This phase involves the propagation of colonies from the selected wells that showed the highest antibody titer. However, the correct antibody secreting cells are mixed with other cells which may be non-secreting or may not have the highest titers. So we need to separate out the specific antibody producing cells from the other contaminants and expand this particular colony to make sure that the antibodies produced are truly monoclonal. This is the most critical stage of monoclonal antibody production. We use the limited dilution technique and separate the cells in 96 well plates to identify the right clones. Many of the hybridoma cells do not survive this step as they cannot adapt to the sudden changes in concentration. So we use feeder cells to enhance the survivability of the hybridomas. The supernatant from the wells need to be screened again to make sure the right clone is being expanded. Some of the selected hybridomas will be used for invitro or invivo expansion and production of monoclonal antibodies. Most of the correctly identified clones are cryopreserved for future use. At this stage the cryopreserved hybridomas are sent to the customer for long term storage.