Mouse embryonic palatal mesenchymal cells were derived from palatal tissue on 13-day C57BL/6 mice embryos (Henan Laboratory Animal Center of Zhengzhou University, China). All experiments were performed in accordance with the Experimental Animal Center Guide for the Care and Use of Laboratory Animals, and the Institutional Ethical Guidelines for Experiments with Animals. The method of MEPM cell culture was according to the detail by Feng et al.12 (link) The MEPM cells were cultured in flasks with DMEM (Dulbecco's Modified Eagle's medium)/F12 medium (Hyclon, Logan, Utah) supplemented with 10% fetal calf serum (Sijiqing, Hangzhou, China). The MEPM cells were placed in a humidified incubator at 37°C with 5% CO2 atmosphere with media replaced every other day. The third passage cells were seeded. Some cells were treated with 0.5 nM, 1 nM, 5 nM, 10 nM, 20 nM, and 50 nM TCDD, and TCDD concentration was selected according to some reports,13 (link),14 (link) others were treated with 10 nM TCDD (DD-2378-S, Sigma, Saint Louis, Missouri), or 10 ng/mL TGF-β3 (cyt-143, PROSPEC, Zion, Israel), or combination of 10 nM TCDD and 10 ng/mL TGF-β3 for further analysis. Control cells were treated with DMSO (Dimethyl sulfoxide) (D2650; Sigma).
Tgf β3
Manufactured by ProSpec
Sourced in Israel, United States
TGF-β3 is a recombinant human Transforming Growth Factor-beta 3 protein. It is a member of the TGF-beta superfamily and plays a role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (7 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
L ascorbic acid 2 phosphate, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Dd 2378 s, by Merck Group (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
L proline, by Merck Group (2 mentions)
Insulin transferrin selenium, by Merck Group (2 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
24 well tissue culture plate, by Thermo Fisher Scientific (1 mentions)
Rat tail collagen 1, by BD (1 mentions)
α sma, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by ScienCell (1 mentions)
Tissue tek cryo oct compound, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Concentrated hydrochloric acid hcl, by Sinopharm (1 mentions)
Dopamine da, by Merck Group (1 mentions)
19 protocols using tgf β3
1
Mouse Palatal Mesenchymal Cell Culture
2
Chondrogenic Differentiation of FPSCs
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Initially, to remove any residual DMEM all scaffolds were washed and rehydrated in D-PBS.
To enhance seeding efficiency, the scaffolds were placed inside agarose moulds with a diameter of 5mm. The agarose moulds containing the scaffolds were then placed into 24-well tissue culture plates (Fisher Scientific). To examine the ability of the various AC scaffolds to promote the chondrogenic differentiation of FPSCs in vitro, 0.5x10⁶ FPSCs were seeded onto individual scaffolds suspended in 25µl of expansion media. FPSCs were allowed to attach to the scaffolds for 1 hour in an incubator at 37°C. After FPSC attachment, 2.5ml of chemically defined chondrogenic differentiation media (CDM) was added per well. CDM consisted of high glucose DMEM supplemented with Penicillin (100 U/ml) and streptomycin (100μg/ml) (both Gibco), 100µg/ml sodium pyruvate (Sigma), 40µg/ml L-Proline (Sigma), 50 µg/ml Lascorbic acid-2-phosphate (Sigma), 1.5mg/ml bovine serum albumin (BSA-Sigma), 1X insulin transferrin selenium (ITS-Gibco), 100nM dexamethasone (Sigma) and 10ng/ml transforming growth factor beta-3 (TGF-β3 -ProSpec Tany, Israel). The FPSC seeded constructs were maintained in CDM for 28 days with the CDM being replenished three times per week.
To enhance seeding efficiency, the scaffolds were placed inside agarose moulds with a diameter of 5mm. The agarose moulds containing the scaffolds were then placed into 24-well tissue culture plates (Fisher Scientific). To examine the ability of the various AC scaffolds to promote the chondrogenic differentiation of FPSCs in vitro, 0.5x10⁶ FPSCs were seeded onto individual scaffolds suspended in 25µl of expansion media. FPSCs were allowed to attach to the scaffolds for 1 hour in an incubator at 37°C. After FPSC attachment, 2.5ml of chemically defined chondrogenic differentiation media (CDM) was added per well. CDM consisted of high glucose DMEM supplemented with Penicillin (100 U/ml) and streptomycin (100μg/ml) (both Gibco), 100µg/ml sodium pyruvate (Sigma), 40µg/ml L-Proline (Sigma), 50 µg/ml Lascorbic acid-2-phosphate (Sigma), 1.5mg/ml bovine serum albumin (BSA-Sigma), 1X insulin transferrin selenium (ITS-Gibco), 100nM dexamethasone (Sigma) and 10ng/ml transforming growth factor beta-3 (TGF-β3 -ProSpec Tany, Israel). The FPSC seeded constructs were maintained in CDM for 28 days with the CDM being replenished three times per week.
