285 VIABILITY AND GENE EXPRESSION OF MESENCHYMAL STEM CELLS CRYOPRESERVED WITH DIFFERENT CRYOPROTECTANTS

Abstract

Long-term storage of stem cells with self-renewal plays a pivotal role in cell tissue engineering, which could be a novel option for improving regenerative diseases. However, the choice of a selective cryoprotectant is still to be addressed. Dimethyl sulfoxide (DMSO), which in current practice is the most widely used as a cryoprotectant for cell freezing, is known to have toxic side effects (Wang et al. 2007 Cryobiology 55, 60–65). In this study, therefore, the effect of two different cryoprotectants, used alone or in combination, on frozen–thawed porcine mesenchymal stem cells (MSCs) was investigated by evaluating their viability, apoptosis, and gene expression patterns. MSCs isolated from bone marrow were cultured in advanced Dulbecco's modified Eagle medium (ADMEM) supplemented with 10% fetal bovine serum (FBS) and characterized by the expression of alkaline phosphatase (AP) activity and cell-surface antigen profiles (CD105 as positive, CD45 and CD133 as negative markers). Subconfluent cultures of MSCs (1 × 10⁶ cells mL^–1) at 4–5 passages were equilibrated in different cryoprotectants: (1)ADMEM supplemented with 10% DMSO, (2) ADMEM supplemented with 1.5 m ethylene glycol (EG), and (3) ADMEM supplemented with 0.75 m EG and 5% DMSO, and frozen in a programmable freezer. The straws were cooled at –4°C min^–1 from 25°C to –7°C. After being seeded, the straws were further cooled to –35°C at a –0.6°C min^–1 ramp rate, and then immediately plunged into LN2 and stored at least a week. After thawing at 37°C in a water bath, viability and apoptosis of cells were analyzed by flow cytometry using an In Situ Cell Death Detection kit (Roche, Mannheim, Germany). Expression of HSP70, Nanog, and β-actin was analyzed by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR), and compared to that of non-cryopreserved MSCs. Doubling time of cryopreserved MSCs was higher than for non-cryopreserved MSCs. There were no significant differences among cryopreserved groups. The rates of viability and apoptosis of non-cryopreserved MSCs (91% and 4.71%, respectively) was significantly (P < 0.05) higher than for MSCs cryopreserved with 10% DMSO, 1.5 m EG, or the 0.75 m EG and 5% DMSO mixture (71.78% and 3.45%, 70.09% and 3.22%, 68.97% and 2.42%, respectively), but the rates among different cryopreserved treatments did not differ. After thawing, expression of HSP70, Nanog, and β-actin in cryopreserved MSCs showed patterns similar to those of non-cryopreserved MSCs. In conclusion, the present study indicates that EG is an alternative cryoprotectant for cryopreservation of porcine MSCs. Further studies are needed to evaluate the possible effects on the expression of genes related to viability and apoptosis in MSCs cryopreserved with different cryoprotectants.