CRYOPRESERVATION OF RAT BONE MARROW DERIVED MESENCHYMAL STEM CELLS BY TWO CONVENTIONAL AND OPEN-PULLED STRAW VITRIFICATION METHODS

Received: 9/Mar/2009, Accepted: 22/Aug/2009 Objective: Mesenchymal stem cells (MSCs) are obtained from a variety of sources, mainly the bone marrow. These cells have a great potential for clinical research, however they cannot stay alive for long periods in culture. The aim of this study is to determine whether vitrification can be a useful freezing method for the storage of MSCs. Materials and Methods: Mesenchymal stem cells were isolated from rat bone marrow based on their capacity to adhere to plastic culture surfaces. MSCs were cryopreserved using both the vitrification method and open-pulled straw (OPS) vitrification and stored in liquid nitrogen with ethylene glycol ficoll (EFS) as a cryoprotectant for two months. The morphology and viability of thawed MSCs were evaluated by trypan blue staining. Furthermore, pre and post cryopreserved MSCs were induced to osteocyte and adipocyte with corresponding osteogenic and adipogenic medium. Results: After thawing, the viability rates were 81.33% ± 6.83 for the vitrification method and 80.83% ± 6.4 for OPS vitrification, while the values in the pre-vitrification control group were 88.16% ± 6.3 (Mean ± SD, n = 6). Post-cryopreserved cells from both the vitrification method and OPS vitrification also had a similar cellular morphology and colony-formation that was indistinguishable from non-vitrified fresh MSCs. In addition, the resuscitated cells cultured in induction medium showed osteogenesis. Mineral production and deposition was detectable by alizarine red S staining. Moreover, by applying an adipogenic differentiation condition, both pre and post cryopreserved cells differentiated into adipocyte and lipid vacuole accumulation that was stained by oil red O. Conclusion: Vitrification is a reliable and effective method for the cryopreservation of MSCs.


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)(21)(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)(24)(25), hematopoietic progenitor cells (26) and mouse ES cell lines (27). In most protocols, cells are suspended in 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.

Preparation and culture of MSCs
For isolation of rat MSCs; female Sprague-Dawley rats (weighing 200-250 g) with the approval from the Institute for Animal Care were obtained from the Animal Center, Faculty of Medicine, Guilan University of Medical Sciences. Rats were killed by intraperitoneal administration of a lethal dose of sodium pentobarbital. The femurs and tibias were carefully dissected away from attached soft tissue as previously reported with modification (1). The ends of the bones were cut, and the bone marrow was aseptically extruded with 5 ml PBS solution by using a syringe with a 21G needle and flushing the shaft ten times. The marrow tissue was dissociated by pipetting. The cell suspension was then centrifuged at 500 × g for 5 minutes and the supernatant was discarded. Bone marrow mesenchymal stem cells (BMSCs) were then mechanically dispersed into a single-cell suspension so that the density of BMSCs reached 10 6 cells/ml. At this point, marrow cells were plated in a 25 cm² plastic flask in Dulbecco's modified eagle medium (DMEM) containing 20% fetal bovine serum (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 times were morphologically evaluated.

Cryopreservation of MSCs
MSCs at passage 4 of pre-cryopreservation were harvested and centrifuged at 400 × g for 15 minutes as mentioned above. Approximately 1 ×10 6 cells/ml of randomly selected batches were cryopreserved by using the vitrification method or OPS vitrification.

Vitrification and thawing procedure
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 × 10 6 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 × 10 6 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 × 10 6 cells/ml in a 25 cm 2 culture flask and subcultured over seven days in the above described condition.

Adipogenic induction
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 www.intechopen.com as above. The medium was changed every third or fourth day. One week after induction, adipogenic differentiation was evaluated by the cellular accumulation of neutral lipid vacuoles that were stained with oil-red O (Sigma) and observed under an inverted microscope (17). Briefly, after fixation in 5% metanol, induced MSCs were stained in filtered oil red O for 2-3 hours and then rinsed with 60% isopropyl alcohol.

