Effect of Cryopreservation Using Slow Freezing or Vitrification on Viability and Metabolic Activity of Mesenchymal Stromal Cells Encapsulated Within Alginate Spheres with Diameter of 1 mm and More

The aim of the study was to evaluate the effect of exposure duration of alginate encapsulated mesenchymal stromal cells (MSCs) in multicomponent cryoprotective solution DEPS-1 (10% dimethyl sulfoxide, 20% ethylene glycol, 1,2-propane diol and 0.5 M sucrose) on survival and metabolic activity following cryopreservation using vitrification. The spheres of 1mm diameter obtained by dropwise addition of sodium alginate into calcium chloride were used in the experiments. Alginate spheres derived by this method contained more cells as compared to the spheres of (0.5 ± 0.2) mm derived by pulverizing the alginate into calcium chloride. It was shown that using of multicomponent cryoprotective solution DEPS-1 allowed achieving successful cryopreservation of MSCs encapsulated in alginate spheres of different sizes. It was found that saturation with cryoprotective solution of MSCs encapsulated in spheres with 1.2 mm size needed a longer exposure with CPA if compared with spheres of 0.6 mm size.

Previously we studied the possibility to maintain specific properties of MSCs during their low temperature storage within the alginate spheres using slow freezing and protection by 10% dimethyl sulfoxide (DMSO), which resulted in significant preservation of the viability and metabolic activity of the encapsulated cells [6].Moreover we performed a successful modification of MSC suspension vitrification technique using a mixture of cryoprotectants DEPS-1 (10% DMSO, 20% ethylene glycol (EG), 20% 1,2-propanediol (1,2-PD) and 0.5 mol sucrose) for eMSCs within the alginate spheres of (0.5 ± 0.2) mm diameter [7].The advantage of 'small' diameter is the convenience for performing manipulations with the spheres (including the introduction to the recipient organism using standard injection needles).However, the method of obtaining 'small' spheres based on the spraying of alginate into calcium chloride solution, did not allow to achieve a high concentration of the encapsulated cells due to unstable solution viscosity.Development of other methodological approach involving dropwise addition of alginate to the solution of calcium chloride gave the opportunity to partially overcome these limitations and to significantly increase cell concentration in the spheres.Alginate spheres obtained by the second method had larger diameter.Since the size and shape of the samples are essential parameters in cryobiological research, there arises a question whether the conditions for cryopreservation of MSCs within the spheres of 'small' diameter would provide the preservation of their biological properties within the alginate spheres with larger diameter.
Herewith, the aim of this study was to assess the viability and metabolic state of MSCs within the alginate spheres of different diameters after cryopreservation using slow freezing and vitrification.

