Enrichment of human milk lymphocytes by discontinuous density gradient sedimentation

A simple method is described for the enrichment of lymphocytes from human colostrum and early milk. Washed leucocytes from colostrum or milk were suspended in balanced salt solution containing 25% human serum and separated by discontinuous density gradient centrifugation over Lymphoprep (1.077 g/ml) and Nycodenz (1.070 g/ml). The cell fraction harvested from the interface between these two media contained a mean of 56% lymphocytes (9-92%), as compared to 5.6% (0-17%) in the unseparated samples, representing an average enrichment of 12.6 fold. The percentage yield of lymphocytes ranged from 70 to 100%. Enriched preparations of lymphocytes from human colostrum and early milk should prove valuable in studies of the distribution of lymphocyte subpopulations in breast milk and could lead to more definitive studies of their functions in vitro.