Osteogenic induction
To identify osteogenic differentiation, thawed and non-cryopreserved MSCs were cultured in 100 nM dexamethasone, 10 mM ß-glycerol phosphate and 50 μM ascorbic acid-2phosphate in 400 μl DMEM-LG supplemented with 10% FBS on coverslips in a six-well plate for subsequent staining. During the culture period, the medium was changed once per week. After 14 days, osteogenic differentiation was evaluated by staining the coverslips with fresh 0.5% alizarin red solution (1).

Colony-forming unit assays
For these assays, both thawed and non-cryopreserved cells were plated at 1 × 10 6 cells per ml and cultured for 14 days in 25 cm² tissue culture flasks. After 14 days, the cultures were stained with giemsa for 5 minutes. The formations of colonies were considered acceptable until passage 15 (P15) and those less than 2 mm in diameter or faintly stained were excluded.

Statistical analysis
Statistical analysis for comparison of the postthaw survival rate was performed using the χ 2 test. Statistically significant values were defined as p<0.05. All experiments were conducted in triplicate.

Results
The growth and morphology of MSCs appeared rather heterogeneous in primary culture as seen in Fig 1A. Under a phase contrast microscope, the cells appeared fibroblast-like, elongated and spindle shaped with a single nucleus (Fig 1B). These cells showed the ability to form colonies with the occasional cell sphere formation giving an impression of embryoid bodies ( Fig 1C). However, they progressively showed homogenous fibroblast-like features following subsequent subculture (Fig 1D).

Morphology and growth of vitrified-thawed MSCs
The duration of storage in frozen state for MSCs was two months. Post-cryopreserved MSCs from both the vitrification method and OPS vitrification had similar cellular morphology and colony-formation. Resuscitated MSCs first grew as isolated colonies after initial plating. Subsequently these adherent cells grew as typically fibroblastic or spindle shaped.
As the cells approached confluence, they assumed a more spindle-shaped, fibroblastic morphology (Fig 2A, B). The thawed and non-cryopreserved MSCs were subcultured until P15. Until the P9, fibroblast-like morphology was consistently observed in both the thawed and non-cryopreserved MSCs. At the P10, cells in both cultures became large and flat, suggesting senescence. www.intechopen.com

Viability of vitrified-thawed MSCs
Live/dead viability of MSCs was determined by the trypan blue staining test. The number of MSCs was counted and compared to that of the control. After thawing, the viability rates were 81.33% ± 6.83 for the vitrification method and 80.83% ± 6.4 for the OPS vitrification, while values with the pre-vitrification control group were 88.16% ± 6.3, respectively (Table  1). There were no differences in viability between them.

Colony forming unit assay
For these assays, cells of both thawed and noncryopreserved MSCs or cultures were plated at 1× 10 6 cells/ml and MSCs in culture showed a colony formation consisting of 40-80 cells in 25 cm² flasks for 14 days (Fig 3). After P9, the cells showed no colony forming ability which illustrated that colony forming ability decreases with increasing passages.

Differentiation of post-cryopreserved MSCs
After culturing for adipogenic differentiation, the accumulations of numerous neutral lipid vacuoles were detectable in the cytoplasm of vitrified-thawed cells (Fig 4A). Following three weeks of induction, oil Red O staining showed the lipid droplets with orange red color, which demonstrated the committed differentiation of MSCs into adipocytes (Fig 4B). The control cells showed no detectable lipid vacuoles. Under culture with osteogenic induction medium, resuscitated MSCs detached and floated in the medium. After three weeks, mineral accumulations were observed by alizarin red staining (Fig 5A, B). Similar results were observed in the group of non-cryopreserved MSCs when osteogenically induced under the same condition (Fig 5C, D).

