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.