Cryopreservation of avian lymphoid cells

Conditions were standardized for optimum cryopreservation of avian lymphoid cells. Several factors that influenced cryopreservation were examined. The optimum procedure was as follows: a maximum of 50 x 10(6) cells/ml were suspended in the freezing medium, which contained 5-10% dimethyl sulfoxide (DMSO), and the cell suspension was frozen slowly at a rate of -1 C degree/min. The frozen cells were kept at -196 C. Cryopreserved cels were thawed rapidly in a 37 C water bath until the last ice crystal had thawed. The method of diluting out DMSO from thawed cells was critical, and results were best if dilutions were made at room temperature (20-25C) with diluent prewarmed to room temperature. Dilution was begun by adding 0.1 ml diluent to 1.0 ml freshly thawed cell suspension. Thereafter, diluent was added by doubling the volume after each 1-min interval until 12.7 ml of diluent had been added over 6 min. The recovery of viable cells from cryopreserved cells varied from 51.2% to 98.3% (mean 86.0%) for lymphoblastoid line cells and from 24.8% to 87.2% (mean 50.6%) for spleen cells obtained from normal chickens. Viable cryopreserved cells were reactive in a 4-hr Cr-release cytotoxicity assay and responded vigorously to phytohemagglutinin. The standardized method of freezing and thawing avian lymphoid cells may facilitate preservation of large stocks of standard reference cells with predetermined functions for laboratory studies, particularly those involving in vitro assays of cellular immunity.