Osteogenic induction medium can induce the production of mineralized matrix, and cells displaying bone-like nodular aggregates of matrix mineralization can be detected by alizarin reddish S staining (2, 6) while chondrogenic induction medium forms an acidic proteoglycans component which can be detected by alcian blue staining (2, 6, 37, 38)

Osteogenic induction medium can induce the production of mineralized matrix, and cells displaying bone-like nodular aggregates of matrix mineralization can be detected by alizarin reddish S staining (2, 6) while chondrogenic induction medium forms an acidic proteoglycans component which can be detected by alcian blue staining (2, 6, 37, 38). In conclusion, according to the present research, PDT evaluation in two treatments (FFP and non-FFP) showed the last passages had higher PDT in comparison with early passages and the characteristic of MSCs remained MDL 28170 still unchanged in passages 4 to 15. FFP was 22.67 7.01 days and non-FFP was 19.65 2.27 days). Cumulative cell number was significantly different between FFP and non-FFP at P5, 10, 15. AT-MSCs at P4-15 were positive for CD90, CD44, CD105, and CD73, and unfavorable for CD11b, CD19, CD34, CD45, and HLA-DR surface markers. AT-MSCs at P5, 10, 15 had potential toward adipogenic, chondrogenic, and osteogenic differentiation. Therefore, PDT was affected by increased age but no difference was observed in morphology, surface markers and differentiation capacity among passages. Cumulative cell number in FFP was higher in comparison MDL 28170 with non-FFP in P5, 10, 15. Our data suggest that FFP may replace FBS for culturing MSCs. (17). MSCs have a limited life span, undergo senescence on long-term culture MDL 28170 expansion, MSCs need to be able to maintain their character as well as their differentiation and self-renewal capacities (17). The use of platelet rich plasma (PRP) supplement has been described, and applied extensively to increase the expansion of MSCs (6), but it showed no influence on cells’ character and differentiation capacities. The objective of this research was to evaluate the effect of different media namely fresh frozen plasma (FFP) and non-FFP at various passages on cell proliferation, morphology, surface marker characteristics, and differentiation capa-cities. Materials and methods MSCs isolation from adipose tissue AT resulting from liposuction was put into schott bottle (250 or 500 ml) fulfilled with transport medium Minimal Essential Media with alpha modifications (MEM-) 80% (A1049001, Gibco, USA), 1% antibiotic and antimycotic (15240062, Gibco, USA) and FFP (Indonesian Red Cross, Bandung, Indonesia) in ice bag. Sample collection has been MDL 28170 approved by the patient by fulfilling the inform consent. All procedures has been approved by the Institutional Ethics Committee of Padja-djaran University Bandung, West Java, Indonesia (No. 1062/UN6.C1.3.2/KEP/PN/2016). After that, the fats were filtered by cell strainer 100 m (93100, SPL, Korea) and washed with phosphate buffered saline (PBS) (14200075, Gibco, USA), then transferred into 15 ml tube (50015, SPL, Korea). Collagenase type I (30 ml) 0.075% (17100017, Gibco, USA) was added into the tube and centrifuged (MPW-260 R) at 1200 rpm, 10 min at room temperature. Then, the cell pellet was put Ptgs1 into flask with complete medium consisting of 80% MEM-, 20% FFP, 1% antibiotic and antimicotic, and 1% heparin (IH2983, Inviclot, Indonesia) (18). Passaging and cell proliferation analysis Passaging MSCs with FFP treatment were cultured 3-5 days in FFP medium, with the medium being replaced every 2 days. For non-FFP, cells were given a complete medium consisting of 80% MEM-, 1% antibiotic and antimicotic, and 1% heparin but at the last day the cells were treated with a medium without FFP for 24 h to make the cells starve. Cells were counted and passaged at 80% confluence. Briefly, cultured cells were detached by trypsin (25200072, Gibco, USA), then incubated for 1-3 min at 37 oC in complete medium consisting of 80% MEM-, 20% FFP, 1% antibiotic and antimicotic, and 1% heparin that were added to stop trypsin, and centrifuged at 1600 rpm for 5 min at room temperature. The pellet of cells was resuspended with trypan blue solution (25200072, Sigma Aldrich, USA) and diluted 1:1. Then, cells were counted with a hemocytometer (Neubauer, 17849). Population Doubling (PD) was counted at every passage with the formula: overexpression. OCT4 and SOX2 transcription factors as adipogenic and osteogenic markers, are naturally expressed in MSCs for pluripotency and self-renewal at low levels in early passages (36). Cells began to show a round shape and most of them contained cytoplasmatic vacuoles, MDL 28170 intracellular accumulated lipids, and small oil droplets in the cytoplasm that were positive with oil red O staining (2, 6). Osteogenic induction medium can induce the production of mineralized matrix, and cells displaying bone-like nodular aggregates of matrix mineralization can be detected by alizarin red S staining (2, 6) while chondrogenic induction medium forms an acidic proteoglycans component which can be detected by alcian blue staining (2, 6, 37, 38). In conclusion, according to the present research, PDT evaluation in two treatments (FFP and non-FFP) showed that this last passages had higher PDT in comparison with early passages and the characteristic of MSCs remained still unchanged in passages 4 to 15. AT-MSCs are capable to differentiate into osteocytes, adipocytes and chondrocytes. PDT was affected by increasing age,.