Isolation of mononuclear cells from bovine milk by continuous-flow and density gradient centrifugation: response of cells to mitogens.

Continuous-flow centrifugation (CFC) and 2 types of discontinuous density gradients (Ficoll-diatrizoate or polyvinylpyrrolidone-coated colloidal silica) were used to obtain bovine mononuclear cells from milk. Determinants considered were efficiency of cell recovery, cell viability, and functional activity of the enriched cell population in a lymphocyte blastogenesis (LBT) assay. Milk leukocytes were recovered by CFC with an efficiency of 46% and a viability of 63%. Selective loss of a cell population due to CFC was not observed. Using density gradients, enrichment was marginally effective (73% mononuclear cells with polyvinylpyrrolidone-coated colloidal silica and 82% mononuclear cells with Ficoll-diatrizoate) and overall mononuclear cell efficiency of recovery was reduced. In comparison, using either density gradient, blood mononuclear cells were harvested with efficiencies of 61.5% and 63.4%, with 85.6% and 89.3% viability and greater than 99% purity. Compared with peripheral blood lymphocytes, milk mononuclear cells were less viable. When evaluated using the LBT assay, milk mononuclear cells were hyporesponsive to mitogenic stimulation, regardless of the density gradient used. The methods used allowed for the collection of a sufficient number of nonelicited milk mononuclear cells for replicate evaluation of several mitogens or antigens, using the LBT assay. Mononuclear cells recovered had a consistently reproducible blastogenic response to T-cell mitogens.