Monoclonal Antibody Production and the Culturing of Mouse Hybridoma Cells.


The production and maintenance of a hybridoma cell begins with the fusion of a specific antibody producing B cell, to a cancer B cell called a myeloma, which does not produce an antibody by itself. Fusion results in an immortalized line called a hybridoma that will faithfully produce a specific antibody against a single epitope called a monoclonal antibody. Once produced, proper maintenance and culturing is required to maximize the performance and continued production of the antibody in question. Have you ever wondered how this is done? Are you fusing cells to produce your own monoclonal antibodies or wondering about your culturing options. This is your opportunity to ask questions about monoclonal antibody production and the culturing of mouse hybridoma cells.

Question 1

I am a manager of a monoclonal Mab core lab for the university of Colorado, we have a history of successful hybridoma production but recently we have had problems with getting hybridomas from a mouse that shows a strong signal. On the first screening we will get strong positives that would traditional result in good hybridomas, but recently I have seen the positives stop producing the Mab after the initial screen. Few make it to cloning but don’t produce Mab after that. Do you have any idea why this is happening? We’re using mice that have had at least 3-4 injections with the protein of interest and are screened by Elisa titration assay and by a western. A traditional fusion is done with PEG to SP2/0 cells and plated into 96well plates. They are normally screened in 7-10 days. Media is IMDM+15% FBS +HAT

Thanks for your question and providing all the details. I think you are writing that this is happening with one animal and not all the animals you are recently harvesting cells from. Assuming this is the case, I think you got a bad mouse . You also mentioned that spleen cells from the animal initially produce antibody but then production stops even before fusion occurs. It just might be that the cells you harvested from this one animal are more unstable than normal. Was this an older mouse? Making spleen cells, and fusing them do create hybridoma's creates non-normal cells and many cells are lost or never grow because fusion is lethal. Hybridoma cells that survive run a range of genetics and have abnormal chromosomes anyway. Maybe your cells rearrange more than normal. It is always suggested that you do a clonal isolation of cells as soon as possible after selection This will minimize the variation in your culture of cells. If you took at some of those hybridoma cells in the 96 well dish that were producing antibody, but now are not,and serial cloned them, I bet some of them are doing what you want. You don't see the productivity since you are sort of diluting out the signal. Try cloning some cells since it is better than starting over with a new mouse. Also if you do single cell cloning make sure to use conditioned medium initially when you first get the one cell in a well.

Question 2

I am looking into protocols for making hybridomas and notice that some people grow their myeloma cells in 8-azaguanine and other don’t. What is the purpose?

Some people grow their myeloma cells (Sp2/0, Sp2/0-ag14, NS-0 and NS-1 cells for example) in 8-azaguanine or 6-thioguanineThe reason for this is to ensure that the cell line is 8-azaguanine or 6-thioguanine and HGPRT negative. Azaguinine and 6-thioguanine are competitors of guanine. This phenotype is necessary for the myeloma cells to be sensitive to the aminopterin in HAT (hypoxanthine-aminopterin-thymidine). Aminopterin blocks the nucleotide synthetic pathway and the HGPRT negative phenotype means that additionally the myeloma cells can not produce nucleotides via the salvage pathway and therefore will die unless fused. I suggest that the use of 8-azaguanine or 6-thioguanine at least some of the time is important to ensure the correct myeloma phenotype is maintained and only fused cells ultimately survive.

Question 3

or cloning hybridomas via the limiting dilution method (1-2 cells per well), what are your thoughts on the following: (1) serum conc=20% (hybridoma qualified FBS) (2) PEC feeder cells-approx. 2000-5000 per well (3) conditioned medium I make conditioned medium from the myeloma fusion partner (P3X or SP2/0). Is there a better cond. medium to use? What experience, if any, do you have with rat-mouse hybridomas? Anything special I need to know about cloning, growing, maintaining these hybrid cells?

