THE GENERATION OF REACTIVE OXYGEN SPECIES BY CORD BLOOD NUCLEATED CELLS DURING CRYOPRESERVATION

100 Cryopreservation of cord blood (CB) nucleated cells (NCs) can cause the changes of their structural and functional properties [1]. This may disorder many biochemical processes, including the cell power supply system, with the following generation of the toxic reactive oxygen species (ROSs). Along with ROS generation during cell vital activity at all the stages of cryopreservation, particularly NCs isolation, treatment with cryoprotectant and freeze-thawing, the reaction of lipid peroxidation (LPO) can be activated, which may potentiate ROSs accumulation and results in the biomolecules’ damage [2, 3]. Maintaining the cells integrity during cryopreservation also depends on the functioning of antioxidant enzymes [4]. The purpose was to determine the ROSs level in various populations (lymphocytes, monocytes, granulocytes) of CB NCs, depending on the method of isolation, treatment with cryoprotectant and freeze-thawing, as well as the activity of antioxidant enzymes before and after cryopreservation while the cells were loaded with exogenous ROSs. Materials and Methods

Cryopreservation of cord blood (CB) nucleated cells (NCs) can cause the changes of their structural and functional properties [1].This may disorder many biochemical processes, including the cell power supply system, with the following generation of the toxic reactive oxygen species (ROSs).Along with ROS generation during cell vital activity at all the stages of cryopreservation, particularly NCs isolation, treatment with cryoprotectant and freeze-thawing, the reaction of lipid peroxidation (LPO) can be activated, which may potentiate ROSs accumulation and results in the biomolecules' damage [2,3].Maintaining the cells integrity during cryopreservation also depends on the functioning of antioxidant enzymes [4].
The purpose was to determine the ROSs level in various populations (lymphocytes, monocytes, granulocytes) of CB NCs, depending on the method of isolation, treatment with cryoprotectant and freeze-thawing, as well as the activity of antioxidant enzymes before and after cryopreservation while the cells were loaded with exogenous ROSs.

Materials and Methods
Object of the research was human cord blood nucleated cells obtained with glucosecitrate solution.Cord blood was obtained from the umbilical vein after mother's informed consent.
The NC concentrates were isolated by several methods: sedimentation in 3% polyglucinum, centrifugation in ficoll-verografin density gradient, [5] and by our own method of two-step centrifugation of the native cord blood with the following obtaining of NC concentrate in autoplasma [6].
Number of survived NCs was determined by standard method using the cell counter as a ratio of exposed cells in the sample to the amount of intact (before exposure) cells expressed as a percentage.
Phenotyping of nucleated (CD45 + )-cells and viability assessment with DNA marker 7-aminoactinomycin D (7AAD) was performed using flow cytometry (FACS Calibur cytometer, Becton Dickinson, USA) and using the BD reagents.To assess CD45 + -cells and their viability we used standard protocol for immunophenotyping with CD45FITC and 7AAD.The populations of leukocytes (lymphocytes, monocytes and granulocytes) were determined with dot-plots in coordinates of the side scatter (SSC) and the first channel of fluorescence (FL1: CD45FITC).The cells, which were unstained with 7AAD, were considered as viable.Flow cytometry data were calculated using the CELLQuest Pro software (BD).
ROS production in CB NCs was assessed by flow cytometry with the dye DCFH 2 -DA, which transforms from non-fluorescent into the highly fluorescent form DCF in the presence of ROSs in a cell [9].The cells were incubated with 5µM DCFH 2 -DA in the darkness for 15 min.Activity of antioxidant enzymes was determined after the addition of exogenous H 2 O 2 in the cell suspension.
The data are presented as M ± m, the differences significance between the samples were evaluated using Student's t-test with 5% significance level.The sample number included at least 5 experiments.

