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Transfer, Expansion, Testing and Cloning of T Cell Hybrids
If the fusion is successful, hybrid growth will be visible by examination of the wells of the 96 well plates using the inverted microscope. If the fusion is a success, the hybrids will
overgrow the wells within 10 - 14 days. The hybrids should be transferred when approximately 50% of the bottom of the well is covered with cells or when the medium begins to turn orange, whichever occurs first. In order to keep track of individual T cell lines and clones it is necessary to have a concise, consistent nomenclature system. In this laboratory T cell hybrids are designated by an alpha numerical system in which block capital letters abbreviate the strain and antigen, and numbers designate the order in which T cell hybrids were transferred to flasks from the 96 well plates. For example the current
fusion used NOD mice and peptide B:9-23 of insulin so the designation NIB-1, NIB-2, NIB-3 etc., will be used. In order to ensure stability and monoclonality all T cell hybrids that are
investigated in detail are cloned by limiting dilution as described below. The system described below has been developed over a period of years to minimize the amount of labor and the potential for cross contamination microbial contamination.
Key features of T cell hybrids
1. At this stage, we have a good deal of labor, reagents and supplies invested in the cells that were fused, we want to protect our investment. Therefore, filter all media to be used for the propagation of the T cell hybrids the day they are to be used,
even if you filtered it the day before.
2. One key to successful and efficient cell culture in general is to have all media and other required components ready so that you can minimize the time the cells spend at ambient temperature. Proceed briskly while the culture plates and flasks are out of the incubator and plan your work to take breaks after the manipulations are performed and the cells are back in the incubator.
3. All hybrids are initially grown in DMEM/10% FBS/HAT, they grow with a doubling time of approximately 16 - 24 hours and have to be diluted in fresh medium approximately 1:20 every 2 days.
4. When we are in the process of transferring and feeding T cell hybrids, the first thing that should be done upon arriving at the lab each day is to carefully examine the plates and flasks of hybrids to ensure that they are not overgrown or too alkaline. In addition, it is best not to transfer hybrids into cold medium therefore a bottle of DMEM/10% FBS should be removed from the refrigerator and placed in the incubator.
5. When they are in HAT medium all tests and media replenishment must be done with either HAT (for the first 3 weeks to ensure the complete death of BW5147) or HT (from 3 weeks after fusion).
6. The HAT must be diluted out by serial feeding with HT at least 1:1000 before nonsupplemented DMEM can be used.
7. The most efficient way to deal with large numbers of small flasks it to tape them together, groups of 5 are convenient because the culture boxes will accommodate 10 flasks in each 3 rows.
Transfer of T cell hybrids from 96 well plates to flasks
DMEM/10% FBS/ HAT prepare in advance
sterile plugged pasteur pipettes
1. Remove the plates from the box and inspect them for growth either by use of the inverted scope or by holding above and looking at the bottom of the plate. Go through all four plates and circle the wells that are ready for transfer with a lab
2. After all four plates have been marked starting at plate 1 and going from left to right, top to bottom number each well that has been circled and make a note of the number of wells to be transferred, place plates back in the box and in the incubator
while the flasks are prepared.
3. Take out as many 25 cm flasks as necessary to have one flask for each well to be transferred, tape the flasks together in groups of 5 label each flask with the appropriate cell line number and add 2.0 ml of freshly filtered DMEM/10% FBS/HAT
to each flask, close the cap snugly.
4. Place the flasks in numerical order at the back of he hood.
5. Take the fusion plates out of the incubator and starting with plate #1, use a sterile plugged pasteur pipette to transfer the contents of each well into the appropriate flask, using a new pipette for each well.
6. When all of the wells have been transferred, place the plates back in the incubator and place the flasks into a culture box with the cap loose enough to allow gas exchange, gas the box and place in the incubator.
Testing T cell hybrids for antigen specificity.
Conventional T cell lines are tested for antigen responsiveness by proliferation assays, due to the fact that T cell hybrids are essentially transformed tumor cell lines that proliferate continuously, it is not possible to use proliferation. Another consequence of stimulation of T cells is the elaboration of cytokines, among which is IL-2, one of the easiest to measure.
There are two steps involved in use of cytokine production as a measure of T cell activation; first, the hybrids are cultured with antigen and antigen presenting cells (APC) overnight to allow for the stimulation of IL-2 production and the release of this into the
medium; second, the culture supernate (medium) is tested for the presence of IL-2. We use a bioassay to quantitate IL-2 that is based on the availability of a cell line, CTLL that is strictly dependent on IL-2 for viability. This is performed by setting up the test wells in the top row of a 96 well flat bottom plate and culturing overnight, the next day 0.1 ml of DMEM/10% FBS/Hat or HT is added to the lower 6 rows, leaving the second row empty. Aliquots of the culture are transferred to the second and third rows and serial dilutions are performed to the bottom of the plate. Meanwhile the CTLL cells are prepared and added to
the dilutions of the culture SN
Please note: Although any manipulation involved in the propagation of T cell hybrids must adhere to the strictest standards of cell culture to prevent microbial contamination as well as cross contamination.
