Introduction: Articular cartilage (AC) repair in the spine requires expansion of chondrocyte number prior to implantation. Cell density when replicating cells are passaged is often poorly reported but evidence suggests that this may be critical for determining cell phenotype.
Objective: To determine human chondrocytic genotype variation at different cell densities during culture.
Materials and Methods: Human chondrocytes were grown in monolayer and serial photographs were taken daily. Cell density was determined for ‘confluence’ (floor of the dish covered completely) and subsequent ‘maximum cell density’. Subsequently, multiple plates were seeded at the same cell density (20%) and the cells were allowed to replicate. Samples were collected at 20%, 30%, 50%, 70%, 90% and 100% of the predetermined cell density at confluence. RNA was extracted and RT-PCR was performed on each sample to determine gene expression levels for collagen types I and II, aggrecan and GAPDH.
Results: Confluence was achieved on ~day 10 while maximum cell density was achieved ~day 22. Collagen I gene expression was maximally expressed at 90% of confluence then drastically decreased at higher density while gene expression for collagen II was maximally expressed at only 50% of confluence. Aggrecan expression remained more constant. Actual results between different patients varied widely.
Conclusion: Cell density must be clearly defined during the various stages of cell culture because cell characteristics differ significantly dependent on cell density.
Significance: Cells implanted to repair damaged articular cartilage in the spine require specific molecular chondrocytic characteristics. These results highlight the importance of cell density during culture to acquire/maintain these characteristics.