Cloning by Limiting Dilutions Cell Propagation and Archiving

1. Every primary hybridoma clone requires at least three consecutive rounds of recloning.

2. Examine 96-well plates containing hybridoma clones on an inverted microscope (low magnification). Reclone all positive wells after primary fusion and only the largest single colonies (5-10 colonies per one primary hybridoma clone) in subsequent rounds of cloning.

3. Reserve two flat-bottom 96-well plates per each well to be recloned; add 100 p,L of complete hybridoma medium 1 h prior to the recloning procedure. This will result in higher plating efficiency due to absorption of serum proteins on the plastic.

4. Using an automatic pipet with a 200-p.L tip, pipet the desired well to generate a single-cell suspension, immediately aspirate 45 p,L of this suspension and mix it with 5 p,L of Trypan Blue. Calculate the concentration and total number of viable hybridoma cells in the well; calculate the volume and dilutions required for plating hybridoma cells at 1 or 0.25 cells per well in one of the two reserved plates.

5. Using an automatic pipet with a 200-p.L tip, mix and then aspirate the needed volumes from the plate, dilute with complete medium if needed, and add the required volumes to a Petri dish containing 10 mL of complete hybridoma medium. Using a multichannel pipet, add 100 p,L to each well of each of the two reserved 96-well plates.

6. Analyze each of the two plates containing 1 or 0.25 cells per well for single colonies using an inverted microscope every 2 to 3 d. On days 8-12, select the largest 15-20 colonies, test them utilizing the screening procedure of choice, select 5-10 of the best colonies, and repeat the recloning procedure. At each recloning step, cryopreserve all colonies that will be included in the next round of cloning.

7. Use complete HAT hybridoma medium for the first round of recloning. In the second round, start replacing HAT with complete HT medium after 3 d of culture by replacing 50% of the well volume every 2-3 d. In the third round of recloning, use HT medium to fill initial wells and start replacing HT medium with regular complete DMEM-based hybridoma medium according to the same schedule. Continue functional testing and cryopreserving each clone at each step.

8. Select at least five of the best clones for each primary hybridoma; transfer cells into 24-well plates and, upon achieving active and stable growth, into T25 and T75 flasks. Test and cryopreserve clones from each selection/adaptation cell culture step. After the growth of mass hybridoma populations is well established in T75 flasks, perform functional testing again, confirm the desired functional parameters, select one clone for future work, freeze down large numbers of these cells, and proceed to mass production in multiwell plates, tissue culture flasks, roller bottles, cell factories, etc.

9. Even highly selected hybridoma cell lines need to be periodically retested to confirm their specificity and recloned to protect the population from overgrowing by hybridoma revertants with "throw-out genes" that produce no MAb immuno-globulin.

10. Do not keep selected hybridoma clones growing for over 2 mo; ideally, each mass production procedure should be initiated using frozen cells from earlier passages.

11. Hybridoma cells are typically cryopreserved using standard tissue culture procedure in a freshly prepared mixture of 90% FBS and 10% tissue-culture-grade DMSO, with at least 106 viable cells per cryovial.

12. In most labs, thawing of hybridoma cultures involves initial transfer of cells into 15-mL conical tubes, drop-wise addition of complete hybridoma medium, gentle centrifugation at 200g for 8-10 min, aspiration of the supernatant, addition of 2 mL of fresh complete medium, gentle pipetting, and transfer of different volumes of the resulting cell suspension into six 24-well plates containing full hybridoma medium. Growth of the thawed cells should be monitored on an inverted microscope.

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