest Pro software (BD).
To determine a delayed death some cells after freeze-thawing were transferred into Hanks' solution at 1:10 ratio and incubated at 37°C for 1 hr.Then the survival, viability and ROS content in cells was determined with flow cytometry.
Our findings were statistically processed with Student-Fisher test using Excel software (Microsoft, USA).Differences were considered as stastistically significant at р < 0,05.

Results and discussion
A therapeutic efficiency of CB cryopreserved preparations mostly depends on a number of survived cells and their functional activity after thawing [1].Therefore a thorough assessment of structural and functional indices of CB hematopoietic stem cells after cryopreservation is necessary.In addition, because of a limited volume of CB dose (no more than 100 ml) and impossible re-sampling, it is important to preserve the quality in the maximum possible amount of NCSs, comprising hematopoietic stem cells inter alia [21].
The freeze-thawing outcome depends on cell state prior to cryopreservation, therefore it is necessary to determine their survival and viability indices prior to freezing in order to estimate the cryopreservation outcome.
As it has been mentioned previously, the DMSO is the main cryoprotectant used for CB NCs cryopreservation [7,19].But, in addition to cryoprotective effect [11] it may affect the properties of membrane surface, structural regularity of lipids, conformation of peripheral and integral proteins, as well as metabolic cell state, antioxidant system inhibition, in particular.This may result in an increased ROS level in cells, which may lead to their further death.Due to this fact, it is very important that the ROS level in cells meets the physiological norm [21].So the determination of ROS content is one of the key integral parameters to assess the NCs state and impact on them of incubation in the cryoprotectant solutions of various concnetrations at equilibration stage [26,27].
In order to study the influences of concentration and time span effect on NCs suspension we used DMSO at 2.5; 5; 7.5 and 10% final concentrations and incubation time from 0 to 60 min.
Отримані дані щодо накопичення АФК у клітинах до кріоконсервування показали, що з підвищенням концентрації ДМСО до 7,5 та 10% і подовженням часу еквілібрації (особливо після 30 хв) збільшувалася кількість клітин із надлишковим вмістом АФК, що може вказувати на розвиток окисного стресу та/або можливе radicals.In this regard, when designing the cryopreservation protocols it is necessary to ensure the minimum time of cell contact with liquid DMSO phase both prior to and especially after cryopreservation.In addition to determine the ROS content in cells we analyzed the effect of different concentrations of DMSO solutions and incubation time span with it on NCs survival and viability.Our findings (Table 1) demonstrate an increase in both cryoprotectant concentration (up to 7.5 and 10%) and exposure time with it (after 45 min) to result in a pronounced reduction of these indices.Of note is the fact, that DMSO in 2.5 and 5% concentrations affected these indices in a much less degree, that might be associated with its low and ultra low concentration.
The cell viability indices in different experimental groups at the stage of incubation with DMSO did not significantly differ.A significant decrease in this index was observed after equilibration with 10% DMSO within 30 min (Table 1).
Since our research demonstrated the augmentation of ROS content during CB NCs equilibration with DMSO, it would be expedient then to use the substance capable to either 'scavenge' free radicals or neutralize the source of their origin.One of these substances is AC, an antioxidant effect of which is stipulated by the presence of free thiol group, interacting with electrophilic groups of free radicals and reactive oxygen metabolites.This substance inactivates virtually all the species of active oxygen metabolites, including the most reactive Рис. 1. Кількість ЯВК КК із надлишковим вмістом АФК залежно від концентрації ДМСО та часу еквілібрації до кріоконсервування: -2,5% ДМСО; -5% ДМСО; -10% DMSO; * -significant differences comparing to zero point of incubation with DMSO, р < 0.05.[8].
Previously, to elucidate the AC optimal concentration the experiments on studying an antioxidant effect of its different concentrations: 5; 10; 15 and 30 mmol/L, were carried out [2,3,17].The findings showed 5 and 30 mmol/L AC concentrations of AC solutions as not manifesting antioxidant action while adding to cell suspension, incubated with different DMSO concentrations, irrespective of cryoprotectant concentration, moreover in case with 30 mmol/L a prooxidant effect was observed.The AC at 10 and 15 mmol/L concentrations reduced a cell number with excess ROS, and as a result, had a cytoprotective effect, as indicated by the survival indices, observed at high concentrated DMSO.For the next series of experiments we used 10 mmol/L concentration, as it occurred to be the most suitable [6].
The performed studies enabled establishing the fact, that the AC supplemented to the medium with DMSO, contributed to reduce a cell number with excess ROS (Table 2), as well as to enhance their survival and viability (Table 3).It should be noted that the augmentation of both incubation time and cryoprotectant concentration results in an increased number of cells with excess ROS.However, in the samples with 10 mmol/L these changes were less pronounced, even after increasing the cryoprotectant concentration up to 7.5 and 10%.Thus, the AC introduction into the NCs incubated with DMSO may play a key role in keeping lipid assymetry Таблиця 1. Збереженість та життєздатність ЯВК КК (%) залежно від концентрації ДМСО та часу еквілібрації до кріоконсервування (M ± SE) Table 1.CB NCs survival and viability (%) depending on DMSO concentration and equilibration time prior to cryopreservation (M ± SE) Примітка: * -результати статистично значущі по відношенню до нульової точки інкубації з ДМСО, р < 0,05.
Note: * -significant differences comparing to zero point of incubation with DMSO, р < 0.05.