Materials and methods
The experiments were performed in human dermal MSCs derived from adult donors after their informed consent.The cells were obtained by explantation of fragments [4].These were cultured in α-MEM (Sigma, USA) supplemented with 10% fetal bovine serum (FBS) (Biolot, Russia), 2 mM L-glutamine, 50 IU/ml penicillin and 50 mg/ml streptomycin at 37°C, 5% CO 2 and 95% humidity.Following reaching 60-70% confluent monolayer the MSCs of passages 2-3 were trypsinized by standard technique and sedimented by centrifugation at 200g for 7 min.The resulting pellet was resuspended in purified solution of 1.2% sodium alginate (Sigma) based on the phosphate buffered saline (PBS; Sigma).The cell suspension in sodium alginate solution was sprayed with specially designed device [5] or was dropped with an injection needle (0.8 mm diameter) into the solution of 100 mM CaCl 2 .After polymerization (during 10 min) the spheres were washed by PBS supplemented with 25 mM HEPES (Sigma) to remove the excess of CaCl 2 .In case of spraying the diameter of the spheres was (0.5 ± 0.2) mm (group of eMSCs-0.5) and in case of dropping it made (1.2 ± 0.1) mm (group of eMSCs-1.2).The resulting spheres were placed for 24 hrs in culture conditions described above.
Slow freezing of eMSCs-1.2 with the controlled cooling rates was performed under protection of 10% DMSO in α-MEM (Sigma), containing 20% FBS.For this purpose, the spheres were transferred to 2 ml cryocontainers (Nunc, USA) (6 spheres per cryovial) containing 500 µl of cryoprotective solution and incubated for 15 min at room temperature.The samples were cooled at 1 deg/min down to -80°C using the programmable freezer ZP-10 (Special Designing and Technical Bureau with Experimental Unit of the Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences, Ukraine), then transferred into liquid nitrogen and stored from 1 to 5 days.The samples were warmed in water bath at 37°C, washed by dropwise addition of 5 ml α-MEM during 10 min solution changing.
To assess the effect of exposure on the viability of MSC suspension the vials containing cells in 100 µl of physiological saline were supplemented with 400 µl of cryoprotectant solution containing 10% DMSO, 20-60% of ethylene glycol, 0-20% of the 1,2-PD and 0.5 sucrose (final concentration).The exposure time was 5 min, whereat the cells were washed.
Frozen-thawed spheres with MSCs were cultured for 24 hrs under conditions described above.We used not less than 100 µl of culture medium per one sphere with MSCs.
Metabolic activity of eMSCs was assessed by the degree of recovery of redox indicator Alamar Blue™ (AB; Serotec, USA) in the same spheres prior to cryopreservation, immediately after thawing and in a day of culture after thawing.For this purpose the wells of culture plate with spheres were supplemented with 100 µl of 5% AB solution.The fluorescence of the reduced AB form was determined after 2 hrs of culture using a spectrofluorometer (Tecan GENios, Australia) at the excitation wavelength of 550 nm and emission wavelength of 590 nm.The data were processed with XFLUOR4 v.4.50 (Tecan GENios).The results were presented as the differences between the values of fluorescence of experimental and blank sample (5% AB solution) and expressed in arbitrary fluorescence units (AFU).To assess the metabolic activity of eMSCs-0.5 and eMSCs-1.2we used the same amount of encapsulated cell material and expressed it as AFU per well.To determine the number of cells the spheres were dissolved in 10 mM of ethylenediaminetetraacetic acid (EDTA; Sigma), the cells were counted in Goryaev's chamber.The effect of cryopreservation on metabolic state of cells involved in eMSCs-1.2 was estimated by AB test in the same spheres and expressed in percentage as AFU per one sphere after cryopreservation/AFU per one sphere before cryopreservation.
were dissolved by mixing with 10 mM EDTA solution.The cells were pelleted by centrifugation at 200 g for 7 min.The obtained pellet was resuspended in saline, the cells were counted in Goryaev's chamber.Viability was de-termined as the ratio of number of cells accumulated formazan to total number of cells and expressed in percents.
The obtained results were statistically processed using the Origin 6.7 software.The data were expressed as (x ± y)%.To assess the significance of differences between the indices we used parametric method of statistical analysis (Student's t-test)

