Effect of Encapsulation into Alginate Microspheres on Viability of Mesenchymal Stromal Cells after Exposure with Penetrating Cryoprotectants

The effect of encapsulation into alginate microspheres (AMS) on viability of mesenchymal stromal cells (MSCs) after exposure to the solutions of penetrating cryoprotectants dimethyl sulfoxide (DMSO), ethylene glycol (EG) and 1,2-propane diol (1,2-PD) with concentrations varying from 1.5 to 9 M during 1.5 and 5 min was studied. It was shown that the viability of MSCs both as a suspension and encapsulated in AMS decreased with a rise in cryoprotectant concentration and exposure time. Encapsulation into AMS significantly protected the cells from cytotoxic damage of the cryoprotectants, e. g. if the 5-min exposure to 9 M DMSO, EG and 1,2-PD resulted in the death of quite all the cells in the suspension, the viability of encapsulated MSCs after exposure under similar conditions made 60, 80 and 52% respectively. It was found that the studied penetrating cryoprotectants had a various cytotoxicity to MSCs, and it decreased in the row DMSO > 1,2-PD > EG. The findings can be used to develop a multi-component vitrifying solution.

of encapsulated mesenchymal stromal cells there is a need in developing new and improving the existing methods of cryopreservation.Therefore the study of the effect of encapsulation on cryopreservation protocols is quite important.
One of the basic approaches to cryopreserve mesenchymal stromal cells (MSCs) within the AMS is vitrification, which is conventionally realised using high concentrations of cryoprotectants [9].Their mixtures are used to reduce the total toxic effects of cryoprotectants [11].Thus, it has been shown [7], that a high viability of mesenchymal stromal cells in suspension (sMSCs) can be achieved with vitrification, which was performed using a cryoprotectant mixture DEPS, comprising dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propanediol (1,2-PD) and sucrose.
During vitrification of the cells enclosed into AMS, it should be considered that the alginate hydrogel structure affects the rate whereat the mass transfer processes proceed [10,20] and, consequently, slows down the penetration of cryoprotectants, as well as the saturation of the cells with them.In this case, a successful vitrification can be achieved in two ways.
The first way is to prolong an exposure with solutions of cryoprotectants.We have found that a high viability of MSCs following vitrification inside AMS required their long exposure with the vitrification solution DEPS compared to the cells being in the suspension.The exposure time enhanced with increasing diameter of the AMS from 0.5 up to 1.2 mm [24].Herewith it is important to have in mind that long term contact of MSCs with the solutions of cryoprotectants can cause a toxic damage to the encapsulated cells.
The second way is to raise the concentration of cryoprotectants.In case of DEPS mixture, we have increased the concentration of cryoprotectants possessing a minimal cytotoxic effect.It should be noted that nowadays there are no reports on the effect of exposure of encapsulated human dermal MSCs (eMSCs) in the solutions of cryoprotectants constituting DEPS mixture rendered on cell viability.
Therefore the aim of this research was to study the influence of exposure to cryoprotectants (ethylene glycol, 1,2-propanediol and dimethyl sulfoxide) on viability of mesenchymal stromal cells in suspension and alginate microspheres.

Materials and methods
Mesenchymal stromal cells were isolated from human dermis by explantation of the pieces [17].Skin fragments were obtained after a written consent from adult donors.The cells were cultured in α-MEM (Sigma, USA) supplemented with 10% fetal bovine serum (Sigma), 2 mM L-glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin at 37°C, 5% CO 2 and 95% humidity.

Результаты и обсуждение
Ранее нами было установлено, что получить высокие показатели жизнеспособности сМСК и MSCs were encapsulated as described previously [18].After reaching 60-70% of confluence in a monolayer the cells of passages 2-3 were detached from culture plastic using Trypsin-Versene solution by the standard methods and centrifuged at 200g for 7 min.The resulted pellet was re-suspended in 1.2% purified sodium alginate solution (Sigma), then the suspension was dropwise added into 100 mM CaCl 2 solution within 10 min for polymerization.An average microsphere size was of 1.2 mm.The obtained eMSCs were washed with a physiological solution (PS), containing 0.9% NaCl and 25 mM HEPES, and were used for further experiments.
Exposure was carried out at room temperature either for 1.5 or 5 min [23].The tubes containing either capsules or cells in 100 ml PS were filled with 900 ml of a cryoprotectant (DMSO, 1,2-PD or EG) at various concentrations (1.5, 3, 5, 9 and 7.5 M).After the exposure with cryoprotectants the samples were placed in a 0.5 M sucrose solution in PS, and then gradually diluted ten-times with the culture medium.
To determine the number of viable MSCs in the suspension or microspheres we used the MTT test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) [3].With this purpose, the cells were incubated for 2 hrs in a medium containing 5 mg/ml MTT.The cells accumulated formazan were counted using a light microscope (CETI, Belgium).The viability index was determined as the ratio of the number of cells that had accumulated formazan to the total number.Samples not incubated with cryoprotectants served as the controls and their viability was assumed as 100%.
For statistical analysis of the results obtained there was used R environment; the data were presented as M ± m.The significance of the differences between the samples was evaluated by parametric method using Student's t-test.

