Clonogenic Capacity of Periodontal Ligament Cells Stored in Different Storage Media

Ab s t r Ac t Objectives: We examined the effects of storage in Hank’s balanced salt solution (HBSS), low-fat milk, soy milk, and aloe vera extract on the clonogenic capacity of human periodontal ligament (PDL) cells. The Dulbecco’s modified Eagle’s medium (DMEM) was used as control. Materials and methods: Human teeth that are extracted for the orthodontic purpose were collected and the periodontal ligament tissue was cultured. Cultured experimental PDL cells were exposed to different experimental solutions for 24 hours. To evaluate the clonogenic capacity of the stored cells, cells were inoculated into 96-well plates at a concentration of one viable cell/well. For each tested group, 96 replicates were plated and grown for 3 weeks in a culture medium at 37°C in humidified air containing 7% CO2. Under these conditions, discrete colonies arising from a single cell that covered 70–100% of the well were counted under light microscopy (×200). The percentage of cells with clonogenic capacity was calculated as the number of colonies formed/number of cells seeded × 100. Thus, the clonogenic capacity reflected the likelihood that each colony arose from a single cell. The experiments for 2 and 8 hours were repeated three times (three 96-well plates), and for 24 hours, five times (five 96-well plates). Results: The highest clonogenic capacities were found in cells stored in HBSS (15.3 ± 1.15) and aloe vera extract (12.3 ± 2.5) followed by low-fat milk (10.3 ± 1.5). Soy milk showed least clonogenic capacity (3.6 ± 1.5). The Dulbecco’s modified Eagle’s medium, HBSS, and aloe vera showed similar statistical significance (p = 0.092). Conclusion: Highest clonogenic capacity was found in cells stored in HBSS, aloe vera extract, and low-fat milk. Soy milk showed least clonogenic capacity.