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Materials

Procedure

  1. Thaw chondrocytes following the recommended cell-thawing protocol.
  2. Adjust cell concentration to 0.5–1 million per mL.
  3. Add to culture flasks or wells at 1–3 x 105 per cm2.
  4. Incubate at 37°C, 5% CO2.
  5. Passage cells just before they reach confluence using 0.25% trypsin-EDTA to remove cells from the culture surface.
  6. Transfer cells to new culture vessels at the same concentration as in Step 3 (1–3 x 105 per cm2). Incubate at 37°C for 1–3 days.
  7. Continue culture as before.
  8. We recommend using the cells soon after thawing, within the first three passages.

Notes

  • Growth factors such as FGF-18 can be used to increase proliferation.
  • Culture supernatant may be used to measure type II collagen production or proteoglycan synthesis. Baseline levels are very high under these culture conditions, so increases may be harder to detect.