Effect of Cryopreservation on Functional Status of Bone Marrow Hematopoietic and Mesenchymal Stem Cells in Animals with Autoimmune Pathology

The application of cryopreserved autologous bone marrow (BM) is the reconstructive therapy tool for autoimmune diseases (AIDs). The BM use efficiency is determined by preservation rate of its hematopoietic and mesenchymal stem cells (HSCs and MSCs, respectively). This fact determines the need to design the optimal cryopreservation regimens for the BM of AIDs donors. Here we assessed a functional status of bone marrow HSCs and MSCs of mice with adjuvant arthritis (AA), initiated by Freund's complete adjuvant administration. The bone marrow of CBA/H mice to day 14 of AA (chronic stage) and that of healthy animals were cryopreserved under dimethyl sulfoxide protection. The content of HSCs of various differentiation extent was determined by spleen colony formation in vivo to days 8 (CFU-S-8) and 14 (CFU-S-14). Functional activity of MSCs in BM was studied in vitro by the content of fibroblast colony forming units (CFU-F). There was established the fact of altering the functional potential of hematopoietic progenitor cells of BM of various differentiation levels (CFU-S-8, CFU-S-14) and their stromal microenvironment (CFU-F) during clinical manifestation of AA. After cryopreservation of AA animals' BM cells with 7% DMSO a functional potential of CFU-S and CFU-F was established to be similar to the healthy animals' BM, cryopreserved with 10% DMSO. The cryopreserved with the proposed regimen autologous BM may be used for a targeted recovery of lymphohemopoietic system in AA animals.

Nevertheless, there are the convincing data that the cryopreservation might be not only a successful tool for long-term storage of biological material, but also the factor of controlling an internal state of biological object [6][7][8].In particular, there is an evidence of a transient change in the expression level of genes, associated with hematopoietic and immune modulatory activity after the effect of certain cryopreservation regimens on HSCs and MSCs of late gestation fetal liver, providing the material with new functional properties [8].Thus there is an evident need of selecting the cryopreservation regimen, which would be able to approach the functional potential of pathologically changed BM cells to the indices of healthy animals.
Due to the mentioned above, our research was aimed to experimentally substantiate the selection of cryopreservation regimen for AA animal BM, able to "restore" a functional potential of hematopoietic and mesenchymal stem cells.

Materials and methods
All the manipulations with animals were carriedout in accordance with the General Principles of Experiments in Animals, approved by the 5 th National Congress in Bioethics (Kyiv, 2013) and consistent with the statements of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Strasbourg, 1986).The experiments were performed in 4-month-old male CBA/N mice weighing 20-22 g, kept under the standard animal house conditions at the Institute for Problems of Cryobiology and Cryomedicine of NAS of Ukraine (Kharkiv).We divided the animals into following groups: group 1 comprised healthy mice (n = 10), and group 2 had the animals with AA (n = 10).The adjuvant arthritis was induced in mice by subplantar administration of 0.1 ml complete Freund's adjuvant (Sigma-Aldrich, USA) [12].The inflammation intensity was assessed to day 14 of AA development by the integral index of clinical manifestation of pathology: arthritis index, which represented a ratio of circumferences of experimental and control ankle joints.In healthy animals the arthritis index was assumed as 1.Derivation of bone marrow suspension.To derive BM suspension the animals were decapitated under light ether anesthesia.The BM cells were washed out with syringe from femurs using medium 199 (Institute of Poliomyelitis and Viral Encephalitis, Russia) supplemented with 10% embryonic calf serum (BioloT, Russia) and 2% sodium citrate (Weifang Ensign, China) (hereinafter the handling medium).The cell suspension was derived by repeated passage through the needles of decreasing diameter (0.9-0.4 mm) with the following passing through a nylon filter.The number of nucleated cells in BM suspension were counted in Goryaev's chamber.
Cryopreservation of bone marrow cells.A solution for BM cryopreservation was prepared with the handling medium supplemented with 14 or 20% dimethyl sulfoxide (DMSO) (Arterium, Ukraine).Solution for cryopreservation was dropwise added in 1:1 ratio at 4°C (final cryoprotectant concentration was 7 and 10%) to the obtained with handling medium BM cell suspensions.The cells were exposed in cryopreserved solution for 10 min at the same temperature.
BM cells of 6.0×10 6 cells/ml concentration were cryopreserved using the programmed freezer (Cryoson, Germany) in 1.8 ml plastic vials (Nunc, Germany): cooled with the rate of 1 deg/min down to -40°C and then immersed into liquid nitrogen [6].The samples were thawed in 38...40°C water bath for 40-50 seconds until disappearance of a solid phase.The cells were washed of DMSO by slow addition of double volume of handling medium with the following centrifugation (200g, 10 min).
(CFU-S-8 were more differentiated, and CFU-S-14 were less differentiated [11]) an index of differentiation (ID) was used, which was the ratio of CFU-S-14 and CFU-S-8 concentrations.
Assessment of the functional capacity of BM mesenchymal stem cells.The BM content of colony forming units of fibroblasts (CFU-F) which according to S. Hirohata [9] correlated with the content of MSCs was determined by BM culturing in vitro with explantation density of 1×10 4 cells/cm 2 with irradiated (5 Gy) BM feeder cells (2×10 5 cells/cm 2 ) of guinea pig [4].The culturing was performed in CO 2 incubator at 37°C on a glass Petri dish (d = 3 cm, Anumbra, Czech Republic) for 14 days in Iscov's medium (Sigma, USA) supplemented with 10% embryonic calf serum.
To the 14 th day, the number of colonies of at least 50 cells was determined with light microscope MIKMED-2 (LOMO, Russia) and inverted microscope Axiovert 40C (Carl Zeiss, Germany).
The obtained data were statistically processed with Student's method using Excel software (Microsoft, USA).The data were presented as the mean ± standard deviation.Differences were considered as statistically significant at p <0.05.

