Summary Of The Culture Protocol

Our cell culture studies have focused on the analysis of the CD8+ T cell subset, the cell type responsible for controlling viral infections, which are a major clinical problem in the elderly. In preliminary experiments, aimed at optimizing long-term growth and viability of human T cells, we verified that the most reproducible growth patterns of human T cells, whether from neonates or adults, were obtained in AIMV serum-free medium. In these early studies, we observed that some batches of fetal calf serum or even human serum inhibited the growth of T cells, particularly T cells derived from neonates (Perillo et al., 1989).

Our initial cell culture protocol used in defining the proliferative lifespan for human T cells involved stimulation of peripheral blood mononuclear cells with an irradiated allogeneic lymphoblasoid cell line in the continued presence of the T cell growth factor, Interleukin-2 (IL-2). After an initial burst of proliferation over approximately 2-3 weeks, the cells reached quiescence, at which time the culture was restimulated with the same antigen and IL-2. This process was repeated until the point at which the culture showed no cell number increase in response to two rounds of antigen stimulation, at which time it was considered to have reached the end stage of replicative senescence.

Since T cells grow in suspension culture, accurate cell counts could be performed at each passage, allowing us to calculate the total number of population doublings achieved by that culture. Using this cell culture protocol, which favors the generation of CD8+ T cell cultures, we followed over 100 T cell cultures, derived from peripheral blood samples from both adults and neonatal donors, and reported that the number of population doublings (PDs) ranged from 11 to 57, with a mean of 23 ± 7 (Perillo et al., 1989). Similar ranges of proliferative potential of human T cells have been reported by other groups as well.

In our experiments, there was a significant difference in the PDs achieved by cells from adult versus neonatal donors (p < 0.025). However, among the adult donors, there was no correlation between in vitro lifespan and chronological age (Perillo et al., 1989). In terms of function, our studies showed that for alloantigen-stimulated cultures, antigen-specific cytotoxic function was retained even at senescence, and the cells in senescent cultures were able to upregulate expression of the alpha chain of the IL-2 receptor in response to specific antigen, despite the inability to enter cell cycle when exposed to the same antigen (Perillo et al., 1993).

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