Results and discussion
We have shown previously that high viability of sMSCs and prevention of ice crystals appearance during rapid freezing and thawing were possible after 1.5 min exposure with a mixture of cryoprotectants DEPS and subsequent vitrification [7].Successful vitrification of eMSCs was achieved after the exposure time with the same solution was increased up to 5 min [22].Therefore, in the present study the influence of cryoprotectants constituting DEPS on viability of eMSCs and sMSCs was studied after exposure to DMSO, EG and 1,2-PD cryoprotectants for 1.5 and 5 min to interpret closely the previous results.
Note: * -differences are statistically significant if compared with cell viability after exposure with cryoprotectant in 1.5 M concentration (p < 0.05); # -differences are statistically significant if compared with viability of cells in suspension after exposure under similar conditions (p < 0.05)
Такая модификация растворов обеспечивает успешную витрификацию иМСК и других типов клеток по протоколам, разработанным для среды «ДЭПС».На основании полученных результатов however, viability level dropped down to 51% after exposure with 9 M 1,2-PD solution.At the same time a significant reduction on sMSCs viability was observed after exposure to EG and 1,2-PD in 3 M concentration.When increasing the concentration of these cryoprotectants the viability level of sMSCs continued to decline.After exposure with cryoprotectants in 9 M concentration the viable cells were virtually absent.
These findings are consistent with the general notion about toxic effects of cryoprotectants, according to which the level of cell viability decreases with increasing a contact time and concentration of cryoprotectant.The reduced toxic effect of cryoprotectants resulted from encapsulation of cells into AMS was shown in this study for the first time, and this should be considered when developing the protocols for vitrification of biological objects.
It could be assumed that the reduction of toxic effects of penetrating cryoprotectants on eMSCs was due to physical properties of alginate hydrogel.It is known that polymerization of alginate hydrogel results in gaining porous structure.Depending on the concentration and viscosity of the alginate the pore size could be 3.6 nm or more [1,6,12].Some authors have demonstrated that the very porosity of alginate carrier affects the exchange of substances between cells and medium.So, A. Lawson et al. [10] established that the penetration rate of substances into an alginate microsphere depended on its radius and porosity.Similar results were reported by A. Gautier et al. [5].They believed the mass transfer through the alginate hydrogel to obey the Fick's law of diffusion.It has been shown that the vitamin B12 mass transfer coefficient was inversely proportional to the coefficient of the substance transfer through the hydrogel which in turn was directly proportional to the AMS size.In particular, for AMS of 1 mm diameter the transfer coefficient was two times bigger than for capsules of 0.6 mm diameter.Based on the above mentioned we can conclude that the slowing-down of mass transfer between an environment and the encapsulated cells enables the avoiding of prolonged exposure to high concentrations of cryoprotectants, which resulted in a sharp osmotic and toxic responses of cells.However, in this case the saturation of cells with cryoprotectants was not disordered, that allowed to successfully use the protocols of vitrification and to obtain bigger amount of viable cells within the carrier after the cycle of vitrification-warming.
The results of this research suggested various cytotoxicity of the investigated penetrating cryoprotectants, which was reduced in a row DMSO > 1,2-PD > EG.
tectants.It is known that the optimum concentration of DMSO to cryopreserve MSCs is 5-10% and the use of higher concentrations (over 20%) leads to the development of cytotoxic injury [13,14,16,19].
Along with this it has been shown [4,21], that 1,2-PD in the concentrations necessary for vitrification had a pronounced cytotoxic effect compared with EG. T. Herskovits et al. [8] found that 1,2-PD in 8.5 M concentration caused the denaturation of more than 50% proteins, and in case of EG the critical concentrations were above 11%.M. Aye et al. [2] showed that EG had less damaging effect on genetic material if compared with 1,2-PD.
This modification of the solutions provides a successful vitrification of eMSCs and other cell types under the protocols developed for the DEPS mixture.On the base of the findings we can assume that the bulk of the multi-component vitrifying solution should comprise EG, but 1,2-PD and DMSO can either act as additives or to be absent at all.

Conclusions
Encapsulation into alginate microspheres enables minimizing the contact time with high concentrations of cryoprotectants, leading to the development of abrupt osmotic and toxic responses of cells to these concentrations of cryoprotectants.However, in this case the saturation of cells with cryoprotectants is not disordered.
The cytotoxicity of cryoprotectants in respect of eMSCs has been shown to be reduced in the row DMSO > 1,2-PD > EG.
It has been suggested that when creating a multicomponent vitrifying solution the EG should be used as the main substance and 1,2-PD and DMSO may be either in small quantities or absent at all.