Protocol For Cloning By Limiting Dilution

1. Dilute the cells for cloning in culture medium (e.g., RPMI 1640 + 15% prescreened FCS or human serum; or serum-free medium such as X-Vivo 15) to which 40 U/ml of IL-2 is added. Set the concentration so that 10 ^l contain 45, 4.5, or 0.45 cells. Then plate 10 ^l of the 0.45 suspension into 60 x 1 mm-diameter wells of culture trays (Terasaki plates) and leave in a vibration-free area for an hour. Check the distribution of cells in the wells visually using an inverted microscope, being careful to look around the edges of the wells. According to the Poisson distribution, only a maximum of 37% of the wells should contain cells. Readjust dilutions if necessary, and recheck.

2. Plate at least five trays with the 0.45 cells/10 ^l, suspension, one with 4.5 and one with 45, and add a constant number of feeder cells to each well. Irradiated PBMC commonly are used as feeder cells at 1 x 104/well. Use autologous PBMC, a mixture of autologous PBMC and autologous B-lymphoblastoid line cells, or other appropriate APC, in the presence of specific antigen. Alternatively, use an antigen-nonspecific stimulus such as 50 ng/ ml of the anti-CD3 monoclonal antibody OKT3 or 2 ^g/ml of the mitogen PHA, together with the same number of allogeneic or autologous PBMC.

3. Stack plates and wrap in aluminum foil for ease of handling and as a precaution against contamination. Incubate for about a week and then examine the plates using an inverted microscope. Transfer contents of positive wells (> one-third full, ca. 1000 cells) to 7 mm-diameter flat-bottom microtiter plate wells with fresh medium and 1 105 of the same feeder cells as before. Check Terasaki plates again at intervals of a few days up to two to three weeks of age to identify late developers and transfer these also. Check microtiter plates every few days, and identify wells becoming crowded within a week post-transfer. These must be split 1:1 into new culture wells and re-fed with medium (but not feeder cells). After one week in microtiter plates, contents of wells with growing cells are transferred to 16 mm-diameter cluster plate wells with two 5 105 of the same feeder cells, and fresh medium. Observe after three to four days and establish which wells are already full or nearly full. The former should be divided into four, the latter into two, with fresh media, but no more feeders. After a total of one week in cluster plates, count the number of cells in each clone and split to 2 x 105/well, again with two 5 x 105 feeders/well and fresh medium. Feed after three to four days with fresh medium, and split again if necessary. Clones successfully propagated in cluster plate wells for this second week are taken to be established. At this point, some (or all) can be cryo-preserved and the remainder cultured under different conditions to establish optimal parameters for each particular clone. Having a frozen stock enables one to test different culture conditions in order to optimize growth, without the fear of losing the whole clone.

4. Test whether established clones can be propagated with the most convenient feeder cells (80 Gy-irradiated B-lymphoblastoid cell lines) instead of PBMC feeders. Most TCC flourish on B-LCL alone, but some appear for unknown reasons to benefit from the presence of PBMC as well (this is especially true during cloning). Propagation of the TCC on PBMC feeders can also be continued, but it may be found preferable to prepare standardizable batches of B-LCL rather than having to repeatedly isolate PBMC. Furthermore, PBMC from the autologous donor may not be freely available in sufficient amounts for large-scale propagation of numerous clones. The international availability of well-characterized MHC homozygous B-LCL makes it possible to match the feeder cell to the specificity of the TCC being propagated and enhance the antigen-presentation function of the feeders.

5. As a matter of convenience, it is easier to grow TCC in scaled-up culture vessels than in cluster plates, but not all clones can be adapted to growth in flasks. This must also be tested for each clone, using 1 x 105 and 5 x 105/ml TCC with an equal number of feeders in tissue culture flasks. Those clones not growing under these conditions rarely can be adapted to flask growth by altering the amounts or concentrations of TCC or feeders seeded or by increasing or decreasing the frequency of stimulation and/or feeding. It remains unknown why some TCC fail to flourish in flasks.

6. Establish restimulation parameters for each clone. T cells require periodic reactivation through the T cell antigen receptor in order to retain responsiveness to growth factors. This can be accomplished specifically or nonspecifically. All clones can be propagated with weekly restimulation; some but not all can be propagated with restimulation only every two weeks. Human T cell clones can be readily cryopreserved using the same protocols as are suitable for freezing resting T cells. Clones developing with different kinetics can thus be collected and conveniently tested for cytotoxicity in the same experiment.

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