Cryopreservation of Theileria-infected lymphoblastoid cells with functional assessment of viability.

Bovine lymphoblastoid cell cultures infected with Theileria annulata were frozen to -60 degrees C in a programmable cooling apparatus using continuous cooling rates of 1 degree C/min, 10 degrees C/min and 120 degrees C/min and a two-step cooling rate with an equilibration period of 20 min at -30 degrees C. The cryopreservative was DMSO at concentrations of 5, 10 and 15%. Aliquots of cryopreserved material were stored in the vapour phase of a liquid nitrogen refrigerator and resuscitated by rapid thawing in a 40 degrees C water bath. The efficiency of the freezing methods was compared by assessing the viability and plating efficiency of cells after resuscitation, dilution and equilibration in cell culture medium. A significant correlation was recorded between the results obtained by these two methods which showed that the two-step method yielded cells with viability of 95 to 97% and a plating efficiency equal to that of unfrozen cells. It was concluded that such a freezing method would be ideal for the cryopreservation and storage of banks of Theileria annulata-infected cells for vaccine purposes.