Human articular chondrocytes were isolated as previously described (Lopez-Ruiz et al., 2013) Chondrocytes were grown in Dulbecco’s modified eagle’s medium (DMEM) – high glucose (Sigma) supplemented with 10% fetal bovine serum (Gibco), 50 microg/microL of l-ascorbic acid 2-phosphate (Sigma), 1% penicillin-streptomycin (Sigma), and 1% ITS (Gibco) in a 25-cm2 cell culture flask. Cells were incubated at 37 ºC humidified atmosphere containing 5% CO2 and expanded in a monolayer for 7–10 days before the experiment. Chondrocytes seeded on glass coverslips were fixed and stained with 0.4% toluidine blue staining for 15 min and then were rinsed with distilled water. For immunohistochemical analysis, fixed monolayers were blocked for 1 h at room temperature (RT) with 5% BSA, 5% fetal bovine serum (FBS) in phosphate-buffered saline (PBS), and then incubated with Col I (SC25974, St. Cruz) and Col II (SC52658, St. Cruz) overnight at 4 ºC. The next day, samples were washed three times with PBS and incubated with the secondary antibodies (Alexa) for 1 h at RT, and finally were washed three times with PBS and mounted with mounting medium with DAPI. Photographs were taken with a Leica DM 5500B (Solms, Germany) microscope.
References
1. López-Ruiz E, Perán M, Cobo-Molinos J, Jiménez G, Pic on M, Bustamante M, Arrebola F, Hernández-Lamas MC, Delgado-Martínez AD, Montañez E, Marchal JA. Chondrocytes extract from patients with osteoarthritis induces chondrogenesis in infrapatellar fat pad-derived stem cells. Osteoarthr Cartil.2013;21:246–58
2. Baena, J.M.; Jiménez, G.; López-Ruiz, E.; Antich, C.; Griñán-Lisón, C.; Perán, M.; Gálvez-Martín, P.; Marchal, J.A. Volume-by-volume bioprinting of chondrocytes-alginate bioinks in high temperature thermoplastic scaffolds for cartilage regeneration. Exp. Biol. Med.2019, 244, 13–21. https://doi.org/10.1177/1535370218821128
| Number | Category | Product | Amount |
|---|---|---|---|
| 1 | - | Dulbecco’s Modified Eagle’s medium (DMEM), high glucose | 1 |
| 2 | - | Fetal bovine serum | 1 |
| 3 | - | L-ascorbic acid 2-phosphate | 1 |
| 4 | - | 10,000 U/ml Penicillin – 10 mg/ml streptomycin solution | 1 |
| 5 | - | 100X Insulin-Transferring-Selenium | 1 |
| 6 | - | 25-cm2 cell culture flask | 1 |
| 7 | - | Human articular chondrocytes | 1 |
| 8 | - | Toluidine blue | 1 |
| 9 | - | Bovine serum albumin | 1 |
| 10 | - | 1X Phosphate buffered saline | 1 |
| 11 | - | Antibody against collagen I | 1 |
| 12 | - | Antibody against collagen II | 1 |