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P039 Tracking intestinal epithelial cells with fluorescent dyes

J. Seidelin*1, F. H. Bergenheim1, O. H. Nielsen1

1Herlev Hospital, University of Copenhagen, Department of Gastroenterology, Herlev, Denmark

Background

Enteroids have been shown to be able to engraft onto injured intestinal mucosa in murine experimental colitis models. This observation may provide an innovative approach to accomplish mucosal healing in patients with inflammatory bowel disease. Nevertheless, there are several issues to be resolved before this approach can be attempted in humans. One such issue is how to label and track transplanted cells. Hence, we investigated the applicability of a panel of non-gene modifying fluorescent dyes and nanoparticles, and whether labelled enteroids could be visualised using the clinically approved imaging modality, confocal laser endomicroscopy.

Methods

Intestinal biopsies were harvested from healthy human colonic mucosa, and enteroids were established using standard protocols. Enteroids were then attempted stained with fluorescein, a carbocyanine dye (CellBrite™), an inert membrane permeable dye, 5-chloromethylfluorescein diacetate (CMFDA; CellTracker™), quantum dots (QTracker™) and PLGA nanoparticles. Only 5–25 µM of CMFDA was found suitable, and staining homogeneity, durability, cell viability and enteroid forming capacity following single cell seeding were evaluated, together with visualisation of stained enteroids in vitro over time using endoscope-based confocal laser endomicroscopy.

Results

CMFDA efficiently and homogeneously stained all enteroids

Abstract PO39 – Figure 1

CMFDA stained enteroids. The viability and enteroid growth appeared to be unaffected by CMFDA staining

Abstract PO39 – Figure 2

Viability and enteroid forming capacity. (a, b) Whereas single cell seeding revealed a significant reduction in enteroid forming capacity with increasing dye concentration (c). No transfer of dye to unstained enteroids in co-cultures was observed. The CMFDA-derived fluorescent intensity of stained cells decreased in a linear fashion, with a t1/2 of approximately 24 h, and approached the background signal intensity after approximately 7 days. Furthermore, stained enteroids were easily identified in vitro using confocal laser endomicroscopy for a duration of at least 3 days (Figure 1b) .

Conclusion

It is plausible to track human intestinal enteroids using common fluorescent dyes (eg, CMFDA) and confocal laser endomicroscopy. This type of approach might clearly be limited to short-term tracking, which, however, may be sufficient to allow for confirmation of engraftment following transplantation.