Continued expression of plant-made vaccines following long-term cryopreservation of antigen-expressing tobacco cell cultures

Production of vaccines in plant cells provides an alternative system that has several advantages when compared to current vaccine production methods. Establishment of stable seed stocks for a continuous supply of a vaccine is a critical part of production systems. Therefore, a vitrification method for cryopreservation was applied to non-transgenic and three different antigen-expressing transgenic Nicotiana tabacum (NT-1) lines. Preculture of the suspension cultures 1 d prior to vitrification was sufficient for cell survival through the cryopreservation process. Inclusion of 0.3 M mannitol in the preculture medium was necessary for maintenance of cell viability. Cultures were also treated with and without heat shock prior to vitrification, and it was found that heat shock was unnecessary for growth recovery post cryopreservation. All cultures survived storage in liquid nitrogen at intervals ranging from 1 h to 1 yr. Antigen expression was measured by enzyme-linked immunosorbent assay for cultures that grew post cryopreservation and those that had never been cryopreserved. Expression levels in cultures derived from cryopreserved material were comparable to cultures that had not been cryopreserved. Transmission electron microscopy showed that the integrity of the cell structure was maintained post cryopreservation.