Human articular chondrocytes (HACs) were isolated from macroscopically healthy zones of osteoarthritic knee joints obtained from 9 donors undergoing total knee replacement. The study was performed in full accordance with local ethics guidelines, national and European Union legislation regarding human sample collection, manipulation and personal data protection (Ethics Committee for research with human samples, CODECOH: DC-2014-2325) and cartilage samples were collected after written informed consent of the donors. Chondrocytes were extracted as previously described [15 (link)]. Briefly, small slices of cartilage were digested in culture medium consisting of Dulbecco’s modified Eagle medium/Ham’s F12 (Gibco Invitrogen) with 0.06% bacterial collagenase A (Roche Applied Science) overnight. The cells were then seeded at a density of 1.5 x 104 cells/cm2 on culture dishes with culture medium supplemented with 10% fetal calf serum (FCS) (Gibco), 100 mg/mL streptomycin and 100 U/mL penicillin (Invitrogen). Thirty-six hours after seeding, medium was refreshed and further supplemented with 5 ng/mL FGF-2 (R&D Systems) and 5 μg/mL insulin (Umuline Rapide, Lilly), namely the FI cocktail. The culture medium was replaced three times a week. At confluence, cells were trypsinized, counted with a hemocytometer and used for 3D culture.
Dulbecco s modified eagle medium ham s f12
Manufactured by Thermo Fisher Scientific
Sourced in United States
Dulbecco's modified Eagle medium/Ham's F12 is a cell culture medium used for the growth and maintenance of a variety of cell types. It is a combination of Dulbecco's modified Eagle medium and Ham's F12 medium, providing a balanced, nutrient-rich environment for cells.
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Lab products found in correlation
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (4 mentions)
Epilife medium, by Thermo Fisher Scientific (4 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (4 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Umuline rapide, by Eli Lilly (2 mentions)
Bacterial collagenase a, by Roche (2 mentions)
Fibroblast growth factor 2 (fgf2), by R&D Systems (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Sigenome non targeting sirna 1, by Horizon Discovery (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Foetal calf serum, by Merck Group (1 mentions)
Plastic tissue culture flasks, by Corning (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Dubeccos modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Retinoic acid, by Merck Group (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
12 protocols using dulbecco s modified eagle medium ham s f12
1
Isolation and Expansion of Human Chondrocytes
2
LRP-1 Silencing in Thyroid Carcinoma Cells
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The FTC133 human follicular thyroid carcinoma cell line was grown in Dulbecco’s Modified Eagle Medium-Ham’s F12 (Invitrogen, Cergy Pontoise, France) with 10% FBS, as previously described (44 (link)). SiRNA mediated silencing of LRP-1 was described elsewhere (9 (link)). Specific LRP-1 targeting sequences were designed by Dharmacon (distributed by Perbio Science, Brebiere, France) as follows: GACUUGCAGCCCCAAGCAGUU (sense), CUGCUUGGGGUGCAAGUCUU (antisense). Control transfection experiments were achieved by using SiGENOME Non-targeting siRNA #1 (D00121001-20) from Dharmacon. For transient transfection assays, siRNA were transfected for four hours by using Lipofectamine 2000 (Invitrogen) according to manufacturer instructions.
3
Isolation and Culture of Mouse Choroid Plexus Cells
4
Exosome uptake in IPEC-J2 cells
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Porcine small intestinal epithelial (IPEC-J2) cells were cultured at 37 °C and 5% CO2 in Dulbecco’s modified eagle medium/Ham’s F-12 in a 1:1 ratio (Invitrogen, Life Technologies, Carlsbad, CA, USA) supplemented with 5% fetal calf serum (FCS; Invitrogen) and 5 ng/mL epidermal growth factor (EGF; Peprotech, Rocky Hill, NJ, USA). IPEC-J2 cells were seeded at 0.5 × 105 cells/mL in a 10 mL volume in plastic tissue culture flasks (75 cm2 Corning, Corning, NY, USA). After reaching confluency (four days)79 (link), the cells were seeded into 6-well tissue culture plates (9.6 cm2/well) at 2.0–2.3 × 105 cells/well in a 2 mL volume. The cells were allowed to adhere for 24 h, and the media were replaced every other day. When the cells were 90% confluent, we added 0.5 mL exosomes or exosome-free supernatants to each well, the equal volume PBS added as control. We determined that a 25% media substitution was optimum. Exosomes suspended in PBS and supernatants were passed through 0.45-μm and 0.22-μm membrane filters prior to incubation. IPEC-J2 cells were harvested after 8 h. we give all the in vitro experiment for three repeats test.
5
Chondrocyte Isolation and Expansion Protocol
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HACs and MACs were extracted as previously described46 (link). Briefly, small slices of cartilage were digested in a culture medium consisting of Dulbecco’s modified Eagle medium/Ham’s F12 (Gibco Invitrogen) with 0.06% bacterial collagenase A (Roche Applied Science) overnight. The cells were then seeded at a density of 1.5 × 104 cells/cm2 on culture dishes with culture medium supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 mg/mL streptomycin and 100 U/mL penicillin (Invitrogen). Forty-eight hours after seeding, the medium was refreshed and further supplemented with 5 ng/mL FGF-2 (R&D Systems) and 5 µg/mL insulin (Umuline Rapide, Lilly), namely the FI cocktail. The culture medium was then replaced three times a week. At confluence, cells were trypsinized, counted with a hemocytometer and used for hydrogel encapsulation.
