Acid, Selenious

SCP-1 (47^th passage, PDL 57), SCP-11 (68^th passage, PDL 82) and hTERT-hMSCs (52^nd passage, PDL 57) were differentiated towards adipogenic, osteogenic and chondrogenic lineages [18 (link)]. Untransduced cells were used as a positive control. As a negative control both untransduced and transduced cells were cultured under identical conditions in standard medium (D-MEM, high glucose, glutamin, pyruvate) supplemented with 10% FBS and 1% Pen-Strep solution without differentiation supplements.
In vitro osteogenic differentiation of hMSCs was performed as previously published [18 (link)] using 100 nM dexamethasone, 10 mM β-glyc-erophosphate, 50 μM L-ascorbic acid-2-phosphate (Sigma). A total of 5 × 10³ cells/well were seeded in a 6-well plate. After 16 days, cells were assayed by von Kossa staining using a standard protocol.
Adipogenic differentiation was accomplished as previously published [18 (link)] by 1 μM dexamethasone, 0.2 mM indomethacin, 0.1 mg/ml insulin, 1 mM 3-isobutyl-1-methylxanthin (IBMX) (Sigma). The maintenance medium consists of 0.1 mg/ml insulin in standard medium. A total of 4 × 10³ cells/well were seeded in a 12-well plate. Stimulation was started when cells reached full confluency. Cells were grown for 5 days in induction medium, thereafter for 2 days in maintenance medium and then switched to induction medium again. After 16 days of stimulation, the cells were assayed by oil red O staining using a standard protocol.
Chondrogenic differentiation was achieved in aggregate cultures as previously published [18 (link)] with 100 nM dexamethasone, 1 mM pyruvate, 195 μm L-ascorbic acid-2-phosphate, 350 μM L-proline, 1.25% (v/v) insulin-transferrin-selenious acid mix (ITS, 100×), 5.35 μg/ml linolic acid, 1.25 mg/ml bovine serum albumine (BSA) (Sigma) and TGF-β₃ (10 ng/ml; R&D Systems, Minneapolis, MN, USA). A total of 2.5 × 10⁵ cells were used per pellet. Sections of the size of 12 μm were cut with a cryostat vacutome HM 200 OM (Microm, Walldorf, Germany). Anionic sulphated proteoglycans were detected by toluidine blue metachromasia. Slices were stained in 1% toluidine blue solution (Sigma, Munich, Germany), 1% sodium tetraborate (Sigma, Munich, Germany).

The multi-differentiation potential of the TSCs was tested in vitro for adipogenesis, chondrogenesis, and osteogenesis. TSCs at passage 1 were seeded in a 6-well plate at a density of 2.4 × 10⁵ cells/well in basic growth medium (10% heat inactivated FBS, 100 U/ml penicillin and 100 μg/ml streptomycin in DMEM-low glucose). To test adipogenic potential, TSCs were cultured in adipogenic induction medium (Millipore, Cat. # SCR026) consisting of basic growth medium supplemented with 1 μM dexamethasone, 10 μg/ml insulin, 100 μM indomethacin, and 0.5 mM isobutylmethylxanthine (IBMX). As a test of chondrogenic potential, TSCs were cultured in basic growth medium plus 40 μg/ml proline, 39 ng/ml dexamethasone, 10 ng/ml TGF-β 3, 50 μg/ml ascorbate 2-phosphate, 100 μg/ml sodium pyruvate, and 50 mg/ml insulin-transferrin-selenious acid mix (ITS) from BD Bioscience (Bedford, MA). Finally, osteogenic potential was tested by culturing TSCs in osteogenic induction medium (Millipore Cat. # SCR028) consisting of basic growth medium augmented with 0.1 μM dexamethasone, 0.2 mM ascorbic 2-phosphate, and 10 mM glycerol 2-phosphate. TSCs cultured in basic growth medium were used for control cells. To assay adipogenesis, chondrogenesis, and osteogenesis of TSCs, Oil Red O, Safranin O, and Alizarin Red S assays (see below), respectively, were used.
In addition to assessing differentiation potential of TSCs, the possible trans-differentiation potential of patellar tenocytes (PTs) and Achilles tenocytes (ATs) was also tested by the same assays used for testing TSCs. PTs and ATs were grown in the same culture conditions as TSCs.

To optimize the experimental parameters, a multifactorial experiment was performed with three input parameters and three levels of variation. Selenious acid, catamine AB, and ascorbic acid concentrations were selected as input parameters, the average hydrodynamic radius of particles and the ζ–potential were selected as output parameters. The levels of variation of variables are presented in Table 1.
Based on data of Table 1 we prepared an experiment matrix shown in Table 2.

The synthesis of Se NPs was carried out by a chemical reduction in aqueous medium in the presence of stabilizers. The synthesis scheme is shown in Figure 1. Alkyldimethylbenzylammonium chloride (catamine AB) (Vitareactive, Dzerzhinsk, Russia) was used as a stabilizer. Selenious acid was used as a selenium-containing precursor (INTRERHIM, St. Petersburg, Russia), and ascorbic acid was used as a reducing agent (Lenreactive, St. Petersburg, Russia).
The synthesis of Se NPs samples stabilized with alkyldimethylbenzylammonium chloride (catamine AB) was carried out in three stages. In the first stage, solutions were prepared with a different ratio between the amount of quaternary ammonium compounds and the amount of selenious acid. For this purpose, 0.036 M of the selenious acid solution was dissolved in 100 cm³ from 0.68 g to 5.24 g of quaternary ammonium compound, depending on the given ratio. In the second stage, 0.088 M ascorbic acid solution was prepared by dissolving 773.8 mg in 50 cm³ of distilled water. In the third stage, a solution of ascorbic acid was added dropwise to a solution of selenic acid and stabilizer under intensive stirring and the resulting sample was mixed for 5–10 min.

Fetal liver cell cultures were performed as previously described (75 (link)). Cultures were grown in 6 wells plates with William’s E Medium (Gibco) and epidermal growth factor 0.2 ug/ml and Cell Maintenance Supplement Pack (#CM4000, Thermo Fisher Scientific) providing 0.1 uM dexamethasone, 6.25 ug/ml human recombinant insulin, 6.25 ug/ml human transferrin, 6.25 ng/ml selenous acid,1.25 mg/ml bovine serum albumin, 5.35 ug/ml linoleic acid, 2 mM GlutaMAX and 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. Cells were harvested with TrypLE Enzyme solution (Life Technologies), stained, and analyzed by flow cytometry.