3
TGF-β Signaling in Smooth Muscle Cells
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The following cells and reagents were used in our experiments: Sprague–Dawley rats (Harlan Labs, Indianapolis, Indiana, USA), human intestinal smooth muscle cells (hiSMC, ScienCell), smooth muscle cell medium (FBS, ScienCell), Dulbecco’s phosphate-buffered saline (DPBS, Thermo Scientific), human TGF beta 1 and TGF beta 2 (TGF-β1 & β2 (100 ng/ml), Biolegend) and TGF-β3 (TGF-β3 (100 ng/ml), Prospec) recombinant proteins, rabbit polyclonal antibody to all TGF-βs (Abcam), mouse monoclonal antibody α-smooth muscle actin (α-SMA, Abcam), cell dissociation solution (Mediatech, 25-056CI), Alexa Fluor® goat anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (Life technologies), Alexa Fluor® goat anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Life technologies), 4’,6-diamidino-2-phenylindole (DAPI, Life technologies) and rat tail collagen I (BD Biosciences). Western blot signals were detected by using Kodak image station 4000R. Micro-RNA materials included: miRNeasy FFPE and miScript II RT kits (Qiagen) and miRNA assays (SA Biosciences, Valencia, CA).
4
Chondrogenic Differentiation of oBMSCs
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Chondrogenesis was assessed using the micropellet formation (2.5x105 cells) technique [30 (link)]. oBMSCs (from 3 independent samples) pellet was cultured in hMSC Commercial Chondrogenic Differentiation Medium (Lonza) with 10 ng/ml of human transforming growth factor (TGFβ-3) (Prospec-Tany Technogene Ltd., Rehovot, Israel) for 21 days, following the manufacturer’s instructions. Chondrogenic differentiation was compared with cell micropellets cultured for the same period of time with 20% FBS/DMEM. After 21 days, two cell aggregates were OCT (Tissue-Tek cryo-OCT compound, Thermo Fisher Scientific) embedded and frozen for histology and three cell micropellets were frozen, without OCT, for molecular analyses.
5
Chondrogenic Differentiation of Rat BMSCs
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Dopamine (DA) and tris-(hydroxymethyl)-aminomethane (Tris) were purchased from Sigma-Aldrich (USA). Concentrated hydrochloric acid (HCl) was obtained from Sinopharm Chemical Reagent Co., Ltd (China). Polymer PCL (molecular weight = 45 kDa) was purchased from Sigma-Aldrich (USA). Gelatin (Gel), hyaluronic acid (HA), sodium alginate (SA), and calcium chloride anhydrous (CaCl2) were purchased from Aladdin Co., Ltd. (China). CTGF (recombinant, rat) was purchased from R&D systems (USA) and TGF-β3 (recombinant, rat) was purchased from ProSpec (USA). Cell culture reagents, such as Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and trypsin were purchased from Life Technologies (USA). The Rat (SD) Mesenchymal Stem Cell Chondrogenic Differentiation Basal Medium was purchased from Cyagen Biosciences Inc. (China). BMSCs were extracted from the bone marrow of Sprague Dawley (SD) rats. SD rats and nude mice were obtained from the Animal Laboratory Center of Shanghai Jiao Tong University (China).
6
Multilineage Differentiation of hWJ-MSCs
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hWJ-MSCs were cultured at the final density of approximately 2 × 104 cells/cm2 in 6-well culture plates coated with 0.1% gelatin.
hWJ-MSCs were induced to osteogenic differentiation by culture in the culture medium with reduced FBS to 5% and supplemented with 100 nM dexamethasone, 0.2 mM L-ascorbate-2-phosphate, and 10 mM β-glycerophosphate. The medium was subsequently replaced every 2 days for 3 weeks. Calcium deposits from the cells were then visualized by Alizarin Red staining.
To induce adipogenic differentiation, hWJ-MSCs were cultured in the culture medium with reduced FBS to 5% and supplemented with 10 μg/mL insulin, 100 μM indomethacin, 1 μM dexamethasone, 0.5 mM isobutylmethylxanthine (IBMX). IBMX was removed from this medium after 1 week of culture. The medium was subsequently replaced every 2 days for 3 weeks. Cells were then stained with Oil Red O to observe oil droplets.
To induce chondrogenic differentiation, hWJ-MSCs were cultured in a completed chondrogenic medium consisting of culture medium with reduced FBS to 2% and supplemented with 1% Insulin-Transferrin-Selenium-Ethanolamine (ITS-X, Invitrogen), 50 μg/mL ascorbate-2-posphate, 40 μg/mL L-proline, 100 μg/mL sodium pyruvate, 100 nM dexamethasone, and 10 ng/mL of TGF-β3 (Prospec, East Brunswick, NJ, USA). The medium was replaced every 2 days for 3 weeks. GAG production was assessed by Alcian blue 8× staining.
hWJ-MSCs were induced to osteogenic differentiation by culture in the culture medium with reduced FBS to 5% and supplemented with 100 nM dexamethasone, 0.2 mM L-ascorbate-2-phosphate, and 10 mM β-glycerophosphate. The medium was subsequently replaced every 2 days for 3 weeks. Calcium deposits from the cells were then visualized by Alizarin Red staining.
