Effect of Cryopreservation Regimens on Behavior of SPEV Cells in Culture

This work demonstrates the ability of using the substrate-adhered SPEV cells as the object for studying the kinetics of osmotic reactions when exposing them to cryoprotective media. Permeability coefficients of SPEV cell membranes for the molecules of water and dimethyl sulfoxide (DMSO) have been determined. Using the physical-mathematical model for cell suspension freezing and calculated permeability coefficients the osmotic SPEV cell behavior during cryopreservation with different cooling rates has been simulated. The experiments on freezing the SPEV cell suspension showed that the cooling rate of 1.5 deg/min could ensure the optimal protection of the cells against damage in 1M DMSO presence. It has been demonstrated that the cooling rate above and below the optimum may cause a cell death as the result of intracellular crystallization or dehydration.

cryoprotectants [3].The main difficulty for observation of the cells under the microscope is the shifting of the cells by a stream when replacing the liquids.We hypothesized that the cells fixed on a microscope slide due to adhesion [1,7], could serve as a target for volumetric studies.Determined in such a way permeability coefficients of cell membranes for molecules of DMSO and water could be used to predict the osmotic behavior of cells during freezing with different rates.Post-thaw survival of the cells could be estimated by monitoring their adhesion and spreading.Comparison of these observations with predicted osmotic behavior of the cells could enable to suggest the causes of cell damage in case of deviation of freezing regimens from optimal ones.The purpose of this work was to elucidate the peculiarities of the permeability coefficients of cell membranes in culture and to compare the data on supposed cell volume changes during freezing and their preservation after thawing.

Materials and methods
Investigations were carried out in SPEV culture (porcine embryo kidney cell line) procured from the collection of PJSC Pharmstandard-Biolik (Ukraine).
Cells were cultured in plastic flasks (Costar, Germany) in 199 medium (Sigma, USA) supplemented with 10% fetal calf serum (BioloT, Russia) at 37°C in air with 5% CO 2 .Cell suspension was procured by treating the cell culture monolayer with the 0.25% trypsin and 0.02% Versene mixture (in 1:5 ratio) followed by washing the cell suspension by growth medium.
The resulted cell suspension was divided to control samples, which were re-suspended in growth medium, and the samples supposed for cryopreservation, which were transfered to 10% DMSO solution in fetal calf serum [8].
The prepared suspension was placed into 1.5 ml cryovials (Costar) and cooled according to the protocols: 0.3 deg/min down to -20°C (1 hr in a fridge), 1.5 deg/min down to -40°C (in a programmable freezer) or 8 deg/min down to -60°C (in KHB-0.5 tank above the surface of liquid nitrogen), in all the cases the samples were then immersed into liquid nitrogen.
After storage at -196°C for 5-10 days the samples were thawed in a water bath at 41°C.
The osmotic behavior of cells was examined in 1 M DMSO solutions with different concentrations of NaCl (0.0375; 0.075; 0.15; 0.5 and 1 M) by means of microscope Axio Observer Z1 (microscopic images were recorded) and dynamics of changes in cell dimensions was assessed using the software Axio-Vision Rel.4.8 (Carl Zeiss).Morphometric data were used to determine the permeability coefficients for cell membranes and theoretical predicting of cell osmotic behavior during freezing [4].
The findings were statistically processed using Excel (Microsoft, USA) and Past Statistic v/3/01 (Sweden).Depending on the distribution character of the data the significance of differences between the indices was assessed using a parametric Student's ttest or non-parametric Mann-Whitney one [5].Differences between the samples were considered as significant at p < 0.05.

