Titus Keller1, Leonie Wengenroth1, Denise Smorra1, Kristina Probst2,3, Leo Kurian4,5,6, Angela Kribs1, Bent Brachvogel2,3. 1. Department of Pediatrics and Adolescent Medicine, Neonatology, University of Cologne, Cologne, Germany. 2. Medical Faculty, Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, University of Cologne, Cologne, Germany. 3. Medical Faculty, Center for Biochemistry, University of Cologne, Cologne, Germany. 4. Cologne Excellence Cluster for Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany. 5. Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany. 6. Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany.
Abstract
BACKGROUND: Human breast milk could be an important stem cell source for the development of newborn and preterm infants, but quantitative data on the stem cell content in breast milk at various gestational stages are needed to determine the clinical value of breast milk as a source of stem cells. Breast milk also contains milk fat globules, lipid droplets of different sizes, debris and dead cells and these components hamper flow cytometry analysis of human breast milk samples. METHODS: Here, we originally used standard protocols for flow cytometry to characterize cell populations in human breast milk but failed to discriminate between cells and noncellular components. We then applied a centrifugation protocol to separate cream and skim milk from the cell-containing pellet and used a novel staining protocol with DRAQ5™ and SYTOX® blue dye as well as antibodies to characterize cells within the pellet fraction. RESULTS: Flow cytometry analysis identified viable DRAQ5™+ /SYTOX® Blue- cells and determined the content of CD11b+ monocytes and TRA-1-81+ putative stem cells in human breast milk samples. CONCLUSIONS: Hence, we developed a novel and reliable flow cytometry based-approach to quantify subpopulation of cells in human breast milk with a high content of milk fat globules, lipid droplets, and particles. This approach will improve the identification and quantification of breast milk cells and allow standardizing the flow cytometry-based evaluation of the stem cell content.
BACKGROUND:Human breast milk could be an important stem cell source for the development of newborn and preterm infants, but quantitative data on the stem cell content in breast milk at various gestational stages are needed to determine the clinical value of breast milk as a source of stem cells. Breast milk also contains milk fat globules, lipid droplets of different sizes, debris and dead cells and these components hamper flow cytometry analysis of human breast milk samples. METHODS: Here, we originally used standard protocols for flow cytometry to characterize cell populations in human breast milk but failed to discriminate between cells and noncellular components. We then applied a centrifugation protocol to separate cream and skim milk from the cell-containing pellet and used a novel staining protocol with DRAQ5™ and SYTOX® blue dye as well as antibodies to characterize cells within the pellet fraction. RESULTS: Flow cytometry analysis identified viable DRAQ5™+ /SYTOX® Blue- cells and determined the content of CD11b+ monocytes and TRA-1-81+ putative stem cells in human breast milk samples. CONCLUSIONS: Hence, we developed a novel and reliable flow cytometry based-approach to quantify subpopulation of cells in human breast milk with a high content of milk fat globules, lipid droplets, and particles. This approach will improve the identification and quantification of breast milk cells and allow standardizing the flow cytometry-based evaluation of the stem cell content.
Authors: Natalia Ninkina; Michail S Kukharsky; Maria V Hewitt; Ekaterina A Lysikova; Larissa N Skuratovska; Alexey V Deykin; Vladimir L Buchman Journal: Hum Cell Date: 2019-04-10 Impact factor: 4.174