To induce adipogenic differentiation, hWJ-MSCs were cultured in the culture medium with reduced FBS to 5% and supplemented with 10 μg/mL insulin, 100 μM indomethacin, 1 μM dexamethasone, 0.5 mM isobutylmethylxanthine (IBMX). IBMX was removed from this medium after 1 week of culture. The medium was subsequently replaced every 2 days for 3 weeks. Cells were then stained with Oil Red O to observe oil droplets.
To induce chondrogenic differentiation, hWJ-MSCs were cultured in a completed chondrogenic medium consisting of culture medium with reduced FBS to 2% and supplemented with 1% Insulin-Transferrin-Selenium-Ethanolamine (ITS-X, Invitrogen), 50 μg/mL ascorbate-2-posphate, 40 μg/mL L-proline, 100 μg/mL sodium pyruvate, 100 nM dexamethasone, and 10 ng/mL of TGF-β3 (Prospec, East Brunswick, NJ, USA). The medium was replaced every 2 days for 3 weeks. GAG production was assessed by Alcian blue 8× staining.
7
Chondrogenic Culture Conditions for Tissue Engineering
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The chondrogenic culture conditions applied in this study are defined as culture in a chondrogenic medium (CDM) consisting of hgDMEM GlutaMAX supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml sodium pyruvate, 40 μg/ml L-proline, 50 μg/ml L-ascorbic acid-2phosphate, 4.7 μg/ml linoleic acid, 1.5 mg/ml bovine serum albumine, 1 X insulin-transferrinselenium, 100 nM dexamethasone (all from Sigma-Aldrich), 2.5 μg/ml amphotericin B, 500 ng/ml of recombinant human bone morphogenetic protein 2 (BMP-2) (Peprotech, EC Ltd) and 10 ng/ml of human transforming growth factor-β 3 (TGF-β 3) (Prospec-Tany TechnoGene Ltd., Israel).
The constructs were primed for 2 weeks at 5% pO2 followed by 2 weeks at 20% pO2 before implantation.
The constructs were primed for 2 weeks at 5% pO2 followed by 2 weeks at 20% pO2 before implantation.
8
Chondrocyte Construct Engineering for Tissue-Engineered Phalanx
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Chondrocytes were suspended in hgDMEM supplemented with 10% v/v FBS, 100 U/mL penicillin–100 μg/mL streptomycin, and 2.5 μg/mL amphotericin B at a density of 100×106 cells/mL. Forty microliters of this cell suspension was pipetted into 4% agarose cylindrical wells (Ø5×3 mm), to give a final concentration of 4×106 cells/construct, and allowed to self-assemble for 12 h. Thereafter, constructs were cultured in a chondrogenic medium (CM) consisting of hgDMEM GlutaMAX supplemented with 100 U/mL penicillin/streptomycin (both Gibco), 100 μg/mL sodium pyruvate, 40 μg/mL l -proline, 50 μg/mL l -ascorbic acid-2-phosphate, 4.7 μg/mL linoleic acid, 1.5 mg/mL bovine serum albumin, 1× insulin–transferrin–selenium, 100 nM dexamethasone (all from Sigma-Aldrich), 2.5 μg/mL amphotericin B, and 10 ng/mL of human transforming growth factor-β3 (TGF-β3) (Prospec-Tany TechnoGene Ltd.) at 20% pO2 for a period of 4 weeks to form the chondral layer of the tissue-engineered phalanx.
9
Chondrogenic Differentiation of hMSCs in Fibrin Hydrogels
10
MEPM Cell Culture and TCDD/TGF-β3 Treatment
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MEPM cells were derived from palatal tissue on 13-day-old C57BL/6 mice embryos (Henan Laboratory Animal Center of Zhengzhou University, China). All experiments were performed in accordance with the Experimental Animal Center Guide for the Care and Use of Laboratory Animals and the Institutional Ethical Guidelines for Experiments with Animals. The method of MEPM cell culture was according to the method by Feng et al.12 The MEPM cells were cultured in flasks with DMEM/F12 medium (Hyclon, Logan, Utah) supplemented with 10% fetal bovine serum (FBS, Sijiqing, Hangzhou, China). The MEPM cells were placed in a humidified incubator at 37°C in 5% CO2 atmosphere, with media replaced every other day. The third passage cells were seeded. Some cells were treated with 0.5 nM, 1 nM, 5 nM, 10 nM, 20 nM, and 50 nM TCDD, and TCDD concentration was selected according to some reports.13 (link),14 (link) Others were treated with 10 nM TCDD (DD-2378-S, Sigma, Saint Louis, Missouri), 10 ng/mL TGF-β3 (cyt-143; PROSPEC, Zion, Israel), or a combination of 10 nM TCDD and 10 ng/mL TGF-β3 for further analysis. Control cells were treated with DMSO (D2650; Sigma).
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