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Spontaneous differentiation and organotypic tissue formation in 2D and 3D cultures of bovine mammary ductal stem‐like cells isolated directly from fresh milk samples

Following up on literature reports of the harvest and culture of stem cells from normal human breast milk, our group at the UWRF Tissue and Cellular innovation Center has focused an ongoing study on extending those earlier reports using bovine samples. And further, to apply the resulting monolayers to 3D hanging drop (HD) spheroid cultures in order to explore the formation of organotypic artificial ductal tissues. To date, we have successfully harvested, cultured and analyzed more than 8 samples from each of two cows in our University Farm herd. Samples were harvested for culture and flow cytometric analysis. During culture, cell morphology and behaviors were observed and documented and samples prepared for further labeling studies to confirm our flow cytometric data. Analysis of 2D cultures demonstrates that a distinctive stem‐like cell population was indeed isolated from each sample, although distinct differences in absolute cell yields were seen between cows and between multiple samples from the same cow. Isolated stem‐like cells were shown to have an overall dendritic appearance and displayed significant motility until arrival in the vicinity of differentiated epithelial cells, at which point these cells rapidly settled and laid down in proximity to the epithelial cells. Over the following 24 hours these stem‐like cells divided and underwent differentiation to increase the adjacent patches of epithelial tissue. Over a two month culture period, all samples retained a significant population of these motile, dendritic cells, suggesting that they actively divide in the culture conditions provided. Subsequent mammary ductal differentiation was observed in all the 2D monolayers formed. These included development of distinct regions containing lactocytes and evidence of milk fat generation, with layers of fat droplets floating in all wells with monolayers. Current efforts are now focused on the generation of hanging drop artificial tissues from our existing monolayer cultures which contain a combination of stem‐like and differentiated cells. Additional flow cytometry studies will also be performed on these monolayers to confirm the distribution of cells loaded into each HD well.