Hybridoma Culture






Hybridomas are relatively sensitive cell lines since they are by definition, hybrids. Interspecies hybrids (eg hamster/mouse) are potentially very unstable. It is common practise in gene mapping experiments to use human/hamster hybrids to locate genes by chromosome loss. Therefore, to ensure clonal stability and reproducible monoclonal antibody production from hybridomas, cloning by limit dilution should be a matter of routine. Cloning should always be done prior to freezing aliquots of the hybridoma. Cells for freezing should always be in mid-log phase (between 1-5x10^5 cells/mL) to ensure maximum viability after thawing.


Limited Dilution Cloning

  • Perform a cell count on a hybridoma culture in mid-log phase. Mid log phase ensures that cells will clone out with high efficiency. If desired, plates can be set up one day previously with 2x10^4 thymocytes or peritoneal exudate cells in 0.1mL per well as a “feeder layer” prior to seeding the wells with hybridoma cells. The feeder layer secretes cytokines (including IL-6) which enhance cell viability at low density.
  • Perform serial 10-fold dilutions so that the final cell concentration is between 1-10cells/mL. Aim for 10mL of the final dilution per 96-well plate. Plate out 100uL per well (0.1-1cell per well). At this density, and assuming 100% cloning efficiency, the percentage of wells with growth will follow Poisson distribution:

1 cell/well: 63% of wells with growth

0.1 cell/well: 10% of wells with growth

  • Clones will be visible after 2-3 days, and ready for screening in 8-14 days.

To guarantee clonality, limit dilution cloning should be performed twice. Since the hybridoma will almost always have to be derived by cloning in the first place, one cloning step should be enough for routine work. However, if you have a particularly difficult hybridoma, be prepared to carry out cloning on a regular basis.


Cell Freezing Protocol

  • Ensure cells are growing in mid-log phase. Harvest cells by centrifugation from one 75cm 2 flast (1-5x10^5 cells/mL in 15mL, ie 1.5-7.5x10^5 cells total)
  • Resuspend cell pellet in 1.5mL 90% FCS/10% DMSO. Dispense 0.5mL into three freezing vials, and freeze at a controlled rate at -70ºC in a “frosty boy” or enclosed in a foam rack.
  • Next day, perform a test thaw on one vial to check for successful freezing. Thaw one vial into 10mL complete medium and wash once. Resuspend cells in 5mL and inoculate a small (25cm 2) flask. Check viability after 24 hours.