Harvested samples were washed once in PBS, and minced into small pieces. The samples were enzymatically digested for one hour in Dulbecco’s modified Eagle’s medium (D-MEM) supplemented with 10 mg/mL Collagenase II (Gibco, Carlsbad, CA, USA) at 37 °C. Collagenase II was deactivated by adding fetal bovine serum (FBS), and the pellet was centrifuged for five minutes at 1000 rpm. The cells obtained were seeded into a 100 φ dish and cultured with D-MEM supplemented with 10% FBS, 50 mM ascorbic acid-2-phosphate (Gibco), and 1% Antibiotic-Antimycotic solution (Gibco) for one week. The culture medium was changed every three days until confluence. Both healthy and tendinopathy cell samples were cultured until passage 3 and used for experimentation.
Ascorbic acid 2 phosphate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Ascorbic acid-2-phosphate is a stable, water-soluble form of ascorbic acid (vitamin C). It is commonly used as a cell culture supplement to promote cell growth and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Thermo Fisher Scientific (6 mentions)
B27 supplement, by Thermo Fisher Scientific (4 mentions)
Ascorbic acid 2 phosphate, by Merck Group (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Tgf β1, by Thermo Fisher Scientific (2 mentions)
β glycerol phosphate, by Thermo Fisher Scientific (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Collagenase 2, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Mcdb 201, by Merck Group (1 mentions)
Antibiotics penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dmem low glucose, by Merck Group (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Eclipse ts100, by Nikon (1 mentions)
10 protocols using ascorbic acid 2 phosphate
1
Tendon Cell Isolation and Culture
2
Isolation and Culture of Corneal Stromal Stem Cells
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CSSCs were isolated from corneas of three donors aged 15 to 45 years old, as described in Supplementary Table S1 and cultured in a stem cell growth medium (SCGM)15 (link) consisting of Dulbecco's modified Eagle's medium (DMEM) low glucose, MCDB-201, insulin-transferrin-selenious acid supplement (Sigma-Aldrich, St. Louis, MO, USA), gentamicin, albumin, fetal bovine serum (FBS) (2%) (Gibco, Gaithersburg, MD, USA), ascorbic acid-2-phosphate, dexamethasone, platelet-derived growth factor, epidermal growth factor, and antibiotics penicillin/streptomycin (Gibco). Cells were cryopreserved in liquid nitrogen using 70% DMEM/HAMs F12, 20% FBS, and 10% dimethyl sulfoxide (DMSO) for 10 years or more. After 10 years, CSSCs were quickly thawed using water bath maintained at 37°C and cultured in SCGM. TrypLE express (Gibco) was used for cell detachment and further upscale. Cells were routinely observed under bright field inverted microscope (Nikon Eclipse TS100; Melville, NY, USA) and medium was changed every third day.
3
Mesenchymal Stem Cell Characterization
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After harvesting stem cells from adipose tissue, we
evaluated their mesenchymal characteristics using two
methods: flow cytometry and differentiation into the
osteoblastic lineage.
Phenotyping of cell surface markers for MSCs at
the second passage involved labeling with anti-human
antibodies, including CD90, CD44, CD10, CD106,
CD34, and CD45, using fluorescein isothiocyanate
(FITC) (Beckman Coulter, Fullerton, CA). Human
isotype antibodies served as controls (Becton Dickinson;
Beckman Coulter).
For differentiation characterization, a density of 5×104cells/ml at the second passage was cultured in a sixwell
plate with DMEM and 10% FBS. Upon reaching
confluence within one week, the medium was replaced with
osteogenic differentiation media to assess the cells’ ability
to differentiate into the osteoblastic lineage. This medium
comprised DMEM, 50 μg/ml ascorbic acid 2-phosphate, 10
nM dexamethasone, and 10 mM ƒÀ-glycerol phosphate (all
obtained from Gibco, United States). Incubation occurred
at 37°C with 5% CO2 for 21 days, with medium renewal
every three days. Subsequently, the cells were fixed with
10% formalin for 10 minutes, followed by staining with
Alizarin Red for 20 minutes at room temperature. Finally,
the cells were examined under an optical microscope, and
photographic documentation was undertaken (22 (link)).
evaluated their mesenchymal characteristics using two
methods: flow cytometry and differentiation into the
osteoblastic lineage.
Phenotyping of cell surface markers for MSCs at
the second passage involved labeling with anti-human
antibodies, including CD90, CD44, CD10, CD106,
CD34, and CD45, using fluorescein isothiocyanate
(FITC) (Beckman Coulter, Fullerton, CA). Human
isotype antibodies served as controls (Becton Dickinson;
Beckman Coulter).
