Optimization of Cell Isolation from Human Milk

Abstract

Breastmilk contains a plethora of bioactive factors, including biochemical and cellular components, providing not only nutritional, but also immunological and developmental support to the breastfed infant. Maternal cells in breastmilk comprise a heterogeneous population of epithelial cells, leukocytes, progenitor and stem cells that dynamically respond to maternal and infant needs. Although cellular analyses have been previously conducted in breastmilk, optimization of the methodology to isolate and process cells from breastmilk is lacking. In this study, we examined the effect of breastmilk storage and centrifugation on the cell content and integrity of breastmilk. Freshly expressed breastmilk was collected from lactating women and was centrifuged within 30 min from expression using 5 different centrifugation speeds (500, 800, 1600, 2300, 3300 g). Cells were washed in sterile PBS after the initial fractionation and were counted using a Neubauer haemocytometer with Trypan Blue exclusion for measurement of cell viability. Of all centrifugation speeds tested, 800 g yielded the highest cell counts as well as cell viabilities, which markedly decreased above this speed. Storage of whole breastmilk at room temperature or in the refrigerator or freezer was also tested. It was shown that cell counts and viability are preserved within the first 3 hours of storage at all these conditions, but then decrease upon further storage. These findings provide a standardization of the methodology to isolate and process cells from breastmilk for future lactation studies.