rASCs were induced to differentiate into adipocytes, osteoblasts, and chondrocytes. For adipogenesis, the cells were cultured in adipogenic induction medium (DMEM supplemented with 10% FBS, 10−7 M dexamethasone (Sigma, St. Louis, MO), 100 μΜ indomethacin (Sigma), 100 μM 3-isobutyl-1-methyl-xanthine (Sigma), and 10 mg/L insulin (Invitrogen)), and the medium was refreshed every 2 days. Two weeks later, the cells were fixed with 4% paraformaldehyde (PFA) and stained with Oil red O (Sigma). For osteogenesis, the cells were maintained in osteogenic induction medium (DMEM supplemented with 10% FBS, 10 mM β-glycerol phosphate (Sigma, CA), 50 μM L-ascorbate-2-phosphate (Sigma), and 5 ng/mL recombinant human bone morphogenetic protein-2 (HumanZyme, Chicago, IL)) and the medium was changed with fresh one every 2 days. One week later, the cells were examined for alkaline phosphatase (AKP) activity by vector blue alkaline phosphatase substrate kit III (Vector, Burlingame, CA). For chondrogenesis, the cells were cultured in chondrogenic induction medium which includes DMEM supplemented with 10% FBS, 10 ng/mL TGFβ1 (HumanZyme, Chicago, IL), 0.1 mol/L dexamethasone (Invitrogen), 50 mg/L L-ascorbate-2-phosphate (Sigma), and 50 g/L ITS (Invitrogen). The cells were cultured for two weeks and fixed in 4% PFA and stained with toluidine blue sodium borate (Sigma).
L ascorbate 2 phosphate
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Canada
L-ascorbate-2-phosphate is a chemical compound that functions as a source of ascorbic acid (vitamin C) in biological systems. It is a stable, water-soluble form of ascorbic acid that can be used in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (13 mentions)
β glycerol phosphate, by Merck Group (6 mentions)
L glutamine, by Merck Group (5 mentions)
β glycerophosphate, by Merck Group (5 mentions)
Oil red o, by Merck Group (4 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (4 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Collagenase, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
L proline, by Merck Group (3 mentions)
Indomethacin, by Merck Group (3 mentions)
Penicillin g sodium, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Merck Group (2 mentions)
Non essential amino acids (neaa), by Merck Group (2 mentions)
α mem, by Merck Group (2 mentions)
Sodium pyruvate, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Ascorbate, by Merck Group (2 mentions)
Penicillin, by Harvard Bioscience (2 mentions)
36 protocols using l ascorbate 2 phosphate
1
Multilineage Differentiation of rASCs
2
Multilineage Differentiation of hUCMSCs
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hUCMSCs were induced to differentiate into adipocytes, osteoblasts and chondrocytes. For adipogenesis, the cells were cultured in adipogenic induction medium (DMEM supplemented with 10% FBS, 10−7 M dexamethasone (Sigma, St. Louis, MO), 100 μΜ indomethacin (Sigma), 100 μM 3-isobutyl-1-methyl-xanthine (Sigma) and 10 mg/L insulin (Invitrogen)), and the medium was refreshed every 2 days. Two weeks later, the cells were fixed with 4% paraformaldehyde (PFA) and stained with Oil-Red O (Sigma). For osteogenesis, the cells were maintained in osteogenic induction medium (DMEM supplemented with 10% FBS, 10 mM β-glycerol phosphate (Sigma), 50 μM L-ascorbate-2-phosphate (Sigma) and 5 ng/mL recombinant human bone morphogenic protein-2 (HumanZyme, Chicago, IL)) and the medium was changed with fresh one every 2 days. One week later, the cells were examined for alkaline phosphatase (AKP) activity by vector blue alkaline phosphatase substrate kit III (Vector, Burlingame, CA). For chondrogenesis, the cells were cultured in chondrogenic induction medium includes DMEM supplemented with 10% FBS, 10 ng/mL TGF-β1 (PeproTech, Cranbury, NJ, USA), 0.1 mol/L dexamethasone (Invitrogen), 50 mg/L L-ascorbate-2-phosphate (Sigma) and 50 g/L ITS (Invitrogen). The cells were cultured for two weeks and fixed in 4% PFA, and stained with toluidine blue sodium borate (Sigma).
