**6. 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).

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