**1. Introduction**

Bone marrow (BM) is a complex tissue containing populations of progenitor and stem cells (1). One type, hematopoietic stem cells (HSCs), can renew circulating blood elements such as red blood cells, monocytes, platelets, granulocytes and lymphocytes. The other is mesenchymal stem cells (MSCs), which possess two important properties of long-term self renewal and differentiate into osteoblasts, chondroblasts, adipocytes and hematopoiesis supporting stroma (2, 3). Their mesenchymal differentiation potential is retained even after repeated subcultivation *in vitro* (4, 5). Besides originating the forming mesenchymal tissue, many studies have demonstrated that MSCs could differentiate into various nonmesenchymal tissue lineages under appropriate experimental conditions *in vitro* and *in vivo*, such as hepatocytes (6, 7), cardiomyocytes (8, 9), lung alveolar epithelium (10), olfactory epithelium (11), inner hair cells (12), neurons and neuroglia (1, 4, 13). MSCs are spindle shaped fibroblast-like cells that are easily isolated, cultured and expanded *in vitro* due to their adherent characteristics, and not associated with any ethical debate (14). Thus, MSCs may be used in the treatment of a diverse variety of clinical conditions (15) such as engraftment of various organs (16). The long-term cultivation of MSCs may fail for many reasons: genotypic drift, senescence, transformation, phenotypic instability, and contamination or incubator failure. The inability to cultivate MSCs will result in the lack of MSCs for both experimental and clinical use (17). Therefore, it is necessary to cryopreserve MSCs as cell seeds. Although increasing telomerase expression of cells may overcome cell senescence (18), cryopreservation of hMSCs may be more practical in order to save time and culture materials (16, 19). Resuscitated MSCs can be subcultivated for many passages without a noticeable loss of viability and capability of osteogenic differentiation (20-22).

Formulating a cryopreservation protocol for hMSCs is required because these cells cannot survive for long periods under *in vitro* culture conditions. Slow rate cooling methods using dimethylsulfoxide (DMSO) as a cryoprotectant have been used for a wide variety of MSC lines established from bone marrow (23, 24), umbilical cord blood (23-25), hematopoietic progenitor cells (26) and mouse ES cell lines (27). In most protocols, cells are suspended in

<sup>\*</sup> Corresponding Author

Cryopreservation of Rat Bone Marrow Derived Mesenchymal

times were morphologically evaluated.

**2.3 Vitrification and thawing procedure** 

over seven days in the above described condition.

**2.4.1 Adipogenic induction** 

**2.4 Evaluation of the differentiation potential of cryopreserved MSCs** 

Pre and post-cryopreserved MSCs were seeded on coverslips in a six-well plate and cultured in DMEM with 10% FBS. Cells with nearly 80% confluency were exposed to DMEM supplemented with 5μg/ml insulin, 1 μM dexamethasone, 100 nM indomethacine, 0.5 mM methylisobutylxanthine (Sigma), and 10% FBS for 48 hours. Cells were then incubated in the same medium without dexamethasone. For control, cells were cultured in regular medium

**2.2 Cryopreservation of MSCs** 

Stem Cells by Two Conventional and Open-Pulled Straw Vitrification Methods 5

(FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. All cells were incubated at 37 °C, in an atmosphere of 5 % humidified CO2. After 48 hours incubation, the nonadherent cell populations were removed and the medium was added and replaced every three or four days for about two weeks. When the cells grew to 80% confluency they were harvested with 0.25% trypsin and 1 mM EDTA (Gibco, UK) for 5 minutes at 37°C, replated and diluted 1:3 on a 25 cm² plastic flask, again cultured to the next confluency and harvested. Prior to their use in inducing differentiation and vitrification MSCs that were passaged approximatly 15

MSCs at passage 4 of pre-cryopreservation were harvested and centrifuged at 400 × g for 15 minutes as mentioned above. Approximately 1 ×106 cells/ml of randomly selected batches

