Chondrocyte progenitor cells were seeded with 1.5 x 105 cells on uncoated 100 mm dishes cultured in a growth medium for 1 day. The next day, after confirming adhesion, the medium was exchanged with a differentiation induction medium and cultured for 6 days; the final concentration contained D-MEM/F12 Ham (1:1) (Sigma-Aldrich, St. Louis, MO, US), stabilized antibiotic–antimycotic solution (100×) (Sigma-Aldrich, St. Louis, MO, US), 0.2 mM ascorbic acid 2-phosphate (AA2P) (Sigma-Aldrich, St. Louis, MO, US), 10−7 M dexamethasone (Sigma-Aldrich, St. Louis, MO, US), 1× ITS-X (Gibco™), and 5 ng/mL IGF (Sigma-Aldrich, St. Louis, MO, US). After removing the supernatant on the seventh day, the cells were washed with a wash buffer and then seeded with 1.5 × 106 perichondrocytes; the final concentration contained D-MEM/F12 Ham (1:1) (Sigma-Aldrich, St. Louis, MO, US) and stabilized antibiotic–antimycotic solution (100×) (Sigma-Aldrich, St. Louis, MO, US). The next day, after confirming adhesion, the medium was replaced with a differentiation induction medium and cultured for 6 days. This was repeated once more to prepare a three-layer cell sheet; 2.7 × 106 cells of perichondrocytes were used for each cultured cartilage sample.
Stabilized antibiotic antimycotic solution
Manufactured by Merck Group
Sourced in United States
Stabilized antibiotic–antimycotic solution is a sterile, ready-to-use solution containing antibiotics and antimycotics. It is designed to prevent microbial contamination in cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f 12 ham, by Merck Group (2 mentions)
Its x, by Thermo Fisher Scientific (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Basic fgf, by Fujifilm (1 mentions)
Pdgf bb, by Thermo Fisher Scientific (1 mentions)
Transforming growth factor β1 tgf β1, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid 2 phosphate aa2p, by Merck Group (1 mentions)
2 protocols using stabilized antibiotic antimycotic solution
1
Chondrocyte Progenitor Cell Differentiation
2
Chondrogenic Differentiation of Auricular Perichondrial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The following three culture methods are described: 2D culture, micro 3D culture,
and macro 3D culture. The expanded auricular perichondrial chondroprogenitor
cells (passage 1) were dissociated into single cells with 0.25% trypsin. For 2D
culture, 30,000 cells were seeded into a well of a 24-well plate containing 300
μL of chondrogenic induction medium comprising DMEM/F12 Ham (1:1)
(Sigma-Aldrich), stabilized antibiotic–antimycotic solution (100×)
(Sigma-Aldrich), 0.2 mM AA2P (Sigma-Aldrich), 10−7 M dexamethasone
(Sigma-Aldrich), 1× ITS-X (Gibco), 5 ng/mL IGF (Sigma-Aldrich), 10 ng/mL basic
FGF (Wako), 10 ng/mL PDGF-BB (Peprotech), 10 ng/mL transforming growth factor β1
(TGFβ1) (Peprotech), and 2% FBS.
For micro 3D culture, 30,000 cells in 300 mL of chondrogenic induction medium
were divided into two wells of a 96-well U-bottomed micropatterned plate
(Corning). Each well of the U-bottomed micropatterned plate had 79 micro-wells.
Thus, 158 micro-wells were used, and each micro-well contained 190 cells. For
macro 3D culture, 30,000 cells in 300 μL of chondrogenic induction medium were
seeded into a well of a 96-well U-bottomed plate (Greiner Bio-One). The medium
in each culture system was carefully changed daily. After a culture duration of
1, 3, and 5 days, cells were collected for analysis in each culture system.
and macro 3D culture. The expanded auricular perichondrial chondroprogenitor
cells (passage 1) were dissociated into single cells with 0.25% trypsin. For 2D
culture, 30,000 cells were seeded into a well of a 24-well plate containing 300
μL of chondrogenic induction medium comprising DMEM/F12 Ham (1:1)
(Sigma-Aldrich), stabilized antibiotic–antimycotic solution (100×)
(Sigma-Aldrich), 0.2 mM AA2P (Sigma-Aldrich), 10−7 M dexamethasone
(Sigma-Aldrich), 1× ITS-X (Gibco), 5 ng/mL IGF (Sigma-Aldrich), 10 ng/mL basic
FGF (Wako), 10 ng/mL PDGF-BB (Peprotech), 10 ng/mL transforming growth factor β1
(TGFβ1) (Peprotech), and 2% FBS.
For micro 3D culture, 30,000 cells in 300 mL of chondrogenic induction medium
were divided into two wells of a 96-well U-bottomed micropatterned plate
(Corning). Each well of the U-bottomed micropatterned plate had 79 micro-wells.
Thus, 158 micro-wells were used, and each micro-well contained 190 cells. For
macro 3D culture, 30,000 cells in 300 μL of chondrogenic induction medium were
seeded into a well of a 96-well U-bottomed plate (Greiner Bio-One). The medium
in each culture system was carefully changed daily. After a culture duration of
1, 3, and 5 days, cells were collected for analysis in each culture system.
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