6
Directed Differentiation of iPSCs into Epidermal Progenitors
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Example 1
To induce differentiation, undifferentiated iPSCs are transferred into a 20% 02 atmosphere environment and treated with mTESR1 or other pluripotent stem cell basal media supplemented with 1 mM ATRA (Sigma-Aldrich) and 25 ng/ml BMP4 (R&D) for 7 days (Induction).
To select for cells with early acquisition of ectodermal fate, the cells are harvested and replated onto freshly prepared 3D HDF ECM or other type of ECM at a density of 5-10×103 cells per cm2 and grown in Dulbecco's modified Eagle's medium/Ham F12 (3:1; Life Technologies) or Keratinocyte media supplemented with serum substitute such as human platelets lyste and with 1 mM ATRA and 25 ng/ml BMP4 for a further 7 days (Selection).
To enrich for putative epidermal progenitors, rapid adhesion to type IV collagen-coated dishes can be used, and the rapidly adhering cells are cultured in defined keratinocyte-SFM or other keratinocyte medium supplemented with 1 mM ATRA for 7 days (Enrichment). After that, the cells are cultured in EpiLife medium (Life Technologies) or other keratinocyte medium for a further 7 days (Expansion) before final harvest and analysis.
7
Stepwise Differentiation of iPSCs into Epidermal Progenitors
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Example 1
To induce differentiation, undifferentiated iPSCs are transferred into a 20% 02 atmosphere environment and treated with mTESR1 or other pluripotent stem cell basal media supplemented with 1 mM ATRA (Sigma-Aldrich) and 25 ng/ml BMP4 (R&D) for 7 days (Induction).
To select for cells with early acquisition of ectodermal fate, the cells are harvested and replated onto freshly prepared 3D HDF ECM or other type of ECM at a density of 5˜10×103 cells per cm2 and grown in Dulbecco's modified Eagle's medium/Ham F12 (3:1; Life Technologies) or Keratinocyte media supplemented with serum substitute such as human platelets lyste and with 1 mM ATRA and 25 ng/ml BMP4 for a further 7 days (Selection).
To enrich for putative epidermal progenitors, rapid adhesion to type IV collagen-coated dishes can be used, and the rapidly adhering cells are cultured in defined keratinocyte-SFM or other keratinocyte medium supplemented with 1 mM ATRA for 7 days (Enrichment). After that, the cells are cultured in EpiLife medium (Life Technologies) or other keratinocyte medium for a further 7 days (Expansion) before final harvest and analysis.
8
Directed Differentiation of iPSCs to Epidermal Progenitors
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Example 1
To induce differentiation, undifferentiated iPSCs are transferred into a 20% O2 atmosphere environment and treated with mTESR1 or other pluripotent stem cell basal media supplemented with 1 mM ATRA (Sigma-Aldrich) and 25 ng/ml BMP4 (R&D) for 7 days (Induction).
To select for cells with early acquisition of ectodermal fate, the cells are harvested and replated onto freshly prepared 3D HDF ECM or other type of ECM at a density of 5-10×103 cells per cm2 and grown in Dulbecco's modified Eagle's medium/Ham F12 (3:1; Life Technologies) or Keratinocyte media supplemented with serum substitute such as human platelets lyste and with 1 mM ATRA and 25 ng/ml BMP4 for a further 7 days (Selection).
To enrich for putative epidermal progenitors, rapid adhesion to type IV collagen-coated dishes can be used, and the rapidly adhering cells are cultured in defined keratinocyte-SFM or other keratinocyte medium supplemented with 1 mM ATRA for 7 days (Enrichment). After that, the cells are cultured in EpiLife medium (Life Technologies) or other keratinocyte medium for a further 7 days (Expansion) before final harvest and analysis.
9
Differentiation and Selection of Epidermal Progenitors from iPSCs
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Example 1
To induce differentiation, undifferentiated iPSCs are transferred into a 20% 02 atmosphere environment and treated with mTESR1 or other pluripotent stem cell basal media supplemented with 1 mM ATRA (Sigma-Aldrich) and 25 ng/ml BMP4 (R&D) for 7 days (Induction).
To select for cells with early acquisition of ectodermal fate, the cells are harvested and replated onto freshly prepared 3D HDF ECM or other type of ECM at a density of 5-10×103 cells per cm2 and grown in Dulbecco's modified Eagle's medium/Ham F12 (3:1; Life Technologies) or Keratinocyte media supplemented with serum substitute such as human platelets lyste and with 1 mM ATRA and 25 ng/ml BMP4 for a further 7 days (Selection).
To enrich for putative epidermal progenitors, rapid adhesion to type IV collagen-coated dishes can be used, and the rapidly adhering cells are cultured in defined keratinocyte-SFM or other keratinocyte medium supplemented with 1 mM ATRA for 7 days (Enrichment). After that, the cells are cultured in EpiLife medium (Life Technologies) or other keratinocyte medium for a further 7 days (Expansion) before final harvest and analysis.
10
Culturing Breast Cancer and Normal Cells
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