Results and Discussion
Previously it was shown that the different populations of NCs possesed different cryosensitivity, for example, after cryopreservation with 5% DMSO the highest number of non-viable cells was observed in the fraction of granulocytes [9,10].
It was topical to investigate ROS production in different populations of NCs under cryoprotectants treatment and freezing conditions.For this purpose the fluorescence of DCF, which was directly-proportioned to the intracellular ROS amount, was measured [11,12].For the results analysis the high and middle fluorescence intensity regions were taken into account (Fig. 1, A).
Direct DCF fluorescence showed that the content of ROSs after the NCs isolation and their treatment with cryoprotectants significantly differed from that in the cells isolated with Ficoll (Fig. 1, B), where we saw a tendency to increasing in ROS level; wherein almost all the fluorescent cells was in high intensity region.
Later we analyzed the relative change in the number of DCF-labeled NCs in regions of high and middle fluorescence intensity, taking it as either an increase or decrease in fluorescence before and after exposure to the studied factor.It was found that the degree of changes in the number of DCF-labeled cells directly depended on the intensity of effect: the greater changes in the relative number of DCF-labeled cells corresponded to the greater increase in antioxidant enzymes activity and ROS generation in these cells.Fig. 2 shows that the NCs treatment with cryoprotectants, irrespectively to the nature of their impact on cells, resulted in no changes in the ROS generation in cells, that corresponded to an insignificant change in relative DCF fluorescence in the studied cells [up to 8% in areas of high (High) and middle (Mid) DCF fluorescence intensity].
A similar pattern was noted for different NCs populations (lymphocytes, monocytes and granulocytes): treatment with cryoprotectants did not cause significant changes in relative DCF fluorescence (Fig. 3).Further the ROS production by NCs after freeze-thawing (Fig. 4) was investigated.Of importance was the fact that DCF fluorescence intensity in the cells was largely related to their viability.Thus, using most effective methods for isolation and cryopreservation we showed that the cells number in the intense light zone remains high, in contrast to the case when the cells were frozen after isolation by Ficoll were observed the redistribution of DCF-labeled cells with decrease of cell number in the High zone and increase in the Mid zone.
Analysis of DCF relative fluorescence of CD45-labeled NCs showed that the more effective method of cryopreservation, the less pronounced divergence from baseline fluorescence (Fig. 5).It should be noted that in total NCs after thawing were characterized by an increased number of cells with a decreased fluorescence intensity in the Mid zone.
Further we determined the ROS generation in NC populations after freeze-thawing taking into account the heterogeneity of CD45 + fraction, including lymphocytes, monocytes and granulocytes (Fig. 6).
The data showed that most effective method of lymphocytes cryopreservation includes their the isolation by dextran and freezing with DMSO, while the isolation with Ficoll and freezing with DMSO as well as freezing with dextran without cryoprotectant were the least effective.Monocyte population was characterized by minimum changes in the ROS generation during freezing with DMSO regard-less of the isolation methods.In granulocyte population after cryopreservation the greatest changes in the relative fluorescence were observed varying within the range of 36-58%, but were not significant due to their heterogeneity.Of importance was to note that for the granulocyte fraction the intracellular ROS generation activity correlated with their high cryolability.

Fig. 1 .
Fig. 1.Data of standard experiment (A) and direct DCF fluorescence of CB NCs (B) in regions of high (High Ō) and middle (Mid Ō) fluorescence intensity depending on isolation method and cryoprotectant type used for cells' treatment: treatment with 5% DMSO of cells isolated either in polyglucinum (1) or Ficoll density gradient (2); treatment with 10% PEO-1500 of cells isolated by either two step centrifugation (3) or in Ficoll density gradient (4)

Fig. 2 .Fig. 3 .Fig. 4 .
Fig. 2. Relative change in number of DCF-labeled NCs in regions of high (Ō) and middle (Ō) fluorescence intensity depending on isolation method and cryoprotectant type used for cells' treatment: here and in Fig. 3: treatment with 5% DMSO of cells isolated either in polyglucinum (1) or Ficoll density gradient (2); treatment with 10% PEO-1500 of cells isolated by either two step centrifugation (3) or in Ficoll density gradient (4)