3
Serum-free Culture Protocols for Chondrocytes and Keratinocytes
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JM-H for CSSC culture: DMEM (1 g/L D-glucose, Gibco 10567-014), MCDB 201 (Sigma-Aldrich M6770), added with insulin-transferrin-selenite (ITS, 0.5×, Gibco 41400-045), AlbuMAX I (1 mg/mL, Gibco 11020-021), L-ascorbate-2-phosphate (0.5 mM, Sigma-Aldrich A8960), recombinant human EGF (10 ng/mL, Sigma-Aldrich E9644), recombinant human PDGF-BB (10 ng/mL, R&D 520-BB), dexamethasone (10 nM, Sigma-Aldrich D4902), penicillin/streptomycin (BioWhittaker, Lonza, Walkersville, MD, USA), and 2% pooled human serum (Innovative Res.).
ERI for CSK culture: DMEM/F12 (Gibco 10565-018), MEM amino acids (Gibco 11130-051), MEM non-essential amino acids (Gibco 11140-050), ITS (0.5×), AlbuMAX I (1 mg/mL), L-ascorbate-2-phosphate (0.5 mM), recombinant insulin growth factor 1 (10 ng/mL, Sigma-Aldrich I3769), Y27632 (10 nM, Chemdea CD0141), human amnion stromal extract (5 μg protein/mL, preparation following the method of Yusoff et al. [24 (link)], and penicillin/streptomycin.
ERI for CSK culture: DMEM/F12 (Gibco 10565-018), MEM amino acids (Gibco 11130-051), MEM non-essential amino acids (Gibco 11140-050), ITS (0.5×), AlbuMAX I (1 mg/mL), L-ascorbate-2-phosphate (0.5 mM), recombinant insulin growth factor 1 (10 ng/mL, Sigma-Aldrich I3769), Y27632 (10 nM, Chemdea CD0141), human amnion stromal extract (5 μg protein/mL, preparation following the method of Yusoff et al. [24 (link)], and penicillin/streptomycin.
4
Immortalized Mesenchymal Stromal Cells and Primary Chondrocytes
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Human immortalized bone marrow derived mesenchymal stromal cell line (SCP1 cells) [49 (link)] was a kind gift from Prof. Dr. Matthias Schieker. Cell were expanded in culture medium (α-MEM (minimal essential medium; GIBCO, Darmstadt, Germany)), 10% FCS (GIBCO, Darmstadt, Germany), and 1% penicillin/streptomycin (Merck, Darmstadt, Germany) 10,000 units/mL penicillin and 10 mg/mL streptomycin) in a cell incubator at 37 °C and 5% CO2 [37 (link)].The isolation of primary human chondrocytes was performed as previously described [23 (link)]. Briefly, chondrocytes were isolated from donated femoral condyle tissues (donor number N = 23; sex m/f = 10/13, respectively; age = 74.86 ± 8.15 years) (BG Klinik, Tübingen) by collagenase (1500 U/mL, GIBCO, Darmstadt, Germany) digestion, and expanded in culture medium (DMEM/Ham’s F12 (1:1), 10% FCS, 1% penicillin/streptomycin, 50 µM L-ascorbate-2-phosphate (Merck, Darmstadt, Germany) at 37 °C and 5% CO2.Different donor samples (N ≥ 3)with passage number 2 were used for each experiment.
5
Isolation of Primary Human Chondrocytes
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Primary human chondrocytes were isolated from the femoral condyles or tibia plateaus of patients who had received a total knee replacement. The isolation of primary human chondrocytes was performed as described before [68 (link)]. Briefly, the cartilage (donor number N = 14; 10 males and 4 females; age = 69.64 ± 8.82 years) was cut into pieces and thoroughly washed with phosphate-buffered saline (PBS) (without Ca2+/Mg2+, Merck, Darmstadt, Germany). Afterward, the pieces were digested by collagenase (1500 U/mL, GIBCO, Darmstadt, Germany) at 37 °C overnight, then, the supernatant was centrifuged in order to remove the collagenase. Thereafter, cells obtained were expanded in culture medium (DMEM/Ham’s F12 (1:1) supplemented with 10% Fetal Calf Serum (FCS, GIBCO, Darmstadt, Germany), 1% penicillin/streptomycin (Merck, Darmstadt, Germany) and 50 µM l -ascorbate-2-phosphate (Merck, Darmstadt, Germany) at 37 °C and 5% CO2. For all experiments, a medium with or without CSE was changed every day and cultures were investigated at 3, 7, and 14 days.