Standard assays conditions
2 wells for each hybrid, one with APC + antigen the other with APC only
1.0 X 105 T cells from the hybrid
1.0 X 105 lymphoma APC OR 1.0 X 106 spleen APCin 0.3 ml of DMEM/10% FBS/ HAT or HT culture 18 - 20 hours
15 ml centrifuge tube
96 well flat-bottomed plates (one for each 6 hybrids to be tested)
0.1 ml DMEM/ 10% FBS/ HAT or HT
1. Keep the cells in numerical order and count the T cells ( or estimate number when you have become proficient) and remove enough for the test (2.0 X 105) and place in 15 ml centrifuge tube (do not forget to label each tube with the cell line # and make absolutely certain that you record this information in your lab notebook as you do the counts), pellet the cells and aspirating off the medium, place the tubes in
numerical order after pelleting.
2. Once you have determined the number of lines that need to be tested count the lymphoma line or spleen cells and take enough cells to give 2.0 X 105 lymphoma cells per line to be tested or 2.0 X 106 spleen cells for each line to be tested and
3. Open the required number of 96 well flat bottomed plates (one for each 6 hybrids to be tested), mark the plates to be used for the assay with the number of the line tested over the two test wells required for each hybrid.
4. The total volume of medium is 0.3 mls therefore time is saved if the T cells are resuspended in 0.5 ml and 0.2 ml added to each of the 2 wells.
5. The APC are resuspended to either 1.0 X 107 cells per ml for the lymphoma or 1.0 X 106 cells per ml for spleen cells and 0.1 ml of this cells suspension is added to each well.
6. The desired antigen is added to the well in 10 - 20 μl and the plates are cultured overnight.
7. After 18 - 20 hours of culture remove the plates from the incubator and add 0.1 ml DMEM/ 10% FBS/ HAT or HT to each well in the lower 6 rows.
8. Using a multi channel pipettor remove an aliquot of 50μl of medium from the culture well and add it to the second row of wells, take another 50μl aliquot and add it to the third row, which also contains 0.1 ml medium, mix the culture SN with the medium by pipetting up and down several times minimizing the formation of bubbles transfer 50 μl of this to the next row down and mix and repeat until you have diluted down to the bottom of the plate.
9. A total of 5.0 X 103 of the indicator cell CTLL are added to every well in the lower 7 rows of the plate, there are a total of 84 wells therefore 4.2 X 105 CTLL cells are needed for each plate.
10. Count the CTLLs and take out enough for all of the plates to be tested, pellet and wash 4 X with 5.0 mls of DMEM/10% FBS, it is necessary to wash extensively to remove traces of the IL-2 that the CTLL are grown in order to get a satisfactorily low
11. The CTLL cells are added to the plates in 20 μl of DMEM/10% FBS and 5.0 X 103 are added per well therefore resuspend the CTLLs to 2.5 X 105 cells per ml and
12. Incubate overnight and assess CTLL viability by visual inspection using the inverted scope.
Freezing T cell hybrids of the desired antigen specificity
It cannot be emphasized enough that as soon as a T cell hybrid is determined to besufficiently reactive to the antigen to be of interest, at least three vials of this line should be frozen. The freezing of cells is described in another protocol
Cloning T cell hybrids
For most purposes it is essential that T cell hybrids be clonal, that is that the population arose from a single cell, in order to accomplish this the T cell hybrids are cloned. The goal of cloning is to have individual wells in 96 well plates that have a single viable hybrid cell. There are two general methods for cloning, first is micromanipulation in which single cells are transferred from a cells suspension into a well of a 96 well plate, the second method achieves a single cell per well by limiting dilution. The method of limiting dilution cloning used in this laboratory is described below.
DMEM/ 10% FBS is used for all cultures Cloning of T cell hybrids is done in 96 well flat bottom plates
96 well plates
DMEM/ 10% FBS
15 ml centrifuge tubes
1. For each line to be cloned four 96 well plates, one each with an average of 10, 3, 1, and 0.3 cells per well are set up.
2. The cells are added to the cloning plates in 0.1 ml of medium, therefore to get an average of 10 cells per well a cell suspension @ 100 cells / ml is required.
3. Count the T cell hybrid line or lines to be cloned and determine the total dilution required to give 15 ml @ 100 cells / ml.
4. Perform the serial dilutions in 15 ml centrifuge tubes and transfer 1500 cells to a 15 ml tube and fill to 15 ml with medium.
5. Distribute the cell suspension into the plate labeled 10 cells / well, this will require approx. 9.6 ml, dilute the remaining 5 ml of cells up to 15 ml by adding 9.6 ml medium, this dilution will give approx. 3 cells per well when 0.1 ml is added to each
well, distribute the cells in the 3 cells/well plate and repeat this dilution and distribution of cells for a 1 cell per well plate and a 0.3 cell per well plate.
6. When all of the plates have been prepared place them in a culture box, gas and place in the incubator.
7. Clone growth is apparent after 7 - 10 days and when the clones have expanded to cover approx. half of the area of the well transfer to a flask with 2.0 ml medium, we generally transfer 20 wells per cloning
8. Always transfer clones from the lowest dilution possible, therefore use the 0.3, and 1 cell per well plates to obtain as many clones as possible.
9. The clones are expanded, tested and frozen in a manner identical to that used for the uncloned lines, in general 3 or 4 of the most strongly reactive clones are selected for freezing and approx. 5 vials of each clone should be frozen.