For differentiation characterization, a density of 5×104cells/ml at the second passage was cultured in a sixwell
plate with DMEM and 10% FBS. Upon reaching
confluence within one week, the medium was replaced with
osteogenic differentiation media to assess the cells’ ability
to differentiate into the osteoblastic lineage. This medium
comprised DMEM, 50 μg/ml ascorbic acid 2-phosphate, 10
nM dexamethasone, and 10 mM ƒÀ-glycerol phosphate (all
obtained from Gibco, United States). Incubation occurred
at 37°C with 5% CO2 for 21 days, with medium renewal
every three days. Subsequently, the cells were fixed with
10% formalin for 10 minutes, followed by staining with
Alizarin Red for 20 minutes at room temperature. Finally,
the cells were examined under an optical microscope, and
photographic documentation was undertaken (22 (link)).
4
Multilineage Differentiation of BMSCs
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Differentiated BMSCs were stimulated to undergo adipogenesis, osteogenesis, and chondrogenesis according to standard procedures35 (link)–37 (link). Briefly, at 90% confluence, the BMSCs were incubated in a differentiation culture medium for a further 21 days. For adipocyte differentiation, the complete culture medium was supplemented with 1 mM (10 nM) dexamethasone (Sigma, St Louis, MO, USA), 100 nM (0.5 mM) indomethacin (Sigma), and 2 mM insulin (Sigma). Adipogenesis was verified by Oil Red O staining (Beyotime, Beijing, China). To induce osteogenic and chondrogenic differentiation, the BMSCs were cultured with osteogenic medium [10 mM β-glycerophosphate (Sigma), 50 mg/ml ascorbic acid 2-phosphate (Sigma), and 100 nM dexamethasone] or chondrogenic medium [50 μg/mL ascorbic acid 2-phosphate, 100 nM dexamethasone, 1% insulin-transferrin-selenium solution (Gibco), and 10 ng/mL TGF-β1 (PeproTech, Rocky Hill, PA, USA)], respectively. Alizarin Red (Sigma), ALP (Beyotime), and safranin O (Solarbio) staining were conducted to visualize calcium accumulation and evaluate chondrogenesis according to the manufacturer’s suggested protocol. All staining results were viewed using an upright microscope (Olympus, Japan).
5
Differentiation of NPC-derived iPSCs into NP-like Cells
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Fibroblast derived iPSCs were generated and identified by the same method. fibroblasts were obtained from osteoarthritis patient, and fibroblast derived iPSCs were characterized [36 (link)]. When we differentiate NPC derived iPSCs into NP-like cells, human fibroblast derived iPSCs were used as a control. To differentiate NPC derived iPSC into NP-like cells, iPSCs at a high density (1 × 107 cells/ml) were dropped into the centre of a culture plate pre-coated with Matrigel, allowed to adhere for 1h, and then differentiation was induced with an NP differentiation medium (high glucose DMEM supplemented with 10ng/mL TGF-β1, 100 nM dexamethasone, 50 μg/mL ascorbic acid 2-phosphate, 1% ITS, 100U/mL penicillin, and 100 mg/mL streptomycin, all from Life Technologies) at 37°C in a 5% CO2 atmosphere. The differentiation media was changed twice per week and cells cultured in high glucose DMEM with 10% FBS served as a negative control (undifferentiatied cells). At Day 21, the cell pellets were then evaluated as described below.
6
Metformin's Impact on DPSC Proliferation and Differentiation
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Considering the dose-dependent effect of metformin, 100 µmol/L metformin hydrochloride (Alborz Pharmaceutical Co., Tehran, Iran) was chosen based on a previous study. 19 (link) The positive effect of this concentration of metformin on the proliferation and differentiation of DPSCs was evaluated. In brief, the cells at a density of 10 4 were seeded in 48-well plates with 100 μmol/L metformin hydrochloride, and either the standard medium was added for the proliferation assay or the osteogenic medium for the differentiation assay. Proliferation was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) (Sigma Aldrich, St. Louis, USA) assay after 1, 3 and 7 days of treatment with or without metformin. Osteogenic differentiation was evaluated using an alkaline phosphatase (ALP) activity kit (Sigma Aldrich) after 3, 7 and 14 days. Similar to the proliferation assay, DPSCs cultured in the osteogenic medium only (DMEM-low glucose (LG), 0.2 mol/L ascorbic acid 2-phosphate, 10 -8 mol/L dexamethasone, and 10 mmol/L β-glycerol phosphate; Life Technologies), without metformin, were used as a control for pairwise comparisons at each time point.
7
DPSC Osteogenic Differentiation with FDBA and Metformin
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Particulate cortical/cancellous mineralized 1-2-millimeter FDBA granules (Tissue Regeneration Corporation, Tehran, Iran) were placed in 48-well plates and a cell suspension including 2.5 × 10 4 DPSCs was loaded into the wells. The plates were incubated at 37°C for 1 h. Then, according to the type of evaluation, 2 kinds of media (standard or osteogenic) were added to the wells. The osteogenic medium included DMEM-LG, 0.2 mol/L ascorbic acid 2-phosphate, 10 -8 mol/L dexamethasone, and 10 mmol/L β glycerol phosphate (Life Technologies). Next, 100 μmol/L metformin hydrochloride was added to the samples. The study groups and the content of the wells are summarized in Table 1.