Низький відсоток клітин із над-
of membranes both at the preparation stage prior to cryopreservation and after freeze-thawing.
Freezing and thawing are the most dangerous stages for cells during cryopreservation [6].So the next task in our study was to assess the efficiency of CB NCs cryopreservation in cryoprotective solutions containing differently concentrated DMSO and 10 mmol/L AC.
На наступному етапі нами була проведена оцінка відстроченої загибелі клітин після розморожування.Для цього ми застосували підхід із перенесення ЯВК КК до умов, наближених до фізіологічних.В експерименті ми використовували просту In the samples cryopreserved with AC we observed a lower number of cells with excess ROS with respect to the corresponding groups with no antioxidant (except 2.5% DMSO).Their number was significantly lower in 2.5 times as compared to the corresponding antioxidant-free groups.The best results were obtained in those samples, cryopreserved with 7.5 and 10% DMSO solution, which completely coincided with the survival and viability data.
The results of cell survival analysis after cryopreservation (Table 4) demonstrated a decrease in this index and an inverse relationship between a number of survived cells and the excess ROS cell amount (except the sample with 2.5% DMSO, where an excess cell disintegration occurred).The lowest survival of cells was observed in the AC-free cryopreserved samples with the determined maximum ROS content.This results from applying an inefficient (either low or extremely low) cryoprotectant concentration in this cryopreservation protocol.The highest survival indices were obtained after using 7.5 and 10% DMSO solution.
The NCs freezing in the media containing 10 mmol/ L AC provided higher indices of CB NCs survival as compared to the corresponding AC-free control groups, excluding those samples, cryopreserved with extremely low (2.5%) cryoprotectant concentration.
Analysis of a percentage of viable cells showed the reduction of this index after cryopreservation, but no significant differences between experimental groups were revealed (Table 4).However, in terms of the absolute number of survived cells (Fig. 2), the cryopreservation with AC was found to significantly increase the total number of viable NCs in the samples cryopreserved with 7.5 and 10% DMSO.
Note: * -significant differences comparing to zero point of incubation with DMSO, р < 0.05.
As a next stage we assessed a delayed cell death after thawing.For this purpose, we used the approach with transferring CB NCs into conditions close to physiological ones.In the experiment we used a simple model, reproducing thereby only the basic features of transfusion: dilution of frozenthawed cell suspension, which proceeded naturally in a recipient's blood stream; medium isoosmolality and incubation temperature (37°C).The maintenance of 37°C temperature within all the period of cell incubation is an important factor enabling to reveal metabolic disorders, since the cell survival under lower temperatures masks a possible imbalance in cell metabolism due to the slowdown of functional activity of virtually all the processes Analysis of the results of cell survival and viability after transferring into physiological conditions for an hour (Table 5) revealed that the indices were minimal in the samples cryopreserved with DMSO in 5 and especially 2.5% concentrations.In these samples the losses of absolute cell number were significantly higher than right after thawing.The best results were obtained with 7.5 and 10% DMSO.With these concentrations a decrease in survival and viability indices was less pronounced as compared to other experimental groups.-без внесення АЦ, -у присутності АЦ; * -різниця статистично значуща по відношенню до відповідної групи клітин, кріоконсервованих без внесення АЦ (р < 0,05).
The minimum number of cells with excess ROS was observed after cryopreservation with 2.5% DMSO, but the maximum one with 10%.This fact may be explained by the loss of a significant number of cells, occurred with low concentrated DMSO use (that coincided with survival and viability data).
The analysis of viable NCs number, with respect to the absolute number of survived cells, demonstrated (see Fig. 3) the cryopreservation with 10 mmol/L AC and subsequent transfer into physiological conditions after thawing to ensure the survival of a higher cell number in all the experimental samples.In this concentration the AC may significantly augment (in 1.5 times) the viability index when using low and ultralow cryoprotectant concentration (5 and 2.5%), i. e. under less favorable cryopreservation conditions.However, the AC supplement to cell suspension with higher concentrations of DMSO (7.5 and 10%) also augmented the level of viable cells in terms of absolute number of survived cells.A cell number with excess ROS in these groups was herewith also lower as compared to the AC-free cryopreserved samples (Table 5).
Thus, our findings may suggest AC to prevent a disordered functioning of antioxidant processes in cells, reducing thereby the ROS level and preventing oxidative stress development.The CB NCs cryopreservation with 10 mmol/L AC, 7.5 and 10% DMSO promotes a significant increase in cryopreservation efficiency.

Conclusions
1. Analysis of ROS accumulation in CB NCs demonstrated the augmentation of cell number with excess ROS to occur with increasing DMSO concentrations and incubation time span.
2. The CB NCs cryopreservation in DMSO presence resulted in an increased cell number with excess ROS, which in turn led to a decrease in survival and viability indices.
3. The N-acetyl-L-cysteine supplement to cell suspension enabled reducing ROS content and, consequently, improving the survival and viability indices of CB NCs at all the stages of cryopreservation.Література

Fig. 3 .
Number of viable CB NCs after cryopreservation and transfer into conditions close to physiological ones with differently concentrated DMSO and 10 mmol/L AC taking into account the absolute values of survived cells: -with no AC supplement, -in AC presence; * -difference is statistically significant in respect to the corresponding group of cells cryopreserved without AC (р < 0.05).* * *

Table 5 .
Survival, viability and number of cells with excess ROS in CB NCs (%) depending on DMSO concentration and 10 mmol/L AC after freezing and transferring into conditions close to physiological ones (М ± SЕ)