Monoclonal Antibody Production – A discussion on the Culturing of Mouse Hybridoma Cells

By on July 9, 2013
Monoclonal Antibody Production

Last week, we finished our Ask the Expert discussion on 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. These were the topics covered in our last Ask the Expert session – Monoclonal antibody production and the culturing of mouse hybridoma cells.

This session, sponsored by Life Technologies and hosted by Timothy Fawcett, Ph.D. was a good discussion and with such a broad topic, we had quite a range of questions.

Dr. Fawcett is Director of the BioTechnical Institute of Maryland (BTI) a non-profit institute located in Baltimore, Maryland. He is also the Founder and Director of BioSciConcepts, a social venture of BTI that provides hands-on training for professional scientists in cell culture, baculovirus based expression, as well as topics such as molecular biology, PCR and real-time PCR. Dr. Fawcett who has been in biotechnology for over 30 years, provided real hands-on suggestions that readers could benefit from right away.

Question topics included:

  • Use of azaguanine
  • Cell viability post cryopreservation
  • Purity of antigen
  • Serum-free culture
  • Problem obtaining hybridomas in spite of strong signal
  • Culture Conditions
  • Rat-mouse hybridomas

I have selected a few of the submitted questions and answers to include below. For a full list of questions and answers, please see Ask the Expert discussion on Culturing of Mouse Hybridoma Cells.


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.

The Answer:

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.


On 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?

The Answer:

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.


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

The Answer:

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 does create 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.

Please visit this week’s Ask the Expert session – “Catch the WAVE a discussion on using the WAVE disposable bioreactor for research and manufacturing operations,” hosted by Christian Kaisermayer, Senior Scientist, GE Healthcare.


  1. bsargent

    9 July, 2013 at 9:41 AM

    With respect to the question on serum-free hybridoma culture. I know that InVitria has an animal-free supplement called Zap Hybridoma that can be added to classic media for serum-free culture of hybridoma cells. See here for more information –

  2. Pingback: The Dish's Weekly News Wrap Up - July 12, 2013 - The Cell Culture Dish

  3. Malu

    13 August, 2013 at 4:45 PM

    I know what people traditionally do is to perform fusion, obtain hybridoma supernatants and then look for cell biding by flow cytometry. What do you think about detecting cell binding directly from immunization bleeds at intervals or purifying the serum by Protein A and then looking for cell binding ahead of time?
    Won;t this help in prioritizing as to which mouse to fuse?
    Also, if not any of the above, how does one usually decide which mouse to fuse, if all of them have good antigen Titers?


  4. Dai Quach

    7 April, 2014 at 12:21 AM

    When I do the fusion, I usually plate 10×96-well plates. And at day 14-post plating I screen all the wells in 10×96-well plates at the same time. I pick the hybridoma based on the OD

    My friend suggest me to plate 20×96-well, more diluted. And screen the wells turn yellowish in stepwise process. It takes more time to finish screening all the wells. My question is the hybridoma I get at eg. the last screening can compare (?) Which way is the right one to do ?

    Thank you so much in advance …


  5. Joanne Smith

    30 January, 2017 at 5:49 PM

    With regards to the question about the number of passages for the SP2/0 cells; the answer given was pretty vague:
    “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.”

    I’m wondering how many passages qualifies as “several”? Thanks.


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