**2.3 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 × 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 over seven days in the above described condition.

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

#### **2.4.1 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

Cryopreservation of Rat Bone Marrow Derived Mesenchymal

cells changed into spindle-like fibroblasts.

cryopreserved MSCs.

Stem Cells by Two Conventional and Open-Pulled Straw Vitrification Methods 7

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

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 non-

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.

### **2.4.2 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-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 fresh 0.5% alizarin red solution (1).

## **2.4.3 Colony-forming unit assays**

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.

#### **2.4.4 Statistical analysis**

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 in triplicate.