Discussion
Cryopreservation is an important method to maintain cells for biological research and medical applications such as tissue engineering, gene therapy, cell transplantation, pharmacological testing and future therapeutic indications (17,28). A study on the longterm storage of BM-derived MSCs is of critical importance (1). The objective of the current investigation was to test the possibility that vitrification could be a useful method for the cryopreservation of MSCs. Thus, in the present study, we isolated MSCs from bone marrow of adult female rats. In culture; MSCs are characterized by their capacity to adhere to a plastic culture surface and form a fibroblast-like shape (Fig 1). Our data corroborated previous findings from other groups which showed homogenous fusiform features with oval vesicular nuclei (36) and the colony forming ability of MSCs, which decreased with increasing passages (37). The achievement of pure fibroblastic clones from murine bone marrow was first reported by Wang and Wolf (38).  cellular densities of colonies that were dependent upon the number of the cells plated per culture dish and was the method employed in this investigation. We also successfully vitrified MSCs and confirmed the morphology, viability rate and differentiation capacity of post-vitrification MSCs as compared with non-cryopreserved controls. Generally, most comprehensive studies on the cryopreservation of MSCs were carried out by using slow-rate cooling methods (17,(22)(23)(24). Slow-rate cooling methods using dimethylsulfoxide (DMSO) as a cryoprotectant are effective for a wide variety of cell lines, including ES cell lines (27,28). This method by decreasing temperature slowly with a low concentration of cryoprotectant is used to balance damage caused by various factors including ice crystal formation, fracture, toxic and osmotic damage (29).
It is difficult to completely eliminate injuries from intracellular ice formation, which is the main source of fracture and damage to the cytoplasm (29). It is also a time-consuming procedure and requires an expensive programmable freezer (31). On the other hand, there are some reports that stem cells are highly sensitive to cryoinjury and the vitrification method is a better choice for HES cell cryopreservation than conventional slow freezing and rapid thawing (28,33). This method has been previously applied for cryopreservation of oocytes, fertilized eggs and embryos of several mammalian species, including humans (32).

