Evaluation of Distinct Freezing Methods and Cryoprotectants for Human Amniotic Fluid Stem Cells Cryopreservation

Figure 4

AFSCs plasticity. AFSCs were seeded in 6-well plates and cultivated with osteogenic, adipogenic, and chondrogenic inductor medium for 3 weeks. (a) Control cells cultivated without inductor medium (magnification: 40x). (b) Osteogenic differentiation was assessed by von Kossa (calcium deposits in black) and (c) Alizarin Red (calcium deposits in red) staining. Adipogenic differentiation was assessed by Oil Red O staining. (d) Differentiated cells without staining (magnification: 40x), (e) differentiated cells stained by Oil Red O (cytoplasmic lipids droplets in red, 400x). (f) Chondrogenic differentiation was confirmed with H&E (magnification: 400x) (g) and immunofluorescence staining with antibodies against collagen type II (400x). (h) Osteogenic gene expression was determined by RT-PCR; products were electrophorezed and visualized under ultraviolet light in 2% agarose gel stained with ethidium bromide (100 bp molecular weight, control (water), osteopontin [OPN], osteocalcin [OCN] and Wnt-1). (i) Typical adipogenic gene expression was determined by RT-PCR (100 bp molecular weight, control (water), PPAr-γ and lipoprotein lipase). (j) Chondrogenic gene expression was determined by RT-PCR (100 bp molecular weight, control (water), collagen type II, perlecan, and syndecan).