Results and discussion
Swelling of joints is one of the clinical signs of AA development, the severity of which was evaluated by arthritis index [20].To the 14 th day after administration of Freund's adjuvant, this index was 1.6 times higher than the control (1.0), that indicated development of AA.
Assessment of the functional state of BM stem cells in animals with AA showed the significant differences in colony-forming activity of CFU-S-8 and CFU-S-14 if compared with those in healthy animals (Fig. 1A).A significant decrease (2.5 times, p < 0.05) in the number of CFU-S-8 formed colonies was accompanied by 1.3 times increase in the content of CFU-S-14 formed colonies, which caused the rise of ID if compared with the control ((3.65 ± 0.19) and (1.0 ± 0.05), respectively, p<0.05).Established in our research redistribution in subpopulations of CFU-S with various differentiation level which accompanied AA development could be the evidence of changes in structural and functional characteristics.For example, in our previous studies we have shown that AA resulted in a lack of the most differentiated hematopoietic progenitor cells (colony-forming units of granulomonocytopoiesis in BM) that could be a possible reason for increased content of more potent CFU-S-14 [6,20].Such redistribution of hematopoietic progenitor cells in BM might arise from developing inflammation in an organism.Indeed, AA development is accompanied with a significant change in cytokine profile in an organism [15,17,18], and haematopoietic system quickly responds to that.According to S.P. Sivalingam et al. [17] the patients with AA have increased levels of IL-6, IL-8 interleukins and granulocyte-macrophage colony stimulating factor.It is known that IL-6 stimulates differentiation of pluripotent HSCs, including high-proliferating CFU-S-14, and IL-8 controls the formation, chemotaxis and activation of neutrophils, being the main cell substrate of leukocytosis at AA.Moreover, HSCs interact closely with MSCs [1,14].It is important that the increasing in content of more potent CFU-S (CFU-S-14) in BM of mice with AA correlated with the rise (in 1.4 times) of CFU-F content in BM of healthy animals (1.56 ± 0.13 and 1.12 ± 0.10, respectively, p < 0.05) (Fig. 1, B).It is possible that regulatory mediators produced by MSCs are more capable of stimulating the growth of more potent HSCs and/or inhibit the function of CFU-S-8.
It is known that a change in the structural and functional characteristics of BM cells affects their resistance to cryopreservation factors [2,6,8].Our assessment showed that cryoresistance of bone marrow HSCs and MSCs in healthy animals and animals with AA was different.
As shown in Fig. 2, A, a freezing regimen with 10% DMSO provided the highest preservation of functional potential of CFU-S-8 and CFU-S-14 of healthy animals' BM (67.50 ± 0.84 and 88.00 ± 6.16, respectively) with some increase of ID (1.30 ± 0.08).
Очевидно, что после криоконсервирования даже КМ здоровых животных показатели колониеобразующей активности обоих типов КОЕс изменялись, что соответствует полученным ранее результатам [4,11].При этом минимальные отклонения иссле-   with AA depended on the concentration of used cryoprotectant.Following freeze-thawing with 10% DMSO the integrity indices of both types of CFU-S and ID were closest to the fresh BM of animals with AA, i. e. with a significant increase of ID.Reduction of DMSO concentration down to 7% provided the maximum balanced ratio of both CFU-S subpopulations of animal bone marrow with AA and preservation of their high colony-forming function (about 60% of CFU-S-8 and 70% of CFU-S-14) if compared with native BM of healthy animals.