6
Quantification of Glycosaminoglycans in Cell Culture
7
Fibroblast Cell Culture Protocols
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PDL fibroblasts were maintained in α-minimal essential medium (α-MEM; Sigma-Aldrich) containing 10% fetal calf serum (FCS; Hyclone Laboratories, Logan, UT), 2 mm L-glutamine (Sigma-Aldrich), 100 μM L-ascorbate-2-phosphate (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 50 U/mL penicillin G and 50 μg/mL streptomycin (Sigma Aldrich) [37 (link)]. IMR90 fibroblasts were cultured in Eagle’s Minimum Essential Medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; HyClone), 0.1 mM non-essential amino acids (Sigma-Aldrich), 1.0 mM sodium pyruvate (Invitrogen) and 1X Pen/Strep (Invitrogen) [39 (link)]. Media were refreshed every 48h for both cell lines.
8
Dental Cell Differentiation on Silk Scaffolds
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DPCs and PDLCs, suspended in alpha Modified eagle’s Medium (α-MEM, Sigma) supplemented with 10% fetal bovine serum (FBS, BioWhitekerTM, Cambrex Bioscience Walkersville, Inc., MD), were seeded onto the silk scaffolds. To obtain a very high cell seeding density, the cells were seeded onto the silk scaffolds under a dried condition. 100 μl of a harvested suspension – containing 2.8 × 104 human dental pulp cells or 1 × 106 human PDL cells – and silk scaffolds were placed in the petri dish in a humidified 5% CO2 incubator, and 3h later, 10 ml of α-MEM was added to the petri dish. The medium was replaced every 3 days for 7 days. After incubation for additional 7 days, the scaffolds were cultured in differentiation medium, which was replaced every 2 days for 3 weeks. The differentiation medium for DPCs was alpha Modified Eagle’s Medium (α-MEM, Sigma) supplemented with 10% fetal bovine serum (FBS, BioWhittakerTM, Cambrex, MD, USA), 1% AA, 100 nM dexamethasone, 0.05 mM Ascorbic acid and 10 mM β-glycerophospate. The differentiation medium for PDLCs was α-MEM containing 10% FBS, 10 mM β-glycerophosphate (Sigma), 50 μM L-ascorbate2-phosphate (Sigma) and 10−7 M dexamethasone (Sigma). The result of cell-seeding procedure was checked with scanning electron microscopy.
9
Lipid Compound Characterization and Cell Signaling
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PAME, palmitic acid (PA), and stearic acid methyl ester (SAME) were purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in 100% methanol. DC260126, dexamethasone, L-ascorbate-2-phosphate, indomethacin, 1,2-bis (2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxymethyl ester) (BAPTA-AM), triethyl phosphate, and Oil Red O were purchased from Sigma-Aldrich. GW1100 was purchased from Cayman Chemical Company (Ann Arbor, MI), AH7614 was purchased from Abcam (Cambridge, England), U73122 was purchased from Millipore (Burlington, MA), and U0126 was purchased from Calbiochem (San Diego, CA).
10
Derivation and Characterization of iPSC-Derived Mesenchymal Stem Cells
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Human iPSC‐MSCs were generated from urine cell‐derived‐induced pluripotent stem cells (U‐iPSCs) which donated by Guangzhou Institute of Biomedicine and Health, Chinese Academy of Science (Guangzhou, China) as described in our previous study.15 (link) In brief, U‐iPSCs were cultured in MSC‐inducing‐culture media containing Minimum Essential Medium Eagle‐α modified (α‐MEM, Gibco, Gaithersburg, Maryland), 10% serum replacement (Stem Cell Technologies, Vancouver, Canada), penicillin/streptomycin, sodium pyruvate, 10 mM l ‐ascorbate‐2‐phosphate, l ‐glutamine and nonessential amino acids (Sigma‐Aldrich, Inc, St. Louis, Missouri) for 2 weeks. The generated iPSC‐MSCs were cultured in Dulbecco's modified Eagle medium supplemented with 15% fetal bovine serum (FBS), 1% penicillin‐streptomycin (Gibco, Gaithersburg, Maryland), 5 ng/mL EGF, and 5 ng/mL β‐FGF (PeproTech, Inc, Rocky Hill, New Jersey) at 37°C, 5% CO2 and 95% humidity. They were positive for CD44, CD73, CD90, CD105, CD144, and CD146, but negative for CD14, CD34, and CD45. Their adipogenic, chondrogenic, and osteogenic differentiation capacity was also confirmed before usage.15 (link)
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