In vitro viability, mitogenicity and clonogenic capacity of periodontal ligament cells after storage in four media at room temperature

Abstract

The choice of storage medium for preserving traumatically avulsed teeth is important for the success of future replantation. The objective of this study was to compare the effectiveness of four recommended storage media (Hank's balanced salt solution [HBSS], culture medium, alpha minimal essential medium [alpha-MEM], and ViaSpan) to preserve cultured periodontal ligament fibroblasts (PDLF) at room temperature (22 degrees C). PDLF were obtained from explants of extracted healthy human teeth. Plates with confluent PDLF were soaked in the various media for 2, 8 and 24 h at room temperature. A control group was incubated with culture medium at 37 degrees C. After incubation, viability of the cells was determined by trypan blue exclusion test. Viable cells were then analyzed for mitogenic (with thymidine) and clonogenic capacity (by culturing one cell/well). Viability of PDLF stored up to 24 h was comparable in all tested media, and the differences were limited to 1%-3%. PDLF stored for up to 24 h in various media had statistically comparable mitogenicity to the control group. After 8 h of storage, the differences were limited to 2%-9%, except for the alpha-MEM group which had 23%-29% lower mitogenic capacity compared to the control group. Increasing the storage time up to 24 h further decreased the mitogenicity of the cells by 22%-47%. The highest mitogenicity after 24 h of storage was found in PDLF stored in culture medium or HBSS, and the lowest in alpha-MEM. PDLF stored for 2-8 h in various media had a comparable clonogenic capacity to the control group. However, after 24 h, the cells' clonogenic ability dropped by 14%-66%. A similar trend of reduction was noted in the mitogenic and clonogenic capacity, although it was statistically significant only in the clonogenic capacity. Culture medium and ViaSpan, followed by HBSS, were the most effective in preserving the clonogenic capacity of PDLF after 24 h of storage. The lowest clonogenic capacity after 24 h of storage was in the alpha-MEM group (66%, P < 0.0025). In conclusion, culture medium, followed by HBSS and ViaSpan, was the most effective media for preserving the viability, mitogenicity and clonogenic capacity of PDLF stored for up to 24 h at room temperature. The lowest functional abilities were found in PDLF stored in alpha-MEM.