Results and discussion
The SPEV cells cooled in an alcohol bath down to -28°C with a 15-min stop at this temperature, followed by immersion into liquid nitrogen, demonstrate a high survival and proliferative potential [3].In the present studies, we have examined the possibility of using other freezing regimens to cryopreserve SPEV cells, in particular, freezing with the controlled rate of 1.5 deg/min down to -40°C, followed by an immersion into liquid nitrogen; non-controlled freezing in a fridge down to -20°C (0.3 deg/min estimated cooling rate) with immersion into liquid nitrogen, and, finally, freezing on cooled in nitrogen vapors metal base with temperature of -60°C (8 deg/min estimated cooling rate) and following immersion into liquid nitrogen.Effect of various freezing protocols was assessed by observing the behavior of SPEV cells in culture.
Following incubation of the intact SPEV cells for 15 min they sedimented to the bottom of a plastic Petri dish, when shaking they easily transited into the suspension.During following 15 min of incubation the part of the cells was loosely attached, only some cells transited to a suspended state when shaking.In the majority of cells the nucleus with nucleoli was clearly visible.Some cells showed the first stage of adhesion (the filopodia appearance).More firm attachment to the substrate was ensured by lamelopodia which were formed in the majority of cells after an hour of incubation.This was evidenced by the fact that the transi-  tion of cells into a suspended state was not provided by slight mechanical shaking.In 1.5-2 hrs the part of cells was getting flattened.On the fifth hour of observation about 10-15% of the cells have acquired the structure characteristic for SPEV cell culture (Fig. 1).
When comparing the behavior of cells in culture after cryopreservation according three freezing protocols it has been found that freezing with the rate of 1.5 deg/min down to -40°C, followed by immersion into liquid nitrogen ensured the maximum preservation of their adhesive properties.Observing the cells during adhesion and spreading showed the same dynamics as in intact cells.After 5 hrs of incubation 10-16% of cells completed spreading (Fig. 2).
The freezing protocol with a rate of 8 deg/min down to -60°C, followed by an immersion into liquid nitrogen was unfavorable for cells (Fig. 3).Within an hour of incubation no tendency to adhesion and spreading of SPEV cells was observed.In 2 hrs the bulk of the cells was easily detached from the substrate when shaking.Cell cytoplasm was vacuolated, about 20% of the cells had a protrusion on the membrane (vesicles).As reported by Ye.A. Porozhan et al.Subjected to freezing with a rate of 0.3 deg/min down to -20°C followed by an immersion into liquid nitrogen (Fig. 4) the cells completely lost their functional activity.Within 5 hrs of observation the cells did not adhere, were of round shape, when being gently shaken easily transited to a suspended state, almost all the cells had vesicles, the cytoplasm was vacuolated.
Based on these results, we predicted the osmotic behavior of cells under the used freezing conditions.Initially we identified the transport characteristics of cell membranes.During culturing the cells were spread, therefore it was important to ensure keeping the shape of adhered cells from the moment of attachment to the substrate up to the spreading start.
As Fig. 5 demonstrates the culturing under the microscope allowed us to observe both the changes in the shape and movement of adherent cells.It should be noted that even up to the beginning of spreading the adhered cells did not change their sizes.This allowed to suggest the cell shape prior to the spreading as a spherical one.
Fig. 6 presents data on osmotic reactions of SPEV cells in 1M DMSO solutions: in the ones isotonic by NaCl (0.15 M NaCl) the cells restored an initial volume after dehydration, in the hypertonic solutions (0.5 and 1 M NaCl) they remained dehydrated and in hypotonic ones (0.075 and 0.0375 M NaCl) they swell.The calculated using the mathematical model coefficients of filtration and permeability for DMSO molecules are shown in the Table .The data of the Table demonstrate that the permeability of SPEV cell membranes for DMSO depends on the medium composition.At the same time, the calculated permeability coefficients of adherent cells for DMSO were of the same order and close to the values of the permeability coefficient, determined for cell suspensions, fixed in a collagen matrix [1].Как видно из рис.5, культивирование под микроскопом позволяет наблюдать как за изменением формы, так и перемещением адгезировавших клеток.Следует отметить, что вплоть до начала распластывания адгезировавшие клетки не изменялись в размере.Это позволило считать форму клеток до начала распластывания близкой к сферической.
Рис. 6. Экспериментальные данные и теоретические зависимости изменения объема сферических клеток СПЭВ от времени в 1 М растворах ДМСО, содержащих 1 М (1), 0,5 М (2), 0,15 М (3), 0,075 М (4) и 0,0375 М (5) NaCl.In the following studies we simulated the cell dehydration in a suspension at various cooling rates using the mathematical model of cell suspension freezing in DMSO presence [3].Fig. 7 represents the calculated data of the relative volume change of SPEV cells during freezing according three protocols.Analysis of these dependencies allowed to suggest that during the freezing with optimal rate (1.5 deg/min) the dehydration protected the cells against intracellular crystallization, at the rates under the optimum (0.3 deg/min) the dehydration can cause the death of the cells, and at those above the optimal one the cells do not dehydrate and are damaged due to intracellular crystallization.

Conclusions
Thus, the substrate-adhered cells retaining the shape close to spherical one are the proper object to study the kinetics of osmotic cell responses which can be used to assess cell membrane permeability parameters for cryoprotectants and water molecules.
normal adhesion.After 5 hrs of incubation only single cells attached and flattened.