|| Checking for direct PDF access through Ovid
The formation of epithelial microfold (M) cells is mediated through cell-to-cell interactions between enterocytes and lymphocytes. Based on this concept, we developed a cell-to-cell model by encouraging interactions between enterocyte C2BBe1 and Raji B cells through a preincubation approach. Raji B cells and C2BBe1 cells were allowed to interact in detached condition for 2 h at ratios of 1:1, 1:2 and 1:4 and then plated in culture plates. Monocultured C2BBe1 cells were used as the control. Flow cytometric analysis of the M cell-specific marker clusterin revealed that the optimum ratio of Raji B to C2BBe1 cells to obtain the maximum number of M cells was 1:1. Scanning electron micrographs exhibiting the lack of microvilli with complete tight junctions and Western blot analysis showing intense expression of clusterin confirmed the unique phenotypes of the formed M cells. Fluosphere® transport studies showed a 7-fold increase in the cell-to-cell model compared to the monoculture control. Importantly, we found that the induction of M cells could be enhanced by the effect of epithelial growth factor (EGF). C2BBe1 cells were pretreated with EGF at 10, 25 and 50 ng/mL before co-culturing with Raji B cells. Flow cytometric analysis of clusterin revealed that EGF significantly increased the formation of M cells. From mechanistic studies, we found an increase in the number of M cells involved the induction of stemness by EGF indicated by a dramatic increase in β-catenin, Nanog, and Oct-4, which in turn up-regulated the cell-to-cell interacting protein Integrin β-1. Furthermore, we confirmed the transport functions of the conventional, cell-to-cell, and cell-to-cell with EGF models using a Fluosphere® transport assay. Overall, we demonstrated an effective novel protocol for the formation of M cells as well as the effect of EGF on enhancing cell-to-cell interaction, which may benefit transport studies in M cells and promote better understanding of the biology of M cells.