Measurement of telomere lenght as a marker of senescence in human cultured chondrocytes

Abstract

Purpose: Autologous chondrocyte implantation is one of the treatments for cartilage repair. It is considered that a telomere shorter than 3 Kbp (kilo base-pairs) is a critically short telomere related to fusion events that lead to genome instability and cell senescence. In this work, the proliferative ability of cultured chondrocytes, in terms of their telomere length, before being implanted to patients with cartilage defects, was studied. Methods: Articular cartilage biopsies (B1, B2 and B3) were obtained from 3 patients (2 males and 1 female) with a similar age (36, 40 and 42 years-old) with cartilage defects in the knee, which were going to be treated with high density autologous chondrocyte (ICC). The chondrocytes were isolated and cultured in DMEM with autologous serum. After the 3rd passage, an aliquot of 1 million cells was removed to estimate the telomere length and the remaining cells were implanted in the patients. In each culture, the doubling time and the estimated number of cell divisions during expansion were calculated. Telomere length measurement was carried-out by quantitative fluorescent in situ hybridization (Q-FISH), studying 10 metaphases from each cell culture. Results: The number of the starting cells obtained was 10000 in B1, 45000 in B2 and 38000 chondrocytes in B3, and after 53, 49 and 35 days of culture, a number of 20, 38 and 26 million cells were obtained, respectively. The doubling time and the number of cell divisions are shown in the table 1. The figure 1 shows a representative Q-FISH from each cartilage biopsy. The median telomere length in each cell culture was 10.3, 10.7 and 10.5 Kbp, respectively (Table 2), being these values statically higher than 3 Kbp (p&lt0.001) in the 3 cases. Conclusions: Telomeres of the chondrocyte cultured for future implantation are larger enough to think that these cells are not senescent.Table 1Doubling time and number of cell divisions during the culturesCartilage biopsyAge (years)GenderTime in culture (days)Dubling time (days)Estimated number of eel divisionsB140Female534.8310.97B236Male495.049.72B342Male353.729.42 Open table in a new tab Table 2Median and 20 th percentile of telomere length of the cartillage biopsiesCartilage biopsyMedian lenght (bp)20 th Percentile lenght (bp)Coeficient of variationB110.2706.8403.94B210.7337.0955.72B310.4677.0593.54 Open table in a new tab