Studies developed by Athanasiu on goat costal chondrocytes demonstrated that the proliferative ability of the cells decreased after passage 5. In the present study, we have demonstrated that human TMJ cells were highly proliferative at most cell passages, with the lowest values found at P9. This could be possibly associated to a senescence process. It is important to highlight that most previous works have been performed in 5 or less cell passages. In terms of cell viability, the classical idea that early passage cells are the most feasible for tissue engineering should be deeply considered, because it has been previously demonstrated that the earliest cell passages are frequently under adaptative conditions to ex vivo environments and they do not display the most accurate conditions to be used in tissue engineering protocols. In this sense, all cell sources should be previously characterized and deeply studied using a combination of highly sensitive techniques to select and determine the most accurate cell passage for regeneration protocols. In this milieu, we recently described an ACVL method to evaluate cell viability of human cells high accuracy. In the present work, we used a combined approach based on the use of 3 different methods to determine a cell viability index at different levels: cell membrane integrity, cytoplasmic metabolism and intracellular ionic content. Thus, our results revealed that the most viable cell passage of cultured TMJF could be P6, although P5 and P7 also showed good cell viability levels. Interestingly, the combination of classical protocols as trypan blue Albaspidin-AA assays and precise techniques such as calcein/AM and ethidium homodimer-1 and electron-probe Xray microanalysis, showed a good correlation Cinoxacin between classical and novel assays to determine the cell behaviour of TMJF cells along nine subcultures. First, the trypan blue assay, a classical method that evaluates the cell membrane integrity evidenced high cell viability levels in all cell passages analyzed here, with the maximum cell viability at P6 which is in agreement with previous results obtained using WHJSC. In the second place, we analyzed the enzymatic esterase cytoplasmatic activity and the membrane integrity by using calcein/AM and ethidium homodimer-1 assays. The results demonstrated that the most viable passage was P7 followed by P6 and P5. In contrast with trypan blue assays, this sensitive method showed that cell viability was very variable among the nine cell passages. In fact, P1 and P2 in junction with P9 were the less viable cell passages that were not efficiently detected by trypan blue assay. This may suggest that early stages of cell death in which the cell membrane has been not damaged could not be efficiently detected by trypan blue. For this reason, we hypothesize that the utilization of classical methods should be always accompanied by more sensitive assays such as electron-probe X-ray microanalysis which allows us to determine qualitative and quantitative intracellular concentrations of key ions involved in cell viability. As we previously described, this method is highly sensitive for determination of the mechanisms of cell death that may occur after sequential cell passaging. The link between intracellular levels of potassium, sodium, chlorine, calcium, sulfur, magnesium, phosphorous and cellular physiology has been deeply studied and it is clear that 
a close relationship exists between them. However, the intracellular ionic contents of TMJF and their relation with cell functions have not been previously described.