In Vitro Stimulation of Whole Milk Specimens: A Field-Friendly Method to Assess Milk Immune Activity

Background: The immune system of milk protects against infections and guides immune system development. A system-level understanding of milk immune activity is critical for research into infant infectious disease risk and lifelong health. Research aim: To describe a protocol to characterize immune activity in human milk via in vitro stimulation for use in population-based (rather than clinical) research. Methods: This study proceeded in two phases, each with a cross-sectional design. Human milk specimens were incubated for 24 hr at 37 °C in mammalian cell culture medium with stimuli (e.g., Salmonella enterica) in a CO₂-enriched environment. Immune responses to stimuli were characterized as the change in cytokine: [stimulated]/[baseline]. Predictors of cytokine responses were evaluated with generalized linear models. Results: Patterns were detectable across mother–child dyads: Interleukin-6 responses to stimuli were generally positively associated with child age and with maternal autoimmune disease. Conclusions: Our method allows characterization of pro-inflammatory milk immune activity in vitro in population-based (rather than clinical) research settings. In vitro activity has a system-level interpretation and is likely to be of broad utility in global health research in settings with high infectious disease risk, where understanding the immune system of milk is critical to understanding maternal and child health.