300 MHz proton NMR study of the differentiation of diploid human epidermal keratinocytes

The nuclear magnetic resonance spin-lattice (T₁) and spin-spin (T₂) relaxation times are closely related to the molecular motions of the molecules in a liquid sample. T₁ and T₂ of human epidermal cells were measured at 300 MHz as functions of harvesting methods (i.e., scraping vs trypsinization) and age in culture. It was found that T₁ and T₂ values have smaller variances when the cell is harvested by trypsinization rather than scraping. The correlation coefficients for both T₁ and T₂, obtained from cells harvested by trypsinization, are much higher than those obtained from cells harvested by scraping. More importantly, this is the first report to monitor in vitro aging through relaxation times measurement. There is a significant increase in the values of T₁ and T₂ from the third to seventh passages. Human keratinocytes slowed down and even ceased to grow the seventh passage. Therefore, the cellular water molecules of human keratinocytes have higher mobility in a more differentiated state. The factors contributing to the change in relaxation times as cells progress toward senescence are discussed.