Aging Aging

58 Pawelecweekly or fortnightly subculture with new feeder cells and fresh medium (seeNote 5).7. Estimate longevity in terms of population doublings (PDs). Score initial limitingdilution cloning wells as positive if at least one third full of growing cells. Onethird of the surface of a Terasaki well is equivalent to about 1000 cells (= 10 PDs).Use the number of clones derived at this stage to calculate the average life-spanof all clones derived, that is, do not include clones achieving less than this numberof PD in the analysis. The number of cells per microtiter plate well prior tocluster plate transfer is approx 1 × 10 5 (= approx 17 PDs). Assume that clonesdying between the Terasaki and microtiter plate stages have undergone 17 PDs,and use this figure in calculations of average longevity. After clones are transferredto 16 mm diameter cluster wells, the number of PDs undergone can beestimated for each clone from the exact number of cells counted at each subculture.Cryopreserve cells at any point of their life-spans. Continue calculations ofPDs undergone on the basis of the number of viable cells replated after thawing,not the number of cells originally present. Take maximum life-span of cells ineach cloning experiment to be the PDs corresponding to the time point of thedeath of the longest living clone in each case (see Note 6).4. Notes1. For many applications where mature T-cells are to be cloned, it is not necessaryto purify them beforehand. PBMCs as the starting population can be so stimulatedthat only T-cells can grow (e.g., with T-cell mitogens or antigens). Whenusing CD34 + cells, purity is, however, critical, because contaminating non-CD34cells most likely have a growth advantage over the CD34 + cells.2. Clearly the culture medium employed is a critical aspect of the technique.For many years, we and others found that although T-cells could be grown forlimited periods in completely chemically defined serum-free media, cloningand long-term propagation in such media was not possible. We and otherswere forced to use a serum supplement, most commonly FCS or HS. In ourexperience, very few batches of FCS prove suitable for human T-cell cloningand extensive propagation. Unfortunately, the same was true for commerciallyavailable HS. We have therefore always obtained material from bloodbanks and prepared the serum ourselves. Because of paucity of AB blooddonors, we have always used nontransfused male blood. Each serum is separatelyheat inactivated (56°C, 30 min) and individually tested for its abilityto support lymphocyte proliferation. Sera supporting acceptable levels of proliferation(usually around 80% of tested samples) are pooled and used at 10–20% v/v with culture medium (RPMI 1640 or IMDMEM). However, note thatCD34 + cells cannot be grown in either FCS- or HS-containing medium. Twofactors recently enabled us successfully to grow these cells and facilitatetheir differentiation into mature T-cells. The first was to use the serum-freemedium X-Vivo 10 without adding any other serum supplement. This mediumis also suitable for the cloning and long-term propagation of TCC derived