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In vitrification method, the concentrations of cryoprotectants seem to be dangerously high at the final phases, it happens at low temperatures, where the real toxic effect is minimal. Moreover, the high cooling and warming rates applied at vitrification provide an unique benefit compared to the traditional freezing (40).
Umbilical cord blood-derived mesenchymal stem cells (UCB-derived MSCs) were vitrified by vitrification and by programmed freezing without dimethyl sulfoxid (DMSO) by Wang et al. (2011). Their results showed that the viability of thawed UCB-derived MSCs was enhanced from 71.2% to 95.4% in the presence polyvinyl alcohol (PVA) for vitrification, but only < 10% to 45% of viability was found for programmed freezing (41). While, Kim et al. reported that Post-thaw colony-formation of embryonic stem cells (ESCs) was detected only after a slow freezing using DMSO by stepwise placement of a freezing container into a -80°C deep freezer and subsequently into -196°C liquid nitrogen, while no proliferation was detected after vitrification (42). Also, hMSCs from pre-and post-cryopreservation by slow freezing had similar colony-formation and cellular morphology similar to our resuls (17). Low survival of human ESCs has been also reported when they are frozen slowly with DMSO (43). There have been several reports to demonstrate the superiority of vitrification to other freezing programs for human ESCs, because it is able to avoid cell injury resulted from ice crystal formation (42,44). Carvalho et al. (2008) reported that viability of frozen BM-MSCs by slow freezing which was to added 5% DMSO was 94.76% and 90.58% viability before versus after cryopreservation (45). Also, the high survival rate (81.8%) is obtained after cryopreservation of human ESCs by programmed freezing (46). Our result showed 81.33% and 80.83% cell viability of two different cryopreserved vitrification methods versus 88.16% viability of non-cryopreserved cells. Nevertheless, we are not able to deny the feasibility of vitrification program for effective cryopreservation of SCs (42).
Also Fujioka et al. vitrified ESCs using EFS40; ethylene glycol, 18% ficoll 70,000 MW and 0.3 M sucrose (similar to this work) and slow-freezed in freezing medium containing 10% DMSO. Their results indicated that the vitrification methods yielded higher cell recovery and survival rates than did the slow-rate freezing methods (28).
DMSO has been well known as a toxic agent for stem and progenitor cells, and particularly for human embryonic stem cells. It is also known as a powerful differentiation agent that may interact with the chromatin structure (47).
Therefore, the selection of suitable cryoprotectants is essential. Cryopreservation procedure ought to become different according to cell type and cellular characteristics. So, all components consisting of freezing and thawing procedures should specifically be determined in each case of cryopreservation (42).
This study was DMSO-free vitrification of MSCs using cryovial and straw. Straws, preferably thin straws, even in sealed form, can be cooled safely with an increased cooling rate (40). There was any report about vitrification of MSCs without DMSO.
So that in the present experiment, we have observed the viability and proliferation capability as well as differentiation potential of cryopreserved MSCs in vitro. Inverted microscope findings showed that, after culturing for seven days, numerous MSCs adhered well to the surface culture dish (Fig 1). In a study by Moon et al. on human amnion-derived mesenchymal stem cells (HAMs), they observed that slow freezing resulted in a lower survival rate compared with vitrification, indicating a high efficiency of the vitrification procedure (21).
Moreover, Heng (48) demonstrated that 39.8 ± 0.9% of the hMSCs could be recovered after cryopreservation using a conventional slow freezing method which was lower than that our result (Table 1). Here, resuscitated MSCs kept a high proliferative potential. They first grew as clones after limiting dilution and then expanded rapidly with the typical features of spindle-shaped cell bodies and confluence after a lag phase of 6-14 days. There were no differences among the pre and post-cryopreservation of colony formation at the same passage (Fig 2). In addition, the results showed that the passage procedure was selective for MSCs and it could be inferred that the passaging of resuscitated MSCs increased cellular homogeneity.
Ji et al. demonstrated that cryopreservation of encapsulated HES cells offers better cellular viability, higher colony recovery, and less differentiation than the slow-freezing techniques most commonly used to preserve HES colonies. Therefore, this difference in recovery may be due to differences in cell lines, freezing and thawing protocols, or growth substrate (49).
On the other hand, previous studies have shown that cyropreservation had no effect on either the proliferation or osteogenic and adipogenic differentiation of human MSCs in vitro (5,22). In agreement with these reports, Liu et al. using slow cooling with Me2SO as a cryoprotectant and rapid thawing demonstrated that thawed cryopreserved human MSCs had higher survival rates in comparison with non-cryopreserved MSCs and differentiated into osteoblasts when cultured in osteogenic media. Also they found that cryopreserved hMSCs could not differentiate into osteoblasts spontaneously when cultured in basic culture media (50). In addition to the characteristics described above, our present study demonstrated that post-cryopreserved MSCs from bone marrow were still pluripotential and differentiated into osteoblasts and adipocyte under appropriate culture conditions (Fig  3, 4). These observations suggest that the "memory" of proliferation and differentiation in MSCs is not affected by the process of vitrification. The ability of frozen BM-MSCs by slow freezing to differentiate into mesenchymal derivatives (such as osteogenic and adipogenic) reported by Lee et al. (4). In this study, we established a two-step vitrification protocol for MSCs using EFS containing 40% v/v EG for the vitrification solution, which is widely used for successful vitrification of mouse embryos (30), human blastocysts (32) and ESCs (34). Our findings are in line with the reports by Gajda et al. who used the same methods for somatic cells which have been proven to be less toxic on bovine skin fibroblast and cumulus cells (35).
EG is the most commonly used cryoprotectant for vitrification due to its low molecular weight and low toxicity (35). In addition, additives with high molecular weights, such as sucrose, can significantly reduce toxicity by decreasing the concentration of permeating agents required for the vitrification solution. We also used ficoll as a macromolecule to promote permeation by cryoprotectants, which seems to have the advantages of lower toxicity, higher solubility and lower viscosity (30). In a study by Moon et al. on HAMs, they observed that the combination of EG with either PROH or DMSO resulted in a very low survival rate of HAMs as compared with EFS alone (21). Also, Kuleshova and Lopata ascertained the advantages of vitrification when compared with earlier applied cryopreservation techniques (51). These advantages include the control of solute penetration and dehydration rates, prevention of prolonged temperature shock and damage from ice formation, and inexpensive equipment and running costs. Vitrification is a process where glass-like solidification of a solution occurs without the formation of ice crystals inside living cells, by exposure to a high concentration of cryoprotectant with a higher cooling rate (52). This procedure is a simple method to circumvent the obstacles of slow freezing without the need for a freezing container to modulate the reduction of temperature in a deep freezer before storing in liquid nitrogen (21).

Conclusion
In the present experiment, it was shown that vitrification can be an efficient storage method for MSCs without losing their activity and usual properties. Such a system will be exceedingly helpful for both experimental research and medical applications.