Results and discussion
Method of spraying the suspension of cells in solution of 1.2% sodium alginate allowed to get spheres with diameter (0.5 ± 0.2) mm, the number of cells within spheres made from 100 to 150.Method of dropping the suspension into the polymerization solution using injection needles allowed to increase the diameter of spheres up to (1.2 ± 0.1) mm and the number of cells in each sphere up to 8,000-12,000.Visual assessment revealed no differences between two options of encapsulation, cell size varied within the same ranges, they were mostly rounded and homogeneously distributed over the volume of carrier (Fig. 1).
In 24 hrs of eMSCs culture we assessed the cell state in the spheres.It has been shown that encapsulation regardless of the used method did not adversely affect cell viability, by MTT test it was above 90%.Cells in the 'small' (eMSCs-0.5)and 'large' (eMSCs-1.2) spheres reduced the AB similarly ((0.17 ± 0.05) AFU/ well and (0.16 ± 0.02) AFU/well, respectively), indicating the same level of metabolic activity.
The approach tested for vitrification of eMSCs-1.2 which was developed for eMSCs-0.5 [7] did not give satisfactory results.Based on the previous experience we assumed that the alginate carrier might decelerate the penetration of cryoprotectant into the cells.Probably the duration of incubation in DEPS-1 solution prior to vitrification established previously as optimal for 'small' spheres was insufficient for cells placed into the carriers of larger diameter.To verify this assump-tion we performed screening and selected two options of longer treatment with cryoprotectant solution: one-step exposure in 100% DEPS-1 for 5 min, and two-step 4-min exposure in 50% DEPS-1 at the first stage and 30 sec in 100% DEPS-1 at the second stage.
As it was found, both options of treatment allowed to increase considerably the indices of viability and metabolic activity of eMSCs following vitrification and warming (Table ).Moreover, we have found an advantage of one-step exposure protocol, i. e. average metabolic activity and viability of eMSCs after vitrification and thawing of the cells treated with DEPS-1 solution for 5 min were higher than those in the case of twostep exposure in a mixture of cryoprotectants for 4 min at the first stage and 30 sec at the second one.Such differences are important from a technological point of view, it is obvious that one-step protocol of exposure Жизнеспособность по МТТ-тесту и метаболическая активность по АВ-тесту иМСК-1,2 после витрификации-отогрева с использованием различных протоколов экспозиции Viability by MTT assay and metabolic activity by AB test of eMSCs-1.2 after vitrification-warming using different exposure protocols Примечание: за 100% приняты значения до криоконсервирования; 1 -различия значимы по отношению к показателям до криоконсервирования (p < 0,05), 2 -после отогрева (p < 0,05), 3 -витрификации с одноэтапной инкубацией в ДЭПС-1 (p < 0,05, n = 6).
in the solution of cryoprotectants is simpler if compared to a two-step one.Interestingly, that such a continuous incubation of non-encapsulated cell suspension resulted in their complete death (viability of (4 ± 2)%), likely due to high concentration of cryoprotectants in solution.It can be suggested that high values of cell viability and metabolic activity obtained after vitrification within the alginate spheres could result from the ability of alginate hydrogel to reduce negative effects on the cells appeared during cryopreservation.This question requires a more detailed individual study, as its solving can significantly improve the efficiency of cryopreservation of biological objects.
Viability and metabolic activity of cells assessed immediately after thawing could not be considered as reliable indices of cryopreservation.It is known that the destructive processes initiated during cryopreservation are developed in the cells during some period of time post warming.For instance, the apoptotic cascade activation after warming of cell suspension that led to a further reduction of cell viability during the following days was reported [9].Also we have established that during reculturing of MSCs after cryopreservation within macroporous carriers the viability of suspension decreased and reached a minimum to the end of the first day and then again increased likely due to proliferation of survived cells [1].In the present research it was revealed that following 24 hr reculturing after vitrification and warming of 'large' spheres the indices of viability and metabolic activity were lower by 7-8% if compared with those immediately after thawing.Therefore, the findings attested the preservation of morphological and functional properties of MSCs undergone vitrification within alginate microspheres, but there is a need in further improvement of cryopreservation methods preventing the decrease in cell viability during reculturing.

Conclusions
Mesenchymal stromal cells can be encapsulated into alginate spheres of different diameters (0.5 ± 0.2) and (1.2 ± 0.1) mm preserving their viability and metabolic activity.
MSC vitrification within the spheres of various diameter is largely determined by the exposure time with the solution of cryoprotectants.With the increase in the diameter of spheres from (0.5 ± 0.2) to (1.2 ± 0.1) mm the exposure time required to maintain viability and metabolic activity of cells after vitrification also increases.
The viability and metabolic activity of MSCs involved into alginate spheres of different diameter can be preserved during cryopreservation both by slow freezing and vitrification.

Fig. 2 .
Viability of eMSCs-1.2mm by MTT assay ( ) and metabolic activity by AB test ( ) after cryopreservation using slow freezing under 10% DMSO protection or vitrification in DEPS-1solution; the values obtained before cryopreservation are denoted as 100%; * -the differences are significant in relation to the indices prior to cryopreservation (p < 0.05, n = 6), # -the indices after vitrification (p < 0.05, n = 6).