Viability of and plasmid retention in frozen recombinant Escherichia coli over time: a ten-year prospective study.

The long-term viability and plasmid retention of recombinant Escherichia coli strains were investigated by real-time testing of master cell banks (MCBs) stored at the Roche Molecular Systems Culture Collection (RMSCC). MCBs at the RMSCC were cryogenically frozen and stored at -80 degrees C for long-term preservation. At regular intervals during a period of 5 to more than 10 years, representative cryovials of each MCB were tested for viability and plasmid retention. Plasmid retention and viability for all 30 MCBs were stable over time. Twenty-seven MCBs maintained high levels of plasmid retention (at or near 100%), while three MCBs showed lower plasmid retention rates (ranging from 13.9 to 96.5%) that were consistent over time. New MCBs with high plasmid retention were created from two of the MCBs with lower plasmid retention by selective pressure with high levels of antibiotics. These new MCBs have shown stable viability and high plasmid retention over the first 5 months of storage. In conclusion, this study shows that properly selected, frozen and stored MCBs retain viability and maintain plasmid retention over time. Moreover, it is possible to recover cultures with high plasmid retention from MCBs with low plasmid retention by selecting clones grown in the presence of high levels of antibiotics.