8
Bovine Synovium and Cartilage Explant Protocol
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Fresh synovial explants (Figure 3A ) and cartilage plugs (Figure 3B ) were harvested from discarded (IACUC-exempt) bovine calf knee joints (2–4 weeks old) [46 (link)]. The thin synovial sheath was extracted from the region adjacent to the medial and lateral femoral condyles. Synovium explants from 3 bovine knee joints were combined and cut into consistent ~1 cm × 1 cm pieces. Osteochondral dowel grafts were harvested using a 4 mm diameter dermal trephine biopsy from the femoral condyle of the joint. The acquired osteochondral plugs were cut 2 mm through the depth of the construct, from the articular cartilage surface to the deep cartilaginous zone, removing the subchondral bone layer. Prior to studies, synovium explants and cartilage plugs were cultured for five days in serum-free media consisting of DMEM supplemented with 50 μg/mL L-proline (Cat. No. P5607; Sigma-Aldrich, St. Louis, MO, USA), 100 μg/mL sodium pyruvate (Cat. No. S8636; Sigma-Aldrich, St. Louis, MO, USA), 1% ITSTM+ Premix (contains insulin, transferrin, selenous acid, BSA, and linoleic acid; Cat. No. 354352; Corning, Corning, NY, USA), 1% antibiotic–antimycotic (AA; Cat. No. 15240062; Thermo Fisher Scientific, Waltham, MA, USA), and 50 μg/mL ascorbic acid-2-phosphate (Cat. No. A8960; Sigma-Aldrich, St. Louis, MO, USA).
9
Directed Differentiation of hiPSCs into Keratinocytes
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Undifferentiated hiPSC (253G1) were seeded at a density of 15,000 cells/cm2 in a 6-well plate coated with iMatrix511 Silk using StemFit supplemented with 10 μM Y-27632, 2 days before the first day of differentiation (day 1). To initiate the differentiation, the medium was changed to Dulbecco’s modified Eagle medium (DMEM)/F12 (Fuji Film Wako Chemical Inc.) and Neurobasal medium (1:1; Thermo Fisher Scientific) supplemented with 0.1 mM non-essential amino acids, 1 mM glutamine, 55 μM 2-mercaptoethanol, 1% N-2 supplement, 2% B-27 supplement (all from Thermo Fisher Scientific), 50 μg/ml ascorbic acid 2-phosphate (Merck), 0.05% bovine serum albumin (Merck), and 100 ng/ml FGF-basic (Nacalai Tesque Inc.). On day 3 and thereafter, the medium was changed to Defined Keratinocyte-SFM (without addition of the attached supplement for day 3 and with the supplement on/after day 5; Thermo Fisher Scientific) with addition of 0.5 μg/ml hydrocortisone, 1 μM all-trans retinoic acid, 25 ng/ml hBMP-4 (Thermo Fisher Scientific), 2.4 μg/ml adenine, 1.37 ng/ml triiodothyronine, 0.3 mM ascorbic acid 2-phosphate, and 2 μM forskolin, every 2 days. Between days 4 and 5, the cells formed self-organized aggregates, and some re-attached to the plate after a few days.
10
Neuronal Differentiation Media Formulations
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N2B27 medium is composed of DMEM/F12 supplemented with modified N2 supplement and Neurobasal medium supplemented with B27 supplement minus vitamin A, in a 1:1 ratio and 50 μM β-mercaptoethanol (Thermo Scientific). FEB medium is N2B27 medium supplemented with 10 ng/mL FGF-2 (Peprotech), 10 ng/mL EGF (R&D Technologies), and 20 ng/mL BDNF (Peprotech). SFA medium is N2B27 medium supplemented with 100 ng/mL FGF-8 (Peprotech), 200 ng/mL sonic hedgehog (Peprotech), and 100 μM ascorbic acid 2-phosphate (Sigma). BGAA medium is N2B27 medium supplemented with 20 ng/mL BDNF, 10 ng/mL GDNF (Peprotech), 500 μM dibutyryl cyclic AMP (Sigma), 100 μM ascorbic acid 2-phosphate, 100 units/mL of penicillin, 100 μg/mL of streptomycin (Pen-Strep, Thermo Scientific), and 2 μg/mL Laminin (Roche).
Cyno NPC differentiation medium is composed of Neurobasal medium supplemented with B27 supplement (Thermo Scientific) and GlutaMAX supplement (Thermo Scientific), 20 ng/mL BDNF, 10 ng/mL GDNF, 100 μM ascorbic acid 2-phosphate, 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 2 μg/mL Laminin.
Cyno NPC differentiation medium is composed of Neurobasal medium supplemented with B27 supplement (Thermo Scientific) and GlutaMAX supplement (Thermo Scientific), 20 ng/mL BDNF, 10 ng/mL GDNF, 100 μM ascorbic acid 2-phosphate, 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 2 μg/mL Laminin.
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