| dc.description.abstract | Breastfeeding supports the developing infant. In the oral cavity, extracellular vesicles in human milk (hmEVs) have been shown to interact with gingival epithelial cells. EVs are nanosized highly heterogeneous spherical particles with a bilipid membrane that facilitate intercellular communication through incorporated proteins, (small) RNA, and lipids. This report is a gathering of findings and learning points concerning the interaction between hmEVs and gingival epithelial cells. hmEVs of three donors were isolated using a combination of differential centrifugation, density gradient ultracentrifugation and size exclusion chromatography (SEC). Isolated hmEVs were stained with MemBright-488 to make them detectable. Next to hmEVs, a negative control hmEV-depleted sample was made. This hmEV-depleted sample was made from the supernatant after the density gradient ultracentrifugation step. The supernatant was loaded on a new density gradient, ultracentrifuged, and further processed the same as was done with the hmEV samples. Gingival epithelial cells (Ca922) were cultured in MEM with Penicillin-Streptomycin, Ultra-glutamine, and Fetal Calf Serum (FCS). For experiments, a part of this cell culture was transferred to a 48-wells plate. These cells attached themselves to the bottom of the 48-wells plate overnight. After attachment, the cells were starved for 2 hours in plain DMEM. Then the medium was replaced and MemBright-488 stained hmEVs/hmEV-depleted were added and incubated with the cells for consecutively 30-, 60-, and 150 minutes. After incubation, the cells were washed and resuspended with trypsin from the wells bottom for flow cytometry measurements using the FACSCanto II. Confocal microscopy with the Olympus /Evident Spin IXplore SoRa was done to test the flow cytometry findings and obtain more specific information about the kind of interaction occurring between hmEVs and gingival epithelial cells. Initial experiments were performed in the absence of FCS in the incubation medium to replicate previously published experiments. Results of these experiments show a significant increase in MemBright-488 signal of the gingival epithelial cells during the first 3 hours of incubation with hmEVs (p < 0.0001). After those 3 hours, the signal stabilises. This suggests that the MemBright-488 stained hmEVs either attached themselves to- or were taken up by gingival epithelial cells. The latter was also confirmed with confocal microscopy. Additionally, incubation conditions including FCS were tested which showed a remarkable difference. When FCS was present in the incubation medium, significantly less increase in MemBright-488 signal was observed in gingival epithelial cells after incubation with hmEVs (p < 0.0001). While trying to identify the mechanistic difference of incubation with- or without FCS, the effect of BSA and LDL were tested. Incubation conditions including BSA showed equivalent results to those in absence of FCS for each of the three donors (p = 0.6322, p = 0.9813, p = 0.9316). Incubation conditions including LDL, on the other hand, showed results like with incubation conditions including FCS (p = 0.9947). From these findings it can be concluded that stained purified hmEVs increasingly interact with gingival epithelial cells during the first three hours of incubation, which includes uptake. The use of FCS in the incubation medium had a profound effect on this stained hmEV interaction. We measured the interaction of hmEVs on gingival epithelial cells in the presence of BSA and/or LDL and observed that especially LDL significantly reduces this interaction. This indicates that components of the milk matrix and FCS strongly affect hmEV interactions in vitro. Therefore, the use of FCS or other components in the incubation medium of hmEV interaction essays should be carefully considered. | |
