UCX isolation and cryopreservation Human UCX cells were isolated according to Santos et al. Briefly, fresh human umbilical cords were obtained after full term natural births, transported to the laboratory facilities Tofacitinib Citrate clinical in a sterile container Inhibitors,Modulators,Libraries containing saline buffer, in a hermetically sealed sterile container, and processed within a period up to 48 h. Identical cord tissue sections were digested, using a precise ratio be tween tissue mass, tissue digestion enzyme activity units, digestion solution volume and void volume using col lagenase. The procedure includes three recovery phases in order to avoid non conformities related to percentage cell recovery. In the first cell recov ery phase, cells dissociated from the tissue are recovered by a static horizontal incubation.
The remaining cells are incubated in a static monolayer culture using basal medium, at 37 C in 7% CO2 humidified atmosphere, Inhibitors,Modulators,Libraries with medium change every 72 h until they reached confluence. Confluent cultures were cryopreserved in 10% dimethylsulphoxide stock solution and FBS, using control rate temperature decrease. UCX expansion and characterization Human cryopreserved UCX cells were thawed and expanded to P5 in complete culture medium with medium change every 72 h. Cells were characterized by their capacity to adhere to plastic in standard MSCs culture conditions. Surface marker expression was analyzed by flow cytometry using a Gallios imaging flow cytometer. The antibodies used and their conjugates were anti human CD105 PE anti human CD73 APC, anti human CD90 PE, anti human CD14 PerCpCy5. 5, anti human CD45 PerCpCy5.
5, anti human CD34 FITC, anti human CD19 pacific blue, anti human HLA DR Pacific blue, isotype Pacific blue IgG1, isotype Pacific blue IgG2a isotype IgG1k PerCp Cy5. 5 isotype IgG2a PerCpCy5. Inhibitors,Modulators,Libraries 5, isotype IgG1k PE, isotype IgG1k APC and isotype IgG1k FITC. For flow cytometry studies cells were incubated for 1 h at 4 C with the antibodies in 2% bovine serum albumin solution, centrifuged and washed with phosphate buffered saline. To induce adipogenic differenti ation, cultured cells were incubated in adipogenic differen tiation medium, for 3 days, consisting of MEM supplemented with 20% FBS, 2 mM L glutamine, 10 ug mL insulin, 200 uM indomethacin, 0. 5 mM isobutylmetylxantine, and 1 uM dexamethasone and subsequently 1 day Inhibitors,Modulators,Libraries in adipogenic main tenance medium, consisting of MEM supplemented with 20% FBS, 2 mM L glutamine and 10 ugmL insulin.
Medium replacement Inhibitors,Modulators,Libraries cycles were repeated during 21 days after which histochemical staining was performed. Belinostat buy For chondrogenic differentiation, cells were grown in sus pension as pellets, incubated with chondrogenic differ entiation medium consisting on DMEM LG, 1% FBS, 2 mM L glutamine, 6. 25 ugmL in sulin, 10 ngmL TGF B1, and 50 uM ascorbate 2 phosphate. The medium was replaced every 3 days during 21 days and histochemical staining was performed.