MSCs were cryopreserved by using a two-step exposure to the equilibration and vitrification solutions (34). The equilibration solution was 20% ethylene glycol (EG; Sigma) and the vitrification solution was composed of 40% EG, 18% Ficoll - 70 (Sigma) and 0.3 M sucrose (Sigma). All solutions were based on PBS (Sigma) containing 20% FBS. A pellet of ~1 × 106 MSCs (~10 μl) was first suspended in 50 μl equilibration solution for 5 minutes and then mixed with 500 μl vitrification solution for 40 seconds. Suspended MSCs were immediately transferred to 1.2 ml cryovials (Nunc) and plunged directly into liquid nitrogen. The OPS vitrification method was carried out according to Reubinoff et al. (33). For OPC, a pellet of ~1 × 106 MSCs (~10 μl) was first suspended in 50 μl equilibration solution for 5 minutes and then mixed with 500 μl vitrification solution for 40 seconds. Suspended MSCs were at once transferred to 0.25 ml plastic straws (IMV, L'Aigle, France). Immediately afterwards, the straws were immersed in liquid nitrogen for two months. Following storage, the cells were thawed by rapidly immersing the vials and straws in a water bath at 37 °C. After warming for about 7 seconds, (at approx. 1800 °C/min) the contents of the vials and straws were suspended serially in 0.5, 0.25 and 0 M sucrose in PBS containing 20% FBS. After thawing, the survival rate was evaluated by the trypan blue staining method. After removing some of the cell pellet and adding 0.4% trypan blue (Sigma), the cells were plated onto a slide and unstained cells were counted as live cells (26). The remaining cells were centrifuged at 200 × g for 10 minutes and washed three times with DMEM medium supplemented with 20% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were immediately plated at a density of 1 × 106 cells/ml in a 25 cm2 culture flask and subcultured

were cryopreserved by using the vitrification method or OPS vitrification.

freezing medium containing DMSO at 5-20%, transferred into glass or plastic cryovials and then frozen by cooling at 1.0 to 2.0 °C/min (28). Slow freezing reduces ice crystal formation and eliminates toxic and osmotic damage to cells through exposure to low concentrations of cryoprotectants while slowly decreasing temperatures (29). However, it is difficult to completely eliminate injury by intracellular ice formation. Damage by ice crystal formation in the cytoplasm during the freezing process is one of the possible causes of cell death; such conventional methods, are not applicable to hMSC cells because many of these cells die immediately after thawing (28). Alternately, vitrification, a rapid cooling method using a high concentration of cryoprotectant, could also be used. Vitrification can completely eliminate damage caused by ice crystal formation in the cytoplasm of cells during freezing (29, 30). It is also advantageous because the procedure takes a relatively short time and a programmable temperature decreasing container is not required (31).

Vitrification has been used for the cryopreservation of oocytes, fertilized eggs and embryos of several mammalian species including humans in order to prevent ice crystal formation (32). There have been some reports demonstrating that embryonic stem (ES) cells could be successfully cryopreserved by vitrification in recent years (27, 33,34). Moon et al. tested vitrification of the human amnion-derived mesenchymal stem cells (HAMs) by using a twostep exposure to equilibration and vitrification solutions (21). They used an EG-based cryoprotectant and their findings were in line with previous reports that showed the superiority of EG. EG has been proven to be less toxic on fibroblast and other somatic cells in comparison with permeating agents such as DMSO and propylene glycol (PROH) that have been used on murine and human embryos (35). However, as a long-term preservation method for HAMs, a well-defined protocol of cryopreservation needs to be established for a human bone marrow derived mesenchymal stem cell bank. In the present study, to confirm the proliferative capability and pluridifferentiation of cryopreserved adult hMSCs; we chose ethylene ficoll sucrose (EFS) 40 that contained 40% v/v EG for the vitrification solution. hMSCs that were cryopreserved for two months were resuscitated and cultivated for 15 passages. An analysis of their expansion, morphological and pluridifferentiation characteristics was undertaken. Finally, under induction conditions, adipogenic and osteogenic potentials have been discussed.
