What media do I use to grow hybridomas?

We use DMEM complete (dipeptide glutamine, sodium pyruvate) media plus HT supplement, Pen/Strep, 55mM 2-mercaptoethanol and 10 – 20% FBS for growing both rat and mouse hybridomas. RPMI-1640 can also be used instead of DMEM. Media should be HEPES buffered. Use 20% FBS for enriched growing conditions, which may be necessary if the cells are at low density or are growing poorly. If a cell line is growing slowly (and is mycoplasma negative) and does not respond to an increase in FBS content (maximum to 20% FBS) then HT (hypoxanthine, thymidine) supplement may be beneficial.

What are the growth conditions for hybridomas?

5% CO2, 37°C with humidity. Tissue culture flasks and plate lids must allow CO2 to enter the cell wells. Flasks should not be filled past the half way mark and plates should not be so full that the lid makes contact with liquid media. Flask lids are left a quarter turn open (loose) so CO2 can enter the flask (plug seal cap) or left closed (vented lid flask).

How do I grow hybridomas?

Hybridomas are relatively easy to grow. They do not secrete toxic by-products and therefore can be diluted in fresh media in the same flask they were seeded in. Most tissue culture plastic ware is “tissue culture treated” which allows adherent cells to attach to the plastic. Hybridomas are suspension cells so they do not significantly attach to the plastic and are easily resuspended by gentle pipeting. Hybridomas are at mid log density between 2 – 4 x 105 cells/mL.  Maintain hybridomas between 1 x 105 and 1 x 106cells/mL. Hybridoma cell lines divide approximately every 18 – 24 hours. Some may grow faster than others. Therefore a slow grower will recover from a 1/4 split differently than a faster growing cell line. Hybridomas need to be in a certain amount of conditioned media (“lived in” media) to be happy. This conditioned media contains growth factors that the cells secrete and then are auto-stimulated by. Therefore, if cells are split too harshly (1/20) they may struggle or die. If the cells don’t look good, they may NOT need more media (which would only dilute out the growth factors) but they just need to sit another day to increase the content of conditioned media. If they are really struggling and may have possibly been over-diluted in media, just spin them down and resuspend them in a smaller volume of media with 20% FBS and let them recover for a few days.

What do hybridomas look like?

Healthy cell membranes appear round and smooth under the microscope. Do not allow plasma membranes to become granulated – this means the cells are not healthy. Cells may be unhealthy for a number of reasons: overfed (diluted out too far in fresh media), underfed (waste products in media outnumber the fresh nutrients), stressed (media too alkaline), or just a poor growing cell line.

First image (mouse hybridoma) cells are a bit too dense (>4 x105 cells/mL) and are starting to struggle. Second image cells are much happier (mid log density 2 x 105 cells/mL) and dividing nicely.

Dense:

Less Dense:

How do I freeze/cryopreserve hybridomas?

For a detailed printable pdf, please click here.

Most hybridomas are frozen in either growth media with 20% fetal bovine serum (FBS) and 10% dimethylsulfoxide (DMSO) or 90% FBS plus 10% DMSO and stored in liquid nitrogen. IPA freezes hybridomas in 90% FBS plus 10% DMSO in 1mL (2 x 106 cells/vial) aliquots. Always wash DMSO away when thawing. Thaw frozen vials directly from liquid nitrogen in a 37°C water bath for exactly 2 minutes. Immediately transfer cells to 10mL DMEM complete media plus 10% FBS in 15mL conical tube, mix gently by inverting several times, spin down at 200 x g for 6 minutes. Transfer pellet to 10mL DMEM complete media plus 20% FBS in pre-gassed 25cm2 flask. FBS content is reduced to 10% once cells are recovered (2-3 days post thaw).

Freeze in 1mL aliquots of 90% FBS and 10% DMSO at 2 x 106 cells per vial. Split cells ½ for at least 2 days before freezing while maintaining them at mid log. Freshly divided cells have stronger membranes that withstand the freezing process better. Keep freezing media ice cold during the freezing process as DMSO is toxic to cells at warmer temperatures.

What is the concentration of monoclonal antibody in tissue culture supernatant?

  • Mouse IgG range: 2 – 20µg/mL      Average = 15µg/mL
  • Mouse IgM range: 2 – 120 µg/mL   Average = 57µg/mL

*determined by SPR analysis using 62 samples taken from mid-log cultures.

What is mycoplasma?

Mycoplasma is a contaminant that is frequently found in tissue culture. It is a tiny prokaryotic organism (smaller than bacteria) that can be easily spread throughout a laboratory and pass from cell line to cell line. It is thought that the main source is the upper respiratory tract of man. It can render cell cultures worthless as it can cause severe cytopathic changes such as morphological transformations and degradation of DNA.It is important to test your valuable cell lines for mycoplasma.  At IPA we routinely test all in-house cell lines and hybridomas for the presence mycoplasma. We also provide mycoplasma testing for your own valuable cell lines. Contact us for more information.