Aging Aging

Human T-Cell Clones 59from mature T-cells. The second factor was to use a suitable cytokine cocktailthat supported the viability of the stem cells and also allowed T-cell growth totake place. This consisted of stem cell factor (SCF), flt3-L, and IL-3, togetherwith IL-2 and either oncostatin M (OM) or IL-7.3. Use autologous PBMCs, a mixture of autologous PBMCs and autologousB-lymphoblastoid line cells, or other appropriate antigen-presenting cells (APC),in the presence of specific antigen. Alternatively, use an antigen-non-specificstimulus such as 50 ng/mL of the anti-CD3 monoclonal antibody OKT3 or2 µg/mL of purified or 1% crude PHA, together with the same number of allogeneicor autologous PBMCs, or pooled PBMCs (irradiated at 30 Gy).4. Clones successfully propagated in cluster plate wells for 2 wk can be taken to beestablished. They can at this point be cryopreserved, although it is advisable toretain some of each clone in culture to test different conditions to establish optimalparameters for each particular clone. Human TCCs can be readily cryopreservedusing the same protocols as are suitable for freezing resting T-cells.Having a frozen stock enables the different culture conditions to be tested tooptimize growth, without risking the loss of the whole clone. Restimulationparameters should be established for each clone. T-cells require periodic reactivationthrough the T-cell antigen receptor to retain responsiveness to growth factors.This can be accomplished either specifically or nonspecifically. All clonescan be propagated with weekly restimulation; some but not all can be propagatedwith restimulation only every 2 wk. It should be established whether each clonecan be propagated with the most convenient feeder cells (80 Gy-irradiatedB-LCL) instead of PBMC feeders. Most TCCs flourish on B-LCLs alone, butsome need the presence of PBMCs as well (this is especially true during cloning).Propagation of the TCCs on PBMC feeders can also be continued, but for practicalreasons it may often be more convenient and easier to grow large amounts ofB-LCLs than to isolate the PBMCs.5. For convenience, it is also easier to grow TCCs in scaled-up culture vessels thanin 16 mm-diameter culture wells. However, not all clones can be adapted togrowth in flasks. This has to be tested for each clone, using between 1 × 10 5 and5 × 10 5/ mL of TCCs with an equal number of feeders in tissue culture flasks.Clones not growing under these conditions can rarely be adapted to growth inflasks by altering the amounts or concentrations of TCCs or feeders or by increasingor decreasing the frequency of stimulation and/or feeding.6. Longevity estimation in PD is an extremely conservative measurement indicatingthe absolute minimum number of cell divisions achievable by each cell. This isbecause it simply assumes that all daughter cells at each cell division are viableand themselves capable of dividing and generating two viable progeny. In reality,it is highly likely that this is not the case.AcknowledgmentsWork in the author’s laboratory is supported by the Deutsche Forschungsgemeinschaft,the Dr. Mildred Scheel Foundation, the Dieter Schlag Founda-