Toxoplasma gondii from liquid nitrogen for continuous cell culture: methods to maximise efficient retrieval

This study aims to increase the efficiency of continuous growth of Toxoplasma gondii in HeLa cells from tachyzoite stocks frozen in liquid nitrogen. Freezing and retrieval of tachyzoites for continuous cell culture requires more stringent protocols than those published for animal culture. The freezing and retrieval conditions are optimised so that a quality harvest (greater than or equal to 1 x 10(6) tachyzoites/mL, greater than or equal to 90% viability) can be produced using T gondii recovered from liquid nitrogen as fast and reliably as possible. Retrieval success rate increased from 36% to 100%. An improved freezing procedure using chilled reagents and freshly harvested parasites, and adoption of an effective recovery protocol with retrieval of 3 x 10(7) tachyzoites into 75cm(2) flasks, change of maintenance media after six hours and subsequent blind passage all contributed to this success. The result is faster and more dependable production of T gondii for diagnostic and experimental use.