High concentration type I collagen, and growth factor reduced Matrigel (MG) were purchased from Corning (Corning, NY). Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) were purchased from Corning. Oncostatin M (OSM) was purchased from R&D Systems (Minneapolis, MN). Tissue culture plates (24-well) were purchased from Greiner Bio-One (Kremsmünster, Austria). DMEM/F-12 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS (MP Biomedicals, Santa Ana, CA), 10 mM nicotinamide (Sigma-Aldrich), 1 × 10−7 M dexamethasone (Dex, Sigma-Aldrich), and 1× ITS (Gibco, Grand Island, NY) was used as the basic medium. The growth medium was prepared by adding 5 ng/ml EGF and 5 ng/ml HGF to the basic medium. The differentiation medium was prepared by adding 1% DMSO (Sigma-Aldrich) to the basic medium.
High concentration type 1 collagen
Manufactured by Corning
High concentration type I collagen is a purified and highly concentrated form of type I collagen, a major structural protein found in connective tissues. Its core function is to provide a natural extracellular matrix material for various cell culture and tissue engineering applications.
Automatically generated - may contain errors
Lab products found in correlation
Epidermal growth factor (egf), by Corning (1 mentions)
Dmem f12 medium, by Merck Group (1 mentions)
Oncostatin m, by R&D Systems (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Nicotinamide, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by MP Biomedicals (1 mentions)
Growth factor reduced matrigel, by Corning (1 mentions)
Type 1 collagen gel, by Corning (1 mentions)
3 protocols using high concentration type 1 collagen
1
Hepatic Differentiation via Collagen-Matrigel
2
3D Collagen-Based Cell Culture Protocol
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High Concentration type I Collagen (from rat tail tendon; 8–11 mg/ml; Corning) was chosen as the 3D extracellular matrix for this study. 3D cells + collagen constructs were prepared by diluting high concentration collagen to a final concentration of 3 mg/ml with 1 N NaOH, 10× DPBS, and sterile distilled water. This concentration was chosen based on previously published research38 , 39 and dilutions were calculated according to manufacturer's protocols. A number of 1.5 × 106 cells were then mixed with the collagen solution, immediately placed in custom built silicon molds and allowed to gel for 30 min at 37°C. Collagen handling and sample preparation was performed on ice and collagen solutions were used immediately following the dilution procedure. Complete cell culture medium was then added to the molds and images of cells+collagen constructs were taken at 24‐h intervals and processed by ImageJ (NIH). Cells+collagen constructs were cultured for a maximum of 6 days at 37°C, 5% CO2, and 95% humidity inside the molds before being used in various testing platforms. Culture medium was changed every 2–3 days. Initial thickness is calculated based on the volume of the collagen solution in each mold. Initial mold dimensions were 60.75 mm (length) × 16.70 mm (width) × 3.94 mm (thickness) for 4 ml of collagen solution.
3
Hepatobiliary Organoid Induction Protocol
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A step-by-step protocol describing induction of the HBTO can be found at Protocol Exchange32 . Cholangiocytes were resuspended in the growth medium and plated in 24-well plates coated with 200 μl of 4 mg/ml type I collagen gel prepared from high concentration type I collagen (Corning) at a density of 50,000 cells/well. Five days after plating, SHs were added to each well at a density of 50,000 cells/well. Following a further two days of incubation, the medium was replaced with differentiation medium supplemented with 10 ng/ml OSM and then overlaid with collagen gel containing 20% MG (Col-MG), which was prepared by mixing 2 mg/ml type I collagen gel and MG (v/v = 4:1) on ice. The plate was incubated at 37 °C for 3–4 h to form a gel, and then the differentiation medium was added. Culture medium was replaced with fresh medium every four days.
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