HYBRIDOMA PRODUCTION

Materials
  1. Tumor cells that have been treated with 8-azaguanine for 48 hours are removed from the drug and grown to a maximum concentration of 500,000 cells per ml.
  2. Rats or mice that were previously immunized and then boosted IV 72 hours prior to hybridization.
  3. Media: Auto-Pow (powdered MEM from Flow Labs) with sodium bicarbonate plus non-essential amino acids, penicillin-streptomycin, L-glutamine and HT. For the serum-containing media (used for the final plating), add 5 - 10% newborn calf serum.
  4. 40% PEG: Aldrich 1000. Made up in serum-free medium (SFM). The stock may be aliquoted and stored at -30°C.
  5. Other materials include: 96-well plates, sterile Petri dishes, conical 15 and 50 ml tubes, round bottom (3 and 12 ml) tubes, sterile dissection instruments, 37°C water bath, spleen crusher, 70% ethanol, syringe and needle.
Procedure (Keep all cells and solutions at room temperature or 37°C)

Day 0
  • Preparation of Tumor Cells
    1. Check water bath for cleanliness. Correct water volume and temperature (equilibrated to 37°C with the lid off).
    2. Soak the spleen crusher in 70% ethanol for 5 - 10 minutes and let it dry sterilely in the hood.
    3. Count the tumor cells. Begin washing the tumor cells with SFM as follows:
    4. Centrifuge the cells out of serum containing media in 50 ml conical tubes (1200 RPM for 5 minutes). Discard the supernatant.
    5. Carefully resuspend the cell pellet in approximately 1 ml of SFM. Transfer the cells to a new 50 ml conical tube. Avoid vigorous pipetting to reduce transferring proteins from the inside of the tube to the cell suspension. You want to transfer as little serum protein as possible into the new tube.
    6. Repeat the last two steps, transferring all of the tumor cells into a new tube.
    7. Add 30 - 50 ml of SFM to the cell suspension and centrifuge again. Discard the supernatant.
    8. Repeat the last step two more times.
  • Preparation of Spleen Cells
        While washing the tumor cells, prepare the spleen cells as follows:
    1. Bleed the animal for antisera and let the blood clot at room temperature for 1 - 2 hours. Transfer the blood overnight to a 4°C refrigerator before removing the clot.
    2. Remove the spleen and place it in a sterile Petri dish containing 10 ml of SFM. Move this Petri dish into the hood.
    3. Transfer the spleen with sterile forceps into new sterile Petri dish containing 10 ml SFM. This step reduces the nonsterile contaminants that may be present in the first Petri dish).
    4. Crush the spleen with the sterile spleen crusher. Push down hard on the entire spleen once only. Do not mash in a circular motion. If the spleen is crushed more than once or twice, fibroblasts will be released into the spleen cell mixture.
    5. Carefully pipet the spleen cell mixture up and down in the Petri dish to break up large cell clumps.
    6. Transfer the cell suspension to a 15 ml conical tube (you may wash out the Petri dish with a few ml of SFM, if desired).
    7. Let debris settle out of the cell solution (2-4 minutes).
    8. Transfer clean supernatant into a new 15 ml conical tube.
    9. Centrifuge the cells for 5 minutes at 1200 RPM.
    10. Resuspend the cell pellet in 10 ml of SFM and count 5 µl on a hemocytometer.
  • Cell Fusion
        The ideal cell ratio of spleen to tumor cells is 5:1. Fuse a maximum of 1.5 - 2.5 x 10    8 spleen cells per tube. If fusing a larger number of spleen cells, divide them into two tubes. Mix the appropriate volumes of each cell suspension together and centrifuge the cells.
    1. In the water bath, bring the following to 37°C (for each tube of spleen cells):
      small pop top tube containing 1 ml 40% PEG
      small pop top tube containing 1 ml SFM
      50 ml conical tube containing 20 ml SFM
      empty 50 ml conical tube
    2. Resuspend the spleen-tumor cell pellet carefully in 0.5 ml of SFM and transfer to a 12 ml round bottom tube. Centrifuge at 700 RPM for 8 minutes to form a loose pellet. Monitor the centrifuge speed and time.
    3. Remove all of the supernatant from the pellet.
    4. Ideally the fusion will be done in a tissue culture hood that has access to containers of 37°C water. Otherwise, the fusion is done in the 37°C water bath outside of the tissue culture hood. Take particular care to keep all sterile tubes, solutions and pipets uncontaminated. Do not let water from the bottom of tubes drip into other tubes.
    5. Disrupt the cell pellet by flicking and/or tapping the bottom of the tube.
    6. Perform the following steps described below. Timing is critical. Use a stop watch to keep track of the time for each step. Whenever possible, perform the procedure over the entire time period, not merely in the first few moments of the time period.

      Time (use stopwatch) Procedure
      00-30 seconds: Add 0.5 ml PEG; tap the bottom of the tube to mix.
      30-60 seconds: Add remaining 0.5 ml; tap tube to mix.
      1-2 minutes: Over this time period, slowly add 1 ml of SFM while agitating the tube.
      2-6 minutes: Add 20 ml of SFM over the remaining 4 minutes. As the volume increases in the original round bottom tube, transfer the contents into an empty 50 ml conical tube.

    7. The longer the cell solution remains in contact with the PEG, the more cells will fuse. Because PEG is toxic to the cells, it must be slowly diluted with SFM. The dilution of the cell-PEG solution should be slower in the beginning of the time course than at the end. Rumor has it that the contaminants in the PEG decrease the efficiency of fusions with different lots of PEG. These usually have an odor, therefore a quick way to screen PEG is to smell it and not use the smelliest batches.
    8. Centrifuge the cells out of SFM and resuspend them in serum-containing medium. For each tube of fused cells, plate the cells into 4 - 6 96-well plates at 0.1 ml per well (Ten ml of cell solution are needed per plate).
    9. Incubate at 37°C.
    Day 1
            Feed the cells by adding an additional 0.1 ml per well with media containing serum and HT plus 2x methotrexate. Process and store the antisera.

    Day 3
            Replace 0.1 ml of media from each well with 0.1 ml of fresh HT media.

    Day 7
            Repeat the Day 3 procedure.

    Day 11
            Repeat the Day 3 procedure, and continue to feed every 7 days.

    Screen the wells when the media just begins to turn from the usual orange color to yellow. This insures that the cells have grown to a number adequate to produce detectable antibody but that they not in danger of overgrowing. The screening typically occurs between days 11 - 14.
        Send comments and updates to  Dr. Bart Frank, Arthritis and Immunology Program, OMRF
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    Copyright 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
    Proper citation for data acquired from this document is: "Ducey, C. M. and Frank, M. B. Hybridoma Production. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/fill-in.html). 1997. Oklahoma City. Revision Date: October 2, 1997."