**7. Discussion**

8 Current Frontiers in Cryopreservation

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 85 81 82 2 93 92 90 3 78 77 70 4 96 81 79 5 89 72 81 6 88 85 83 Mean ± SD 88.16(SD ± 6.30) 81.33 (SD ± 6.83) 80.83(SD ± 6.40)

Table 1. Percentage of cell viability of non-cryopreserved and two different cryopreserved

For these assays, cells of both thawed and noncryopreserved MSCs or cultures were plated at 1× 106 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

Fig 3. Phase contrast images of typical MSCs colony morphology before (A) and after (B) cryopreservation. Cell sphere formation in the MSCs culture produced colony formation

which illustrated that colony forming ability decreases with increasing passages.

**Vitrification method (%)**

**OPS method (%)** 

**4. Viability of vitrified-thawed MSCs** 

**No**. **Before vitrification** 

OPS: open-pulled straw, student t-test (p<0.05).

units that stained with giemsa (A and B: x 40).

**5. Colony forming unit assay** 

vitrification methods by the trypan blue staining test.

1). There were no differences in viability between them.

**(%)**

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).

Furthermore, Eslaminejad et al. obtained an average of 15-17 clones, each one consisting of several fibroblastic cells per 24-well plate (39). This approach yielded both the number and

Cryopreservation of Rat Bone Marrow Derived Mesenchymal

benefit compared to the traditional freezing (40).

Stem Cells by Two Conventional and Open-Pulled Straw Vitrification Methods 11

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

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).

rate (40). There was any report about vitrification of MSCs without DMSO.

and survival rates than did the slow-rate freezing methods (28).

may interact with the chromatin structure (47).

determined in each case of cryopreservation (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

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

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

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

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

Fig 5. Differentiation potency of nonvitrified and vitrified-thawed MSCs observed under a phase-contrast microscope. (A) Nonvitrified MSCs after osteogenic induction. (B) Alizarin red staining, x 100. (C) Vitrified MSCs after osteogenic induction. (D): Alizarin red staining, x 100. After induction of differentiation for 2-3 weeks in respective induction media, the mineralized extracellular matrix of pre and post-vitrification MSCs stained positively with alizarin red (arrows).

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, 22-24). 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).

Fig 5. Differentiation potency of nonvitrified and vitrified-thawed MSCs observed under a phase-contrast microscope. (A) Nonvitrified MSCs after osteogenic induction. (B) Alizarin red staining, x 100. (C) Vitrified MSCs after osteogenic induction. (D): Alizarin red staining, x 100. After induction of differentiation for 2-3 weeks in respective induction media, the mineralized extracellular matrix of pre and post-vitrification MSCs stained positively with

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, 22-24). 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,

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

alizarin red (arrows).

toxic and osmotic damage (29).

(32).

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

Cryopreservation of Rat Bone Marrow Derived Mesenchymal

before storing in liquid nitrogen (21).

**8. Conclusion** 

**9. Acknowledgments** 

**10. References** 

conflict of interest in this article.

2007; 417(3): 281-285.

2004; 320(1): 273-278.

(22): 3479-3484.

Cell Dev Biol Anim. 2004; 40(3-4): 76-81.

418: 41-49.

Stem Cells by Two Conventional and Open-Pulled Straw Vitrification Methods 13

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

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

This research was a part of proposal supported by a grant (3/132/8162 /P) funded by the Guilan University of Medical Sciences and was done in the Cellular and Molecular Research Center at the Faculty of Medicine. The authors wish to thank both centers. There is no

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[4] Lee MW, Choi J, Yang MS, Moon YJ, Park JS, Kim HC, et al. Mesenchymal stem cells

[5] Bruder SP, Jaiswal N, Haynesworth SE, Growth kinetics, self renewal, and the osteogenic

[6] Kang XQ, Zang WJ, Song TS. Xu XL, Yu XJ, Li DL, et al. Rat Bone marrow mesenchymal

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[8] Baharvand H, Mathae KI. Culture condition difference for stablishment of new

removed medium of rat's bone marrow primary culture and their differentiation

improve the cognitive ability of an Alzheimer's disease rat model. Neurosci Lett.

from cryopreserved human umbilical cord blood. Biochem Biophys Res Commun.

potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem. 1997; 64(2): 278-294.

stem cells differentiation into hepatocytes *in vitro*. World J Gastroenterol. 2005; 11

Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002;

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exceedingly helpful for both experimental research and medical applications.

into skeletal cell lineage. Yakhteh. 2008; 10(1): 62-72.

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).
