Projects / Programmes
Characterization of in vitro model to study MAmmary Gland mIcrobial Colonisation (acronym - MAGIC)
| Code |
Science |
Field |
Subfield |
| 3.01.00 |
Medical sciences |
Microbiology and immunology |
|
| Code |
Science |
Field |
| B230 |
Biomedical sciences |
Microbiology, bacteriology, virology, mycology |
| Code |
Science |
Field |
| 3.01 |
Medical and Health Sciences |
Basic medicine |
mammary gland microbiota, epithelial cells, transcriptomics, mastitis, mammary gland dysbiosis, in vitro cell line models, immune response
Researchers (1)
| no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
| 1. |
31910 |
PhD Primož Treven |
Animal production |
Head |
2017 - 2019 |
75 |
Organisations (1)
Abstract
Human milk is one of the main sources of bacteria to the gut of breastfed infant. The process of colonisation is still debatable and most probably the bacterial biofilm is formed in mammary gland ducts. Despite established concept of mammary gland as microbial habitat with its own microbiota (not as contamination), to our knowledge, there are no studies of mammary gland microbial colonisation and possible biofilm formation, probably also due to the lack of appropriate well-characterised in vitro models. Currently no in vitro models are available to study microbial colonisation of the mammary gland and hypothetically formation of bacterial biofilm. The objective of this study is to build up a tool for a research of mammary gland microbial colonisation, i.e thoroughly characterised in vitro cell culture model for study of mammary gland microbial colonisation (MAGIC model). In addition, the purpose of this project is also to test the applicability of the MAGIC model to study the influence of probiotic and mastitis causing bacteria on adherence of bacteria from human milk microbiota.
In order to achieve the objectives of the study we will use immortalized cell line MCF10A, which is already known and well-characterised in the conditions of culturing on permeable support, where they express strong polarized phenotype similar to mammary gland ductal epithelium. This cell line has not been used for the research of host-microbiota relationships, before. The project will be divided in several work packages. In the first work package, we will optimise culturing conditions and determine reaction of the cells after exposure to pure bacterial cultures and human milk. In the core experiment (work package 2), we will seed MCF10A cell line on porous membrane and cultivate them until they reach confluence. Afterwards we will change the apical side medium with sterilised (control) or raw milk. In order to mimic suckling time, we will replace milk sample with fresh one every 4-8 hours. After 24-48 hours, we will perform various analysis. In the third part, we will test the applicability of the MAGIC model to study the influence of probiotic and mastitis causing bacteria on adherence of bacteria from human milk microbiota. For characterisation of the MAGIC model and evaluation of the potential of probiotics to influence the epithelium we will use several state-of-the-art technologies: for determination and/or quantification of adhered bacteria and investigation of bacterial composition of milk – deep sequencing of 16S rDNA amplicons and by qPCR, for analysis of bacterial adhesion and potential biofilm formation – scanning electron microscopy, and for quantification of response of epithelial cell line to milk samples and adhered bacteria – transcriptomic analysis with RT-qPCR and/or deep sequencing of collective bacterial RNA.
The results will give new knowledge about which bacteria bacteria from human milk microbiota can adhere to ductal epithelium, whether these adhered bacteria can form biofilm and what is the influence of milk microbiota on epithelial cells. Thoroughly characterized cell model to study mammary gland microbial colonisation will also open further possibilities for research in other fields of mammary gland physiology, like influence of bioactive milk components on mammary gland microbiota and epithelial cells or influence of pathogenic bacterial strains on bacterial colonisation. Furthermore, as recent studies showed, that resident mammary gland bacteria might be another important factor in breast cancer development, such in vitro tool would also open new possibilities in breast cancer research.
Significance for science
Mammary gland microbiota may be one of the important factors, which was overlooked in the past. Understanding the mechanism of mammary gland colonisation represents the key for understanding several processes, which occur in mammary gland. Thoroughly characterised cell model would represent novel in vitro tool for research of mammary gland microbial colonisation process. Consequently, such a tool will represent solid foundation for further research in many fields of mammary gland physiology, like studies of:
influence of bioactive milk components (milk fractions, human milk oligosaccharides, bioactive peptides, antimicrobials,…) on mammary gland microbiota development and epithelial cells;
influence of pathogenic bacterial strains on bacterial colonisation of mammary gland and its immune response;
influence of probiotics and development of new probiotic products for prevention and treatment of human mammary gland bacterial dysbiosis;
biofilm formation directly on epithelial cells;
a novel way for administration of probiotics to suckling infants via retrograde flow;
the role of mammary gland microbiota and specific bacterial strains on breast cancer development;
co-culture models where studies of interplay between bacteria, mammary gland epithelial cells and immune cells can be assessed.
Moreover, this project is designed to go beyond the state-of-the-art and will provide new data which would help to understand microbial colonisation of the mammary gland and the occurrence of dysbiosis and dysbiosis-related diseases. This knowledge will represent the basis for future research in order to modulate mammary gland microbiota in a way to promote health of mammary gland. Since recent studies show, that resident mammary gland bacteria may be another important factor in breast cancer development, such an in vitro tool would also bring new possibilities in cancer research and make even broader impact on scientific community.
Significance for the country
In order to create awareness of the importance of research to society, several outreach activities associated with the project development are planned. Even though the topic of this project lies in fundamental rather than applied science the topic has a great potential for public and economic outreach since the results would open new possibilities to develop, for example, new probiotics not only for treatment of mammary gland infections, but also to influence infant intestinal microbiota and to microbiota related diseases. The project will enable the development of new ingredients/probiotics for infant formula development and increase the knowledge of functional compounds presents in human milk. Thereby the project will have a direct impact on long-term health benefits for mothers, their infants and the European society. This proposal will reinforce the hypothesis that breast milk compounds might be exploited to promote microbiological, immunological and metabolic programming of infant health and will inform the development of new dietary strategies, functional foods and therapies. Moreover in long term, the conclusions from research based on MAGIC model, could improve the understanding of breast cancer development and consequently its treatment and prevention.
Most important scientific results
Final report
Most important socioeconomically and culturally relevant results
Final report