This is a good set of questions that are all related around how to isolate a single cell into the well of a 96 well dish. The goal of course is to get that single cell to grow into many. The problem is that initial single cell in that big well gets lonely and doesn't get the proper signaling to promote cell division and survival. The odds are against its survival. To answer your specific questions... 1) I don't think adding FBS above 10% is helpful and any excess above that may cause problems later when you try and drop the concentration down to 10% for growth at a higher cell density. Incidentally, I suggest moving away from serum altogether for many reasons including purification of the MAb from the growth medium. There are a lot of great serum-free and even protein-free hybridoma media out there such as CD-Hybridoma or Hybridoma SFM. 2) PEC-feeder cells are peritoneal exudate feeder cells and the idea is to put some PEC-cells in the wells along with your single cell you want to clone. This was/is a lot of work, it involves irradiation or a chemical means to stop the growth of the feeder layer when necessary. It turns out that IL-2 or/and iL-6 is what is needed. My suggestion is to supplement your cloned cells with IL-6 (known as a myeloma growth factor) or something like briclone an IL-6 enriched cloning medium. 3) I am a big fan of conditioned medium. Conditioned medium is partially used medium and it has some factors secreted by cells, but it still has some nutritional value. I use conditioned medium for cloning a single cell and I usually use a 50% fresh /50% conditioned media mixture. I also make the conditioned media using the same cells I want to use in fusion such as the SP2/0 cells or the NS0 cells.

Question 4

I am looking for the most inexpensive way to culture hybridoma cells in serum free conditions. I am starting with classic media, is there anything that is serum free that I can add to classic media to allow for serum free culture or do I need to use a pre-made serum free medium for hybridoma cells.

Moving cells from serum-containing to serum-free media is not so easy since adaptation is required. To my knowledge there are no additives such as a non-animal derived substitute that can be added to a basel (classic) medium that will make it perform like a serum containing medium. For your information there are media such as D-MEM or RPMI-1640 which are used for hybridoma culture, that have modified formulations (Advanced-DMEM or Advanced-RPMI1640 from GIbco for example) that work very well for hybridoma culture using only 2% FBS this could reduce your cost somewhat without losses in performance. SInce you asked specifically about the expense of hybridoma culturing...I think about several issues when I think about cost. First, a bottle of D-MEM supplemented with 10% FBS might cost $40-$50 (currently the cost of serum is low) compared to a serum-free medium such as Gibcos PFHM II, Hybridoma-SFM or CD-Hybridoma which might range from $70-$80. (If the cost of serum gets higher then the price gap decreases further). The cost increase for using a serum-free media is in my mind, is a small price to pay for the many resulting benefits. Second, all of the serum-free media designed for hybridoma culture out-perform when compared to classic media in terms of cell growth, viability and MAb production. third, using a serum-free medium make for much easier downstream purification of your product and finally if you are making your MAbs for diagnostic or clinical work the regulatory hurdles may be simplified.

Question 5

Do i need to inject a 100% pure antigen in order to get a desired results or can I use a 80% purity antigen for injection, as the antigen I am dealing with will only grow along with the bacteria. I’m also using non spf female balb/c mices for the injection. I’m wondering if all these factors may affect to get the desired results.

About your antigen, if you were making polyclonal antibodies the purity would be a problem. Technically, by making monoclonal antibodies you can still get the one you want. But I do envision some problems. Obviously your search for the MAb you want will be made more difficult due to the 20% non-antigen directed Abs. The major problem will be with the search itself. I am not sure how you will find your specific MAbs if you don't at least have pure epitope to facilitate your search. I would suggest that after you do the fusion, subtract the hybridoma cells that are producing MAb against the bacteria as a first screen by using pure bacteria without your antigen. This will help speed things in your search.

Question 6

Hi, I would like to know about the mouse myeloma cell line P3X63Ag8.653 azaguanine resistancy period upon subculture. My cells have undergone 9 passages in total and then I decided to cryopreserve it. Upon revival last month they show 70% viability. My question is,even though the cells has been sensitized with 8 azaguanineprior to buying , do i still need to sensitized it because the cells has undergone few passages? I’m also wondering if myeloma cells can undergo infinite passages and will the cells loose the sensitization to azaguanine along the way? What would be the maximum passage for the myeloma cells because i will need to revive the cells again for fusion and it will undergo few passages again.

P3X63Ag8.653 is a subclone P3X63.Ag8 which was originally selected based with 8-aza-guanine sensitivity. Periodic growth in 8-asa-guanine ensures cells are HGPRT negative and thymidine kinase negative. Growth on 6-Thio-Guanine is useful for periodic selection for HAT sensitivity. Also remember that cells for fusion should not be passaged for a long times so keep frozen stocks and do not maintain the cells for more than several passages prior to fusion. It is also good practice to dilute the myeloma cells to about 200,000 cells/ml the day before fusion. Finally, I think your viability should be greater than 70% after you thaw the cells. Try bringing a cup of 37C water to your frozen cells and thawing as quickly as possible at 37C until the cells are completely thawed.

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