**3. 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).

#### **3.1 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.

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

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-2 phosphate 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

For these assays, both thawed and non-cryopreserved cells were plated at 1 × 106 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 for comparison of the postthaw survival rate was performed using the χ<sup>2</sup> test. Statistically significant values were defined as p<0.05. All experiments were conducted

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

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.

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,

Subsequently these adherent cells grew as typically fibroblastic or spindle shaped.

oil red O for 2-3 hours and then rinsed with 60% isopropyl alcohol.

**2.4.2 Osteogenic induction** 

fresh 0.5% alizarin red solution (1).

**2.4.3 Colony-forming unit assays** 

following subsequent subculture (Fig 1D).

**3.1 Morphology and growth of vitrified-thawed MSCs** 

**2.4.4 Statistical analysis** 

suggesting senescence.

in triplicate.

**3. Results** 

Fig. 1. Morphology and growth of MSCs. (A) Primary (x 40), (B) Passage 4 (14 days, x 100), (C) Passage 4 (35 days, x 100) and (D) Passage 4 (40 days, x 100). In primary culture, cell growth was scattered with some colony formation. Followin subsequent subculture, the cells changed into spindle-like fibroblasts.

Fig. 2. Morphology and growth of vitrified-thawed MSCs. Phase contrast images of MSCs two months after thawing from: (A) vitrification method (x 100) and (B) OPS vitrification (x 40). MSCs had a similar morphology to fibroblasts and were indistinguishable from noncryopreserved MSCs.

Cryopreservation of Rat Bone Marrow Derived Mesenchymal

**6. Differentiation of post-cryopreserved MSCs** 

under the same condition (Fig 5C, D).

red without hematoxiline, x 100), (B).

marrow was first reported by Wang and Wolf (38).

**7. Discussion** 

Stem Cells by Two Conventional and Open-Pulled Straw Vitrification Methods 9

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

Fig 4. Evaluation of adipogenesis potential of MSCs under a phase-contrast microscope. Both the non cryopreserved MSCs and vitrified-thawed MSCs after treatment by adipogenic medium showed numerous neutral lipid vacuoles which accumulated in the cytoplasm. (A) Confirmed by oil red O staining (oil red O + hematoxiline, which one is the top right one: oil

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

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
