Ight microliters of FS were added into six-well culture plates along with 3.20 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/20460822 ?105 rat MSCs that had been cultured and characterized as described previously, followed by the addition of complete culture medium. The plates were kept in an incubator at 37.5 in a humid atmosphere with 5 CO2. The maintenance medium was replaced every 5 days.Sealant samples were cultured with MSCs and fixed in Karnovysky's fixative (1 paraformaldehyde, 2.5 glutaraldehyde in 0.1 M phosphate buffer solution, pH 7.3) for 24 hours. After fixation, the samples were washed with 0.1 M phosphate buffer, pH 7.3 (3?5-minute washes), subjected to post-fixation in 1 osmium tetroxide diluted in the same buffer for 3 hours, dehydrated in increasing concentrations of ethanol up to a final concentration of 100 , immersed in acetone for 1 hour, immersed in acetone/resin (2:1) for 12 hours, immersed in acetone/resin (1:1) forGasparotto et al. Stem Cell Research Therapy 2014, 5:78 http://stemcellres.com/content/5/3/Page 4 ofhours and embedded in Durcupan?ACM Fluka resin (Sigma-Aldrich). Embedded samples were polymerized in an oven at 50 for 48 hours. Sections were cut with appropriate blades and stained as follows: 0.3 m semi-thin sections were cut with a glass knife and stained with 1 Toluidine Blue on a hotplate, and 50 nm ultra-thin sections were cut with a diamond knife and stained with Manila/ lead citrate (SPI Supplies. West Chester, PA. USA). The analyses were performed with a TECNAI BIOT WEEN G2 SPIRIT Transmission Electron Microscope (FEI Company, Hillsboro, OR. USA) at a voltage of 80 kV and were recorded as digital photographs by the same apparatus.Figure 1A,B,C. Figure 1D shows the cell population selected for analyses. No difference was observed in the cell PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/10572343 growth rate after 12 days and 90 confluence regardless of the presence of FS. The average number of MSCs isolated in the first passage was approximately 6.41 ?105.Differentiation into the adipogenic lineageResultsFibrin sealantThe material formed a dense fibrin network that resulted in a stable clot approximately 2 minutes after the reconstitution of its components at room temperature.Mesenchymal stem cell expansion and characterizationLipid-rich vacuoles formed within the first-passage MSCs after 7 days of culture in specific differentiation media. These changes were noted by the presence of red-colored vacuoles that confirmed the adipogenic lineage (Figure 2). There was no significant labeling of intracellular lipids in MSCs after 7 days of culture in the presence of 2-Chloro-3-methoxyaniline FS, which indicated that s16122021 the biomaterial alone could not induce the differentiation of MSCs to the adipogenic lineage.Differentiation into the chondrogenic lineageMSCs from the primary culture were able to adhere to plastic, as confirmed by flow cytometry after the first passage. The flow cytometry results indicated that 1.36 of the cells expressed CD34 and 89.06 and 81.93 expressed CD90 and CD44, respectively, as shown inProteoglycans produced by first-passage MSC chondrocytes N-BOC-3-Fluoro-D-phenylalanine were observed after 16 days of culture in specific differentiation media. These changes were noted by the presence of blue staining that confirmed the chondrogenic lineage (Figure 3). There was no significant chondrogenic cell labeling after 16 days of MSC culture in the presence of FS,Figure 1 Flow cytometric characterization of mesenchymal stem cells with negative and positive markers. Flow cytometric characterization of mesenchymal stem cells (MSCs) with (.