It was obvious that even after cryopreservation of BM from healthy animals the indices of colony-forming activity of both CFU-S types were changed, that corresponded to the previous results [2,5].Herewith, minimum deviations of the studied indices from the control were obtained when using 10% DMSO.The outcomes of cryopreservation for CFU-S of the animals BM with AA were unequal and depended on their differentiation level.In particular, all the used cryoprotectant concentrations variously inhibited colonyforming potential of CFU-S-14 if compared with nonfrozen-thawed BM of animals with AA.At the same time, CFU-S-8 activity even increased in case of using 7% DMSO.It is important that using of 7% DMSO caused the effect of "restoration" of colony-forming potential of pathologically modified BM, i. e. HSCs indices were "harmonized".
In regard to MSCs pool, especially CFU-F (Fig. 2B), their maximum preservation in BM of healthy mice was obtained after using 10% DMSO.In BM of animals with AA cryopreservation provided the preservation of 30 to 40% CFU-F, irrespective of cryoprotectant concentration.
It was found that preservation of the functional capacity of CFU-S and CFU-F of healthy animal BM and those with AA required different "optimal" conditions of cryopreservation.For example, 10% DMSO was the best for BM cryopreservation of healthy animals, while 7% DMSO for animals with AA.For BM of animals with AA a selected regimen allowed the ratio of stem cells of various differentiation level to approach the physiological value.Generally speaking, selective effect of cryopreservation factors on different subpopulations of CFU-S of healthy donors' bone marrow and those with AA is not completely studied.There are recent experimental data testifying to the change in expression level of some genes in HSCs and MSCs after cryopreservation [7,8], which can affect the perception of regulatory signals of hematopoietic microenvironment with receptor apparatus of HSCs [5] and result to a distortion of their colony-forming potential.Moreover, microenvironment cells (as shown in CFU-F) undergo the КМ разного уровня дифференцировки (КОЕс-8, КОЕс-14) и их стромального микроокружения (КОЕф) в период клинической манифестации АА.
Thus, the obtained results confirm the preservation of functional activity of HSCs and MSCs to be depending on their initial state and cryopreservation conditions.

Conclusions
1.There was established the fact of changed functional capacity in BM hematopoietic progenitor cells of various differentiation level (CFU-S-8, CFU-S-14) and their stromal microenvironment (CFU-F) during the AA clinical manifestation.
2. There was experimentally proven the selection of regimen for cryopreservation of BM from animals with AA, which was capable to "restore" functional potential of stem cells.It was found that cryopreservation of BM cells from animals with AA using 7% DMSO resulted in the approaching of functional potential of CFU-S and CFU-F to the indices of healthy animals' BM cells, cryopreserved with 10% DMSO.

Fig. 2 .
The number of colonies formed by CFU-S (A) and CFU-F (B) of cryopreserved BM of healthy animals and those with AA: -CFU-S-8; -CFU-S-14;-ID.The number of colonies formed by native BM of healthy animals was assumed as 100%; ID of native BM of healthy animals was assumed as 1.Differences are statistically significant (p < 0.05) if compared with: * -BM CFU-S-8 of healthy animals cryopreserved with 10% DMSO; # -BM CFU-S-14 of healthy animals